A device and a method for seminological diagnostics

Abstract

A device for in vitro seminological diagnostics and spermatozoa selection, comprising: a selection system (110) that comprises: a body (111) made of plastic; an input chamber (112), located in the body (111), for receiving an ejaculate sample; a sorting channel (113), located in the body (111), having a first end (114) and a second end (116) and comprising a grid (200) of pillar obstacles (201) between the first end (114) and the second end (116); wherein the first end (114) of the sorting channel (113) is connected to the input chamber (112); and an output chamber (115), located in the body (111), connected to the second end (116) of the sorting channel (113), for receiving spermatozoa of the ejaculate sample that have migrated from the first end (114) to the second end (116) of the sorting channel (113); and an incubation chamber (120) for generating a temperature gradient in the selection system (110), such that the temperature at the input chamber (112) is lower than the temperature at the output chamber (115). In another aspect the invention relates to a method for spermatozoa selection using the device according to the invention.

Description

[0001] A DEVICE AND A METHOD FOR SEMINOLOGICAL DIAGNOSTICS

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a device for seminological diagnostics and a method for carrying out seminological diagnostics. It is specifically concerned with the diagnosis of male infertility and the positive selection of spermatozoa capable of normal fertilisation, characterised by significantly better DNA quality than the average for spermatozoa derived from the original ejaculate (without selection). It also pertains to a method for performing in vitro seminal observation of spermatozoa.

[0004] BACKGROUND

[0005] Sperm quality assessment is a crucial procedure used both for diagnosing male fertility disorders and for selecting sperm for further use, such as in assisted reproductive techniques (ARTs) using in vitro fertilisation (IVF). The procedure for assessing sperm quality can be carried out using various methods, considering different sperm parameters, such as morphology, motility testing, sperm count in the semen, or the sperm's response to various chemical and physical agents. Commonly used semen preparation techniques, referred to as the 'gold standard', involve either density gradient centrifugation (DGC) or swim-up. Both methods generate reactive oxygen species (ROS) during centrifugation, which increases the percentage of sperm with fragmented DNA. Poor sperm quality for in vitro fertilisation correlates with an increased risk of genetic defects in the embryo, lower embryo implantation rates, and lower live birth rates after IVF (Vasilescu et al., 2023).

[0006] The most widely used method of assessing ejaculate quality, with a particular focus on sperm quality, is semen microscopic evaluation, which allows the assessment of basic semen and sperm parameters. However, this method does not allow an easy and reproducible assessment of all important sperm parameters, such as chemotaxis or thermotaxis. Furthermore, this method does not allow effective selection from the ejaculate of sperm with the highest DNA quality, which can be used in further medical procedures.

[0007] Consequently, a number of methods and devices are being developed to more accurately assess the spermatozoa present in the ejaculate, as well as methods and devices for their effective selection. Such solutions make use of sperm parameters, such as the 'swimming upstream ability', different sperm morphology, or the presence of apoptosis markers. Recently, devices using taxis (chemotaxis and thermotaxis) have also been developed.

[0008] Spermatozoa that undergo capacitation (conditioning) in the female genital tract in vivo or in vitro by being placed in a medium containing human albumin (GM501 ) react to a temperature gradient. This response, called thermotaxis, also occurs in other cell types. Thermotaxis in mammalian spermatozoa plays an important role by directing sperm from a cooler location (the uterus and proximal section of the oviduct) to a warmer location (the bulb of the oviduct), where fertilisation occurs. The temperature difference is about 0.2°C. One of the changes in the structure of the sperm cell membranes that occurs during capacitation is an increase in the fluidity of the I ipid-protein bilayer of the sperm, due to the action of albumin, which causes the removal of cholesterol, other sterols, and non-covalently bound glycoproteins from the cell membranes. The presence of albumin further reduces the amount of sialic acid, gangliosides, and triglycerides present in the sperm cell membrane. During capacitation, which is necessary for the spermatozoon to respond to the temperature gradient, hyperactivation of the spermatozoon occurs, involving an increase in its motility leading to a change in the trajectory of the male gamete. Hyperactive spermatozoa move in a curved trajectory, allowing them to bind to the transparent envelope at a specific angle and then penetrate and fertilise the egg. Sperm that move in a rectilinear motion in the seminal fluid do not have the ability to fertilise the egg (Kratz and Achcihska, 2011 ).

[0009] From the data available in the literature, there is a weak negative correlation between the ability of sperm to move progressively (normal movement) and sperm DNA quality (r=-0.474, p<0.01 ). Fertile, healthy men have lower levels of DNA fragmentation relative to sperm characterised by abnormal movement (asthenozoospermia) from infertile men. Selection based on motility alone does not guarantee the selection of sperm with the highest DNA quality (and thus does not reduce the risk of miscarriage). The following documents describe a state of the art close to the invention presented here, but do not affect its novelty and inventive step.

[0010] International patent application WO2017127775A1 describes a microfluidic system designed for self-sorting highly motile, morphologically normal sperm from a fresh semen sample. The microfluidic system has one or more exit chambers for collecting sperm, and the centre of the channel through which the sperm travels contains variously microfabricated structures serving as sperm obstacles, allowing active and healthy sperm to move into the exit chambers. The distribution and profile of the obstacles designed into this type of microfluidic system should promote the progressive spiral movement of normal sperm and impede the movement of abnormal sperm. The disadvantage of this solution, however, is that it does not allow assessment of the thermotaxis of the tested sperm population, which is as important a qualitative parameter as sperm motility. Thus, the sorting results may not be fully satisfactory, and the selected spermatozoa may not be of sufficiently good quality.

[0011] International patent application WO2020212695A1 describes a device for sorting motile cells, in particular spermatozoa. The device includes a chamber, an inlet, and an outlet connected flowwise to the chamber, and a plurality of separate obstacles placed in the chamber. Each obstacle includes at least one wall and at least one sharp edge facing the outlet. As with the previously discussed solution, here too the structure of the device does not allow the assessment of sperm thermotaxis. This makes sperm selection insufficiently precise and does not allow for the isolation of a pool of sperm with the best parameters.

[0012] International patent application WO2020148474A1 relates to a sperm selection device that has heaters at the ends of the selection unit, which produce a temperature gradient allowing sperm to migrate from the loading compartment to the recovery compartment over a predetermined period of time. This makes it possible to select the spermatozoa with the highest migratory capacity relative to the spermatozoa from the original ejaculate (without selection). This solution allows the selection of the spermatozoa that are most responsive to the temperature gradient, but does not sufficiently investigate the regularity of the method of sperm movement. This makes the selection of spermatozoa using this device insufficiently accurate, and there is a risk that the pool of selected spermatozoa will contain spermatozoa that do not have a sufficiently good fertilising capacity.

[0013] European patent application EP1672977A1 relates to a device, system, and method for generating a sperm subpopulation enriched with high-quality sperm by exposing the sperm population to an appropriate temperature gradient and recovering the enriched sperm subpopulation for further applications such as diagnostics or infertility treatment. This solution only assesses the motility of the spermatozoa in the temperature gradient without allowing the evaluation of their additional parameters. This means that the selected sperm subpopulation may be of insufficient quality, which may significantly affect the efficiency of further medical procedures. Chinese utility model CN210886071 U relates to a medical device for assisted reproductive technology, in particular temperature gradient sperm screening. This device tests sperm motility, separating good quality sperm from the rest, thus improving the safety and efficiency of assisted reproductive technology. Unfortunately, as with the previously mentioned solutions using only sperm motility by thermotaxis to assess sperm quality, this solution has insufficient accuracy in selecting high-quality sperm.

[0014] In conclusion, the spermatozoa selection devices currently available on the market do not solve all the problems associated with in vitro spermatozoa preparation for use in assisted reproductive techniques (ARTs). Furthermore, the diagnostic utility of current solutions is quite limited. The inconveniences and disadvantages associated with these devices include insufficient efficiency of the selection process and not very accurate spermatozoa selection. Usually, one comes at the expense of the other - devices that have a high quality of spermatozoa selection are not very efficient, while those with a good efficiency of the selection process tend to be inaccurate in terms of the quality of the selected spermatozoa.

[0015] SUMMARY

[0016] In view of the drawbacks of the prior art, there is a need to develop a device that allows the accurate selection of spermatozoa that can later be used for further medical procedures and whose number indicates the real quality of the DNA contained in the patient's semen, thus allowing reliable diagnosis and improving the effectiveness of the ARTs procedures carried out. Such a solution should use several spermatozoa characteristics for selection that correlate well with the quality of the spermatozoa and, in particular, with the quality of the genetic material (DNA) they carry. Such a device will make it possible to select high-quality spermatozoa, which will translate into a reliable assessment of the quality of the ejaculate and into the effectiveness of medical procedures carried out using them.

[0017] The object of the invention is a device for in vitro seminological diagnostics and spermatozoa selection, comprising a selection system that comprises: a body made of plastic; an input chamber, located in the body, for receiving an ejaculate sample; a sorting channel, located in the body, having a first end and a second end and comprising a grid of pillar obstacles between the first end and the second end; wherein the first end of the sorting channel is connected to the input chamber; and an output chamber, located in the body, connected to the second end of the sorting channel, for receiving spermatozoa of the ejaculate sample that have migrated from the first end to the second end of the sorting channel. The device further comprises an incubation chamber for generating a temperature gradient in the selection system, such that the temperature at the input chamber is lower than the temperature at the output chamber.

[0018] The device according to the invention offers several significant benefits that collectively demonstrate its inventive step and distinguish it from existing solutions for spermatozoa selection. At its core, the device integrates a selection system comprising a sorting channel with a grid of pillar obstacles and an incubation chamber capable of generating a temperature gradient, where the input chamber is cooler than the output chamber. This innovative combination enhances the selection of high-quality spermatozoa by leveraging both physical obstacles and thermotactic stimuli, effectively selecting spermatozoa based on motility and thermotaxis — critical factors for successful fertilisation. By simulating physiological conditions, the device mimics the natural thermal environment of the female reproductive tract, encouraging only the most motile and thermotactically responsive spermatozoa to migrate toward the output chamber. This approach leverages the innate behaviours of spermatozoa, leading to a more physiologically relevant selection process and improving the chances of successful fertilisation.

[0019] Furthermore, the integration of physical and thermal barriers represents a significant advancement over existing spermatozoa selection methods. The grid of uniquely designed pillar obstacles within the sorting channel physically challenges spermatozoa, allowing only those with adequate motility to navigate through, while the temperature gradient induces a thermotactic response. This synergistic effect enhances the selectivity of the device beyond what is achievable with either method alone, showcasing a non-obvious solution not suggested in prior art. Additional benefits include the device's non-invasive and chemical-free operation, reducing the risk of spermatozoa damage that can occur with methods involving centrifugation, magnetic fields, or chemical additives. By minimising external interventions, the device preserves the viability and DNA integrity of the selected spermatozoa, which is crucial for maintaining their fertilisation potential.

[0020] The device also offers efficiency and speed, allowing for rapid sorting of spermatozoa and reducing the time between sample collection and availability for use in assisted reproductive techniques. Its design facilitates customisable parameters, such as adjustable temperature gradients and variable obstacle configurations, enabling optimisation for different spermatozoa samples or species. Ease of manufacturing and accessibility are further advantages, as the device is constructed from readily available biocompatible plastics, making it cost-effective and suitable for single-use applications, thereby reducing the risk of cross-contamination between samples. Additionally, the transparent components of the device allow for real-time microscopic observation of spermatozoa movement during the selection process, providing valuable insights for diagnostics and research.

[0021] When compared to similar solutions used for spermatozoa selection, such as density gradient centrifugation, swim-up techniques, microfluidic devices without temperature gradients, magnetic-activated cell sorting (MACS), and zeta potential selection, the device according to the invention stands out due to its unique combination of physical obstacles and a temperature gradient within a single, easy-to-use system. While other methods may select spermatozoa based on density, motility, or surface charge, they often lack the ability to simulate physiological thermotactic conditions or may involve processes that can damage spermatozoa or introduce foreign substances. In contrast, the device according to the invention enhances the selection of high-quality spermatozoa by replicating natural selection mechanisms, providing a non-obvious and effective solution that significantly advances the field of spermatozoa selection.

[0022] Preferably, the input chamber and the output chamber has a volume between 20 pl and 200 pl. This provides precise control over sample and medium volumes, ensuring consistent experimental conditions and efficient use of biological materials. This optimal volume range is critical for balancing sufficient sample size with practical microfluidic design, an advantage not suggested in prior art, thereby contributing to the inventive nature of the device.

[0023] Preferably, the sorting channel has a length between 10 mm and 50 mm, a width between 2 mm and 20 mm, and a height between 40 pm and 1000 pm. These characteristics offer significant advantages in optimising spermatozoa transit time and selection efficiency. These specific dimensions ensure that spermatozoa have adequate space to navigate while maintaining the physical constraints necessary for effective selection based on motility, which is not obvious from existing devices and demonstrates an inventive step in enhancing performance.

[0024] Preferably, the pillar obstacles are shaped as sections of a straight elliptical cylinder. The axis of symmetry is the major axis of the ellipse underlying the elliptical cylinder. The length of the major axis ranges from 500 pm to 1000 pm, while the minor axis ranges from 50 pm to 300 pm. The ratio of the major axis length to the minor axis length is between 1 and 20. The height of the elliptical cylinder is between 40 pm and 1000 pm, and the internal angle of the elliptical cylinder slice ranges from 20° to 75°, with the apex pointing towards the output chamber. Pillar obstacles shaped as sections of straight elliptical cylinders with precise geometric parameters selectively impede spermatozoa with abnormal motility while facilitating the movement of motile spermatozoa toward the output chamber. The innovative geometry, including specific ratios of major to minor axes and internal angles, is not disclosed in prior art and provides a non-obvious solution to improving spermatozoa selection accuracy, thus contributing to the inventive step.

[0025] Preferably, the pillar obstacles are spaced at intervals of 10 pm to 300 pm with respect to each other. Specifying that pillar obstacles are spaced at intervals of 10 pm to 300 pm creates optimal pathways that challenge spermatozoa motility without causing blockages. This precise spacing enhances the selection of high-motility spermatozoa while preventing less motile spermatozoa from passing through. Such detailed control over obstacle spacing is not suggested in existing technologies and represents an inventive approach to improving the device's selectivity and efficiency.

[0026] Preferably, the pillar obstacles are arranged in quantities of 3 to 15 per mm2. This provides an optimal obstacle density that balances effective spermatozoa selection with the prevention of channel clogging. This range allows customisation based on sample variability and is critical for achieving desired selection outcomes. Considering obstacle density as a functional parameter is a non-obvious advancement over prior art, demonstrating an inventive contribution to the field.

[0027] Preferably, the pillar obstacles occupy 60 to 95% of the sorting channel. The device in that configuration maximises interaction between spermatozoa and obstacles, enhancing the selection process. This substantial obstacle coverage reduces unintended fluid flow that could carry less motile spermatozoa, ensuring only spermatozoa with sufficient motility reach the output chamber. Such a high coverage is not evident in prior devices and represents an inventive feature that improves selection accuracy.

[0028] Preferably, the grid of pillar obstacles has an arrangement selected from: a rectangular topology, a hexagonal topology or a random topology. Provision for various grid arrangements — rectangular, hexagonal, or random topology — offers adaptability to different selection needs, influencing spermatozoa movement patterns and allowing the device to be tailored for specific diagnostic or research requirements. This flexibility in grid design is not suggested in prior art and demonstrates an inventive approach to optimising spermatozoa selection, contributing to the device's overall effectiveness.

[0029] Preferably, the incubation chamber is configured to generate the temperature gradient in the selection system such that the temperature at the input chamber is lower than the temperature at the output chamber by at least 0.5°C and at most 7°C. Specifying that the temperature in the input chamber is lower than in the output chamber by at least 0.5°C and at most 7°C fine-tunes the thermotactic stimulus, promoting the movement of high-quality spermatozoa without causing thermal stress. This precise and narrow temperature range is not obvious from existing methods and enhances the device's effectiveness in selecting motile spermatozoa, representing an inventive step.

[0030] Preferably, the incubation chamber is configured to generate the temperature gradient in the selection system such that there is a linear temperature gradient between 0.02°C / mm and 0.7°C / mm between the input chamber and the output chamber. This ensures a controlled thermotactic environment conducive to spermatozoa migration. This specific gradient closely simulates physiological conditions, improving the relevance and effectiveness of the selection process. Implementing such a precise gradient is not suggested in prior art and represents an inventive feature of the device.

[0031] Preferably, the incubation chamber comprises: a plate of a precise temperature gradient, configured to receive the selection system and made of a thermally conductive material; temperature control means located on opposite sides of the plate, perpendicular to a longitudinal axis of the sorting channel. Furthermore, the temperature control means is configured to produce a first temperature at the input chamber and a second temperature at the output chamber, wherein the first temperature is lower than the second temperature. The incubation chamber with a plate of a precise temperature gradient made of thermally conductive material and temperature control means on opposite sides provides enhanced temperature control accuracy essential for maintaining the linear gradient across the sorting channel. The innovative structural integration of these components is not evident in existing technologies and contributes to the inventive step by improving device reliability and selection consistency.

[0032] Preferably, the temperature control means is a ceramic heater, a Peltier cell or a liquid jacket. This provides optimal methods for temperature control.

[0033] Preferably, the incubation chamber comprises a liquid container configured to maintain the humidity of the air in the incubation chamber at a higher level than the humidity of the air surrounding the incubation chamber in such as to reduce evaporation of the liquid from the selection system. This reduces evaporation from the selection system, ensuring consistent medium concentration and volume. This environmental control enhances the reliability of the selection process and is not suggested in existing devices, representing an inventive solution to a technical problem associated with microfluidic systems.

[0034] Preferably, the device according to the invention is configured to allow microscopic observation of the spermatozoa during the examination. This configuration offers realtime monitoring of spermatozoa behaviour, integrating selection and observation within a single device. This design improves efficiency over prior art that requires separate equipment, and the innovative facilitation of optical accessibility contributes to the inventive step by enhancing diagnostic capabilities.

[0035] Preferably, the selection system comprises at least two sorting channels, each of sorting channels has a dedicated input chamber and a dedicated output chamber. This design enhances efficiency in clinical and research settings and allows for comparative studies within the same device. Including multiple channels with dedicated chambers is a non-obvious modification over single-channel devices, demonstrating an inventive advancement in device functionality.

[0036] In another aspect, the invention relates to a method for selecting spermatozoa using the device according to the invention. The method comprises the following steps: heating the input chamber and the output chamber by the temperature control means to a temperature between 35°C and 45°C, so that the temperature in the input chamber is lower by 0.5°C to 7°C than the temperature in the output chamber, and a linear temperature gradient is formed in the sorting channel; filling the selection system with a liquid medium; introducing the ejaculate sample into the input chamber; incubating the ejaculate sample under the temperature gradient for a time duration of between 10 and 60 minutes; and collecting, from the output chamber, the liquid medium containing spermatozoa of the ejaculate sample that have passed through the sorting channel.

[0037] The benefits of this method are multifaceted and stem from its precise control over environmental conditions, which closely mimic the natural physiological processes involved in spermatozoa migration within the female reproductive tract. By establishing a controlled temperature gradient, the method exploits the thermotactic behaviour of spermatozoa, a natural mechanism wherein spermatozoa cells move in response to temperature changes, typically moving from cooler to warmer environments. This thermotactic selection ensures that only spermatozoa with optimal motility and responsiveness to temperature cues progress through the sorting channel, thereby enhancing the overall quality of the selected spermatozoa population.

[0038] An additional benefit of this method is its ability to preserve the integrity and viability of spermatozoa. The controlled temperature range prevents thermal stress that could damage spermatozoa cells, and the incubation times are optimised to allow sufficient selection without exposing the spermatozoa to prolonged periods that could lead to the generation of reactive oxygen species (ROS), which can cause DNA damage. By limiting the incubation time to a maximum of 60 minutes, the method reduces the risk of oxidative stress, thereby maintaining the genetic integrity of the spermatozoa, which is crucial for successful fertilisation and embryo development.

[0039] Furthermore, the method is non-invasive and does not require the addition of chemical agents or physical manipulations that could harm the spermatozoa. The use of a medium that supports spermatozoa viability and capacitation, along with the gentle thermal and physical environment provided by the apparatus, ensures that the selected spermatozoa are in optimal condition for use in assisted reproductive technologies. This contrasts with other spermatozoa selection methods that may involve centrifugation, exposure to magnetic fields, or the addition of chemicals, all of which can introduce stress or potential damage to the spermatozoa cells.

[0040] The method's specific steps, including precise temperature control and incubation times, allow for reproducibility and consistency across different samples and settings. This standardisation is particularly beneficial in clinical applications, where reliable and predictable outcomes are essential. By providing detailed protocols, the method reduces variability and enhances the efficiency of spermatozoa selection, making it a valuable tool in both clinical and research environments.

[0041] Distinguishing features of this method include the use of a linear temperature gradient in combination with a physical selection mechanism provided by the grid of pillar obstacles within the sorting channel. This dual-selection approach is not evident in prior art and represents an innovative means of enhancing spermatozoa selection. The method leverages both thermotaxis and motility, two critical factors in natural spermatozoa migration, to select spermatozoa that are not only motile but also responsive to temperature cues, which may correlate with better fertilisation potential.

[0042] Compared to similar solutions used for spermatozoa selection, this method offers several advantages. Traditional methods such as density gradient centrifugation and swim-up techniques primarily select spermatozoa based on motility and density but do not account for thermotactic behaviour. Moreover, these methods may subject spermatozoa to physical stresses that can compromise their viability. Magnetic-activated cell sorting (MACS) and zeta potential selection involve labelling spermatozoa with antibodies or exploiting surface charge differences, respectively, which may not directly correlate with the spermatozoa's functional capabilities and could introduce foreign substances.

[0043] Microfluidic devices have been employed for spermatozoa selection based on motility, but they often lack the incorporation of a temperature gradient and may rely solely on physical constrictions or flow dynamics. The method of the present invention uniquely combines the physical challenges of navigating through a grid of obstacles with the biological stimulus of a temperature gradient, providing a more comprehensive selection process that aligns closely with natural spermatozoa behaviour.

[0044] Below is a more detailed overview of specific elements of the system, including a non-limiting discussion of its possible alternatives and equivalents.

[0045] The device for in vitro seminological diagnostics and spermatozoa selection according to the invention is designed to select high-quality spermatozoa by evaluating their motility and thermotaxis, crucial factors for successful fertilisation. The essence of the device lies in its ability to simulate natural conditions that favour the movement of healthy spermatozoa, thus enhancing the chances of successful in vitro fertilisation. Alternative forms of this device could include variations that cater to different animal species or integration with automated systems for higher throughput.

[0046] The selection system is the core component where the actual spermatozoa selection occurs. Its essence is to provide a controlled environment that physically and thermally challenges spermatozoa, allowing only those with optimal motility and thermotaxis to progress. Alternative forms might involve different channel geometries or obstacle configurations to adjust the selection criteria.

[0047] The body of the selection system might be made of biocompatible plastic like PDMS or PMMA. The body houses the sorting channel and chambers. Its essence is to offer a sterile, inert, and transparent structure suitable for biological assays and microscopic observation. Alternative materials could include glass or other polymers with similar properties. Possible equivalents are any structural materials used in biomedical devices that ensure biocompatibility and transparency, such as cyclic olefin polymers. The input chamber is where the spermatozoa sample is introduced into the selection system. Its essence is to serve as the starting point for spermatozoa migration under controlled conditions, including temperature and medium composition. Alternative forms might include chambers with varying volumes or shapes to accommodate different sample sizes or facilitate loading. Equivalents could be sample wells or reservoirs in other cell-sorting devices.

[0048] The sorting channel connecting the input and output chambers contains the grid of pillar obstacles. Its essence is to create a pathway that selectively allows motile spermatozoa to navigate through, effectively filtering out less motile or defective spermatozoa. Alternative forms could involve channels with different lengths, widths, heights, or obstacle arrangements to customise the selection process.

[0049] The output chamber collects spermatozoa that have successfully navigated the sorting channel, representing a selection of high-quality spermatozoa. Its essence is to serve as the collection point for spermatozoa suitable for further medical procedures. Alternative forms might include chambers with integrated sensors for real-time analysis or outlets for immediate downstream processing. Equivalents could be collection reservoirs in other cell separation systems or microfluidic outlets connected to analytical instruments.

[0050] The incubation chamber encloses the selection system and generates a linear temperature gradient essential for assessing spermatozoa thermotaxis. Its essence is to mimic the natural thermal environment that influences spermatozoa movement towards the ovum. Alternative forms might include chambers with different heating mechanisms, such as infrared heating or fluidic temperature control. Equivalents might be any environmental control units used in biological assays that provide precise temperature regulation, like incubators with gradient capabilities.

[0051] The precision temperature gradient plate made of thermally conductive material like copper ensures a uniform temperature gradient across the sorting channel. Its essence is to facilitate a stable and linear temperature difference from the entrance to the output chamber, critical for accurate thermotaxis assessment. Alternative materials could include aluminium or graphene-enhanced composites. Equivalents might be any device that can create a controlled temperature gradient, such as thermoelectric modules arranged to produce a linear gradient.

[0052] Temperature control means are devices like ceramic heaters or Peltier cells mounted on or near the precision temperature gradient plate. Their essence is to precisely regulate the temperatures at both ends of the sorting channel, establishing the necessary gradient. Alternative forms might include resistive heating elements or liquidbased thermal baths. Equivalents could be any thermal control systems capable of maintaining precise temperature differentials, such as digitally controlled heating blocks.

[0053] The liquid container located within the incubation chamber holds water to maintain higher humidity, reducing evaporation from the selection system. Its essence is to preserve the medium's volume and concentration, ensuring consistent experimental conditions. Alternative forms could include integrated humidifiers or sealed environments with controlled humidity. Equivalents might be moisture control systems used in cell culture incubators or desiccator cabinets with adjustable humidity.

[0054] Situated within the sorting channel, the grid comprises multiple pillar obstacles designed to impede spermatozoa with abnormal motility. The essence of the grid is to create a physical barrier that selects for spermatozoa with optimal motility patterns. Alternative forms could involve different obstacle shapes, densities, or arrangements, such as using microfabricated posts or labyrinthine pathways. Equivalents might be microstructures in cell sorting devices that use physical barriers to influence cell trajectories, like deterministic lateral displacement arrays.

[0055] These individual obstacles are shaped as slices of straight elliptical cylinders, influencing spermatozoa movement through their geometry. The essence of the pillar obstacles is to create an environment that challenges spermatozoa, allowing only those with sufficient motility and directional movement to pass. Alternative shapes could include circular, rectangular, or custom-designed forms tailored to specific selection pressures. Equivalents might be any microfabricated structures that interact with cells to influence movement, such as nanopillars or microgrooves.

[0056] In some embodiments, multiple sorting channels are included for parallel analysis or increased throughput. The essence of these additional channels is to provide flexibility, allowing simultaneous processing of multiple samples or control experiments. Alternative configurations might involve channels with different orientations or properties, such as varying obstacle designs for comparative studies. Equivalents could be multi-lane microfluidic devices used in other applications, like lab-on-a-chip systems for multiplexed assays.

[0057] These are separate input and output chambers for each additional sorting channel. Their essence is to prevent cross-contamination between samples and ensure independent operation of each channel. Alternative forms might include shared chambers with fluidic barriers or valves. Equivalents could be individual wells in multiwell plates or separate inlets and outlets in parallel microfluidic networks.

[0058] The essence of the method according to the invention is to replicate natural spermatozoa selection processes by combining physical and thermal challenges, thereby isolating spermatozoa with superior motility and genetic integrity. Alternative methods might adjust the temperature gradient, incubation time, or medium composition to optimise selection for different conditions.

[0059] In summary, each element and step of the invention is thoughtfully designed to enhance the selection of high-quality spermatozoa by simulating natural selection mechanisms within a controlled environment. The alternative forms and equivalents provide flexibility in adapting the device and method to various applications, species, or research needs, ensuring that the core essence of selecting optimal spermatozoa remains central to the invention's purpose.

[0060] BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The invention is illustrated with embodiments in the figures:

[0062] FIG. 1 schematically shows an embodiment of a device for seminological diagnostcis and in vitro spermatozoa selection;

[0063] FIG. 2 schematically shows an embodiment of the selection system;

[0064] FIG. 3A-C show examples of embodiments of a pillar obstruction in horizontal section;

[0065] FIG. 3D shows an embodiment of a pillar obstruction in vertical section;

[0066] FIG. 4A-C show embodiments of pillar obstruction grid;

[0067] FIG. 5A-C show embodiments of selection systems;

[0068] FIG. 6 shows schematically the method according to the invention;

[0069] FIG. 7 shows the results of DNA fragmentation analysis of spermatozoa before selection compared to spermatozoa obtained after 30 minutes selection using the device according to the invention;

[0070] FIG. 8 shows the results of DNA fragmentation of spermatozoa before and after selection using a device containing pillar obstacles and a temperature gradient and spermatozoa that were selected using only the pillar obstacle track without a temperature gradient;

[0071] FIG. 9 shows the results of DNA fragmentation of spermatozoa before and after selection with a device according to the invention and a reference device. DETAILED DESCRIPTION

[0072] FIG. 1 shows an embodiment of a device 100 for in vitro seminological diagnostics and spermatozoa selection according to the invention. The device 100 comprises a selection system 110 and an incubation chamber 120, which separates the selection system 110 from the environment and generates a linear temperature gradient in the selection system 110. The device 100 allows for the selection of high-quality spermatozoa by testing motility and thermotaxis. High-quality spermatozoa exhibit normal motility, appropriate thermotaxis, and high genetic integrity, with a DNA fragmentation index (DFI) below 30%.

[0073] The selection system 110, shown in an embodiment in FIG. 2, comprises a body 111 made of plastic, preferably a visible light-permeable, biocompatible plastic (e.g., polydimethylsiloxane (PDMS), poly(methyl methacrylate) (PMMA)). A sorting channel 113 is formed in the body 111 , with an input chamber 112 at the first end 114 and an output chamber 115 at the second end 116. The input chamber 112 receives the ejaculate sample for the sorting channel 113, which contains a grid 200 of pillar obstacles 201. The output chamber 115 collects spermatozoa that migrate through the sorting channel 113. The system may be microfluidic, with a small cross-sectional area that significantly restricts fluid movement.

[0074] The incubation chamber 120 generates a linear temperature gradient, with the input chamber 112 cooler than the output chamber 115. A precision temperature gradient plate 121 , made of a thermally conductive material (e.g., copper), ensures uniform energy distribution for a linear gradient. Temperature control means 122, 123, such as ceramic heaters or Peltier cells, are mounted on or near the plate 121. The temperature gradient may increase linearly along the channel 113. High-precision temperature sensors allow real-time control of system heating. The temperature control means 122, 123 are powered by a controller-controlled source (not shown). A water jacket may also provide a linear gradient. A heat sink and speed-controlled fan may be used for effective temperature control.

[0075] The input chamber 112, sorting channel 113, grid 200 of pillar obstacles 201 , and output chamber 115 can be fabricated by injection moulding, CNC milling, 3D printing, casting, or other known methods. In a preferred embodiment, the input and output chambers have volumes between 20 pl and 200 pl, with each chamber potentially having a different volume within this range.

[0076] In a preferred embodiment, the sorting channel 113 has a length L between 10 mm and 50 mm, a width S between 2 mm and 20 mm, and a height between 40 pm and 1000 pm. Preferably, the sorting channel 113 has a length L between 10 mm and 30 mm (e.g., 10 mm), a width S between 5 mm and 10 mm (e.g., 8 mm), and a height between 50 pm and 200 pm (e.g., 150 pm). It should be noted that as the length L of the sorting channel 113 increases, the time required for spermatozoa to traverse it also increases, potentially extending preparation time and adversely affecting the quality of selected spermatozoa due to the generation of reactive oxygen species (ROS), which may damage the genetic material of the spermatozoa, ultimately reducing their value in subsequent medical procedures. Conversely, a sorting channel 113 that is too short will not allow effective separation of high-quality and low-quality spermatozoa fractions. The width S of the sorting channel 113 affects the throughput of the selection system 110. A greater width S allows more spermatozoa to pass through, but increasing the width S may reduce the selectivity of the device due to the limited number of high-quality spermatozoa present in the processed ejaculate. Therefore, to obtain a higher number of high-quality spermatozoa, it may be preferable to use two or more sorting channels 113 with a smaller width S, each having a dedicated input chamber 112 and output chamber 115, instead of a single sorting channel 113 with a larger width S.

[0077] In a preferred embodiment, the pillar obstacles 201 are substantially shaped as a slice of a straight elliptical cylinder, with the major axis of the ellipse serving as the axis of symmetry. The slice may be centric (i.e., arms extending from the centre of the elliptical cylinder) or non-centric (i.e., arms extending from a point away from the centre, with equal or different lengths). Preferably, the length of the major axis of the ellipse is in the range of 500 pm to 1000 pm (preferably 700 pm to 900 pm, e.g., 800 pm) and the minor axis from 50 pm to 300 pm (preferably 75 pm to 150 pm, e.g., 100 pm). Preferably, the ratio of the major axis length to the minor axis length is in the range of 1 to 20 (more preferably 5 to 10, e.g., 8). Preferably, the height H of the elliptical cylinder is in the range of 40 pm to 1000 pm (more preferably 50 pm to 200 pm, e.g., 150 pm) and the interior angle a of the elliptical cylinder slice is in the range of 20° to 75° (more preferably 30° to 60°, e.g., 45°). The apex of the cylinder slice is directed towards the output chamber 115. In a particular implementation example, the straight elliptical cylinder may be a straight circular cylinder. This shape of the pillar obstacles 201 effectively blocks spermatozoa with abnormal motility and promotes the movement of spermatozoa with proper motility along the sorting channel 113. Examples of pillar obstacles with different dimensions are shown in FIGs. 3A-D.

[0078] In a preferred embodiment, the number of pillar obstacles 201 forming the grid 200 is in the range of 3 obstacles / mm2to 15 obstacles / mm2(preferably 5 obstacles / mm2to 10 obstacles / mm2, e.g., 7 obstacles / mm2). The density of pillar obstacles 201 within this range optimises the spermatozoa selection process.

[0079] In a preferred embodiment, the pillar obstacles 201 may occupy between 60% and 95% of the area of the sorting channel 113 (e.g., 88%). The pillar obstacles 201 may be spaced at intervals of 10 pm to 300 pm.

[0080] In a preferred embodiment, the grid 200 of pillar obstacles 201 has an arrangement selected from: a rectangular topology (Fig. 4C), a hexagonal topology (Fig. 4A), or a random topology (Fig. 4B). Examples of the grid 200 implementations are shown in Figs. 4A-C. Each arrangement is useful for selecting high-quality spermatozoa.

[0081] In a preferred embodiment, during testing, the input chamber 112 has a temperature lower than the output chamber 115 by at least 0.5°C and a maximum of 7°C. This is due to the generation of a linear temperature gradient, which allows the assessment of spermatozoa thermotaxis from the test pool.

[0082] In a preferred embodiment, a linear temperature gradient between 0.02°C / mm and 0.7°C / mm occurs during the test between the input chamber 112 and the output chamber 115. This gradient corresponds to natural conditions for spermatozoa mobility and favours the selection of high-quality spermatozoa.

[0083] The temperature at the input chamber 112 can be set between 35°C and 45°C, and the temperature at the output chamber 115 can be set according to the adopted gradient and channel length.

[0084] In a preferred embodiment, the incubation chamber further comprises a container 124 with liquid, particularly water, configured to maintain higher humidity in the chamber than the surrounding air, reducing liquid evaporation from the selection system.

[0085] In a preferred implementation example, the device 100 is configured to allow microscopic observation of spermatozoa during examination. This is achieved by using translucent (preferably transparent) elements, such as glass or sapphire, in the selection system 110, through which a light beam passes during observation. This solution allows real-time tracking of spermatozoa movement, which may be important for medical reasons.

[0086] In a preferred implementation example, the selection system 110 comprises at least two sorting channels 113A, 113B, each with a dedicated input chamber 112A, 112B, and an output chamber 115A, 115B. This allows for parallel analysis of several samples or increased throughput to obtain more high-quality spermatozoa for a given patient. Examples of the invention's embodiments with one and two channels are shown in Fig. 5A and 5B. In one embodiment shown in Fig. 5C, one of the sorting channels 113C may be perpendicular to the temperature gradient and serve as a channel for performing a negative test to select spermatozoa based on their ability to overcome the grid 200 of pillar obstacles 201 , which may be important in scientific applications.

[0087] In a preferred implementation example, the selection system 110 is filled with a medium. Capacitation media, particularly those containing serum proteins like albumin, or in vitro semen preparation media buffered with HEPES or bicarbonate, may be used.

[0088] Depending on the arrangement of the pillar obstacles 201 and the dimensions of the sorting channel 113, the device 100 can be used for analysing and selecting animal spermatozoa, particularly human spermatozoa, but also spermatozoa from other animal species, e.g., equine or canine.

[0089] In one aspect, the invention relates to a method for spermatozoa selection using the device according to the invention, wherein the method comprising the following steps:

[0090] - heating 401 the input chamber 112 and the output chamber 115 by the temperature control means 122 to a temperature between 35°C and 45°C, so that the temperature in the input chamber 112 is lower by 0.5°C to 7°C than the temperature in the output chamber 115, and a linear temperature gradient is formed in the sorting channel 113;

[0091] - filling 402 the selection system 110 with a liquid medium;

[0092] - introducing 403 the ejaculate sample into the input chamber 112;

[0093] - incubating 404 the ejaculate sample under the temperature gradient for a time duration of between 10 and 60 minutes; and

[0094] - collecting 405, from the output chamber 115, the liquid medium containing spermatozoa of the ejaculate sample that have passed through the sorting channel 113.

[0095] This method for spermatozoa selection yields a pool of high-quality spermatozoa suitable for further medical procedures. Example 1 : Making a selection system with one sorting channel

[0096] The selection system 110 was manufactured using an injection moulding method, which defined an input chamber 112, an output chamber 115, and a sorting channel 113 containing a grid of 485 pillar obstacles 201. The components of the selection system 110 were filled with GM501 medium. The volume of both the input chamber 112 and output chamber 115 was 110 pl. The sorting channel 113 was 10 mm long, 8 mm wide, and 150 pm high. The grid of 485 pillar obstacles 201 had a density of 7 obstacles / mm2and a rectangular topology. All pillar obstacles had the shape of a slice of a straight elliptical cylinder. The major axis of the ellipse was 1000 pm, the minor axis was 50 pm, the height of the elliptical cylinder was 200 pm, and the interior angle of the slice was 75°.

[0097] Example 2: Making a selection system with two sorting channels

[0098] The selection system 110 was fabricated using a 3D printing method, which enabled the creation of two parallel sorting channels 113, each containing a grid 200 of pillar obstacles 201 , with dedicated input chambers 112 and output chambers 115. The individual components of the selection system 110 were filled with an in vitro spermatozoa preparation medium containing human serum albumin. The volume of both input chambers 112 was 200 pl, while the volume of each output chamber 115 was 20 pl. Both sorting channels 113 had the same dimensions: a length of 50 mm, a width of 20 mm, and a height of 1000 pm. The grid 200 of pillar obstacles 201 had a density of 10 obstacles / mm2and a hexagonal topology. All pillar obstacles were shaped as slices of a straight elliptical cylinder. The major axis of the ellipse underlying the cylinder was 500 pm, and the minor axis was 300 pm. The height of the elliptical cylinder was 1000 pm, and the interior angle of the elliptical cylinder slice was 30°.

[0099] Example 3. Making a selection system with two sorting channels and a channel for negative control

[0100] The selection system 110 was fabricated using a CNC milling method, which created three sorting channels 113, each containing a grid of 200 pillar obstacles 201 , with dedicated input chambers 112 and output chambers 115. Two of the sorting channels 113 were parallel, and the third channel was perpendicular to them. The individual components of the selection system 110 were filled with GM501 medium. The volume of all input chambers 112 was 200 pl, while the volume of each output chamber 115 was 200 pl. All sorting channels 113 had the same dimensions: a length of 30 mm, a width of 10 mm, and a height of 200 pm. The grid 200 of pillar obstacles 201 had a density of 15 obstacles / mm2and a random topology. The pillar obstacles were shaped as slices of a straight elliptical cylinder. The major axis of the ellipse underlying the cylinder was 700 pm, and the minor axis was 150 pm. The height of the elliptical cylinder was 200 pm, and the interior angle of the elliptical cylinder slice was 60°.

[0101] Example 4. Making a selection system with two sorting channels with a grid of pillar obstacles with a rectangular topology

[0102] The selection system 110 was fabricated using a 3D printing method, which enabled the creation of two parallel sorting channels 113, each containing a grid 200 of pillar obstacles 201 , with dedicated input chambers 112 and output chambers 115. The individual components of the selection system 110 were filled with a medium for in vitro spermatozoa preparation and / or capacitation. The volume of both input chambers 112 was 200 pl, while the volume of each output chamber 115 was also 200 pl. Both sorting channels 113 had the same dimensions: a length of 20 mm, a width of 8 mm, and a height of 150 pm. The grid 200 of pillar obstacles 201 had a density of 7 obstacles / mm2and a rectangular topology. All pillar obstacles were shaped as slices of a straight elliptical cylinder. The major axis of the ellipse underlying the cylinder was 800 pm, and the minor axis was 100 pm. The height of the elliptical cylinder was 150 pm, and the internal angle of the elliptical cylinder slice was 45°.

[0103] Example 5. Implementation of the device according to the invention

[0104] An incubation chamber 120 of approximately cuboidal shape was made of PMMA. A water container 124 was placed in the chamber to provide higher air humidity than the ambient air during testing, reducing evaporation from the selection system 110. The incubation chamber 120 also contained a precision temperature gradient plate 121 configured to receive the selection system 110. Temperature control means 122,123, in the form of ceramic heaters, were placed on opposite sides of the precision temperature gradient plate 121. These temperature control means 122,123 were configured to produce different temperatures, generating a temperature gradient in the selection system 110. Between the first temperature control means 122 and the second temperature control means 123 was a thermally conductive copper plate (a component of the precision temperature gradient plate 121 ), providing even energy distribution for a linear temperature gradient under the sorting channel 113. The ceramic heaters were connected to a controller, allowing regulation of the temperature generated by each heater between 35°C and 45°C. High-precision temperature sensors placed on the precision temperature gradient plate 121 were used to ensure the correct temperature. A heat sink and a variable speed fan were additionally placed on the input chamber 112 side to increase temperature control efficiency. The ceramic heaters were also connected to an electrical power source in the form of a power supply. The precision temperature gradient plate 121 located in the incubation chamber was configured to accept the selection system 110 described in any of the previous examples and perform the analysis.

[0105] Example 6. Biological experiment preparation

[0106] Prior to the biological experiment, the laboratory diagnostician places the selection system 110 on a precision temperature gradient plate 121 inside the incubation chamber 120. In step 401 , he then heats the selection system 110 using temperature control means 122,123 in the form of ceramic heaters to a predetermined input chamber 112 temperature of 37°C and an output chamber 115 temperature of 42°C. The GM501 medium (Gynemed) used for in vitro spermatozoa capacitation must be warmed to room temperature before use.

[0107] Subsequently, in step 402, the diagnostician uses an automatic pipette to add 60 pl each of the GM501 SpermAir medium warmed to room temperature to the input chamber 112 and the output chamber 115, then protects the fluidic microarray from evaporation. The selection system 110 with the medium-filled input chamber 112 and output chamber 115 is left to incubate for 10 minutes.

[0108] Then, in step 403, the diagnostician takes 40 pl of the medium from the input chamber 112 with an automatic pipette and adds 40 pl of the liquefied semen to the input chamber 112, after which he again protects the microarray from evaporation. The selection system 110 thus prepared, containing the spermatozoa medium, is incubated for 30 minutes in step 404. The dimensions and arrangement of the grid 200 of the pillar obstacles 201 of the sorting channel 113 have been experimentally selected to prevent free movement of the fluid of a given viscosity. Therefore, the flow of spermatozoa in the system results from their thermotaxis-stimulated motility rather than being forced by capillary forces. After the incubation period, at step 405, the diagnostician pipettes all the fluid from the output chamber 115 into an appropriately labelled test tube (OD), from which he can then perform seminological diagnostics on the semen obtained after selection and compare it with the output parameters of the original ejaculate. Fig. 7 shows the results of the DNA fragmentation analysis of the spermatozoa before selection (ejaculate) compared to the average values of the DFI index of the spermatozoa obtained after a 30-minute selection (post-selection) using the device according to the invention. FIG. 8 shows the results (mean values) of the DNA fragmentation of spermatozoa before selection (ejaculate), after selection (using a device containing pillar obstacles and a temperature gradient) compared to the DFI index of spermatozoa that were selected using only the pillar obstacle track without a temperature gradient as a negative control.

[0109] The DNA fragmentation index (DFI index) of post-selection and pre-selection samples was assessed using the spermatozoa nuclear chromatin dispersion (SCD) test with the HT-HSG2 kit (Halotech DNA Pty, Ltd). To carry out this test, the diagnostician places 50 pl of the liquefied spermatozoa solution in an Eppendorf-type test tube containing 100 pl of liquid agarose. He or she then drops 8 pl of this mixture using an automatic pipette onto special coated basal slides provided by the manufacturer, which are then covered with a coverslip (24 mm x 24 mm). The slide with the spermatozoa agarose solution is then transferred to a cooling plate and placed in the refrigerator (4°C) so that the agarose solidifies into a porous gel (5 minutes). After this period, the coverslip is removed, and 4 drops of acid solution are dripped onto the slide for 7 minutes, after which the acid solution is drained from the slide. Lysis buffer is then added to the slide for 20 minutes, after which the slide is washed with distilled water (5 minutes). The slides are then dehydrated by spotting with ethanol (70% and 96%), at each concentration for a period of 2 minutes.

[0110] The dried slides are stored in the dark at room temperature. To stain the cells on the slide, preparate staining is applied using Wright's staining solution (Merck) and phosphate buffer (1 :1 Merck) for 7 minutes, after which the slides are washed with deionized water. The stained spermatozoa are counted using an optical microscope (Olympus CX-23), under an immersion objective (x100). DNA fragmentation analysis of spermatozoa on the slides involves counting cells containing a visible ‘halo’ against spermatozoa without a visible ‘halo’. Spermatozoa cells without a visible ‘halo’ or poorly stained cells with a small ‘halo’ are referred to as having fragmented (degraded) spermatozoa DNA. Spermatozoa having a medium or large ‘halo’ are defined as those containing intact DNA. The DFI index determines the percentage of spermatozoa containing fragmented DNA. According to the manufacturer of the test, a result of up to 15% (DFI) is normal, indicating a high potential for fertilization. Patients whose DFI is between 15-30% also have normal results and their potential for oocyte fertilization is good. From 30% of the DFI value onwards, the result is defined as abnormal with a low potential for fertilization.

[0111] Example 7: Comparative Performance Analysis of the Invention's Device Against a Prior Art Reference Device (ZyMbt ICS I [Fertile Chip])

[0112] To substantiate the superior performance of the device according to the present invention, a comparative analysis was conducted against a commercially available reference device, the ZyMot ICSI. The reference device is a sperm separation device employed in the preparation of motile sperm for assisted reproductive technology (ART) procedures. This reference device operates by segregating sperm based on motility within a microenvironment facilitated by microchannels. It is designed for sterile, singleuse applications and accommodates a processing volume of 2pL per microchannel.

[0113] For the comparative study, ejaculate samples were prepared for selection using the device according to the invention, following the procedure detailed in Example 6. Concurrently, samples for selection using the reference device were prepared in accordance with the manufacturer's protocol. An aliquot of 6.5 pl of medium (Sperm Wash, Irvine Scientific, USA) was applied in each of the apertures of the device. Then, 2 pl of fresh semen were placed in the small apertures; and a small drop of culture oil (Oil for Embryo Culture; Light Mineral Oil; Irvine Scientific, USA) was placed over each of the apertures of the chip. The loaded chip was placed horizontally in a closed heater at 37°C for 30 min. To reach the large apertures at the end of the channel, spermatozoa must swim through the channels. The distance to be travelled enriches for the population of spermatozoa with good motility). The selection process for spermatozoa was conducted over a 30-minute duration. Post-selection, the DNA Fragmentation Index (DFI) was measured for the spermatozoa obtained, as well as for the ejaculate prior to selection, utilizing the methodology outlined in Example 6. The results of the DFI measurements are illustrated in FIG. 9.

[0114] The analysis revealed that the DFI for spermatozoa processed with the device according to the invention was statistically significantly lower than that for spermatozoa processed using the reference device. Statistical tests were conducted between:

[0115] - spermatozoa before selection (ejaculate) compared to spermatozoa obtained after 30 minutes of selection using the device according to the invention (paired Wilcoxon test, p-value = 1.296e-05, n = 25);

[0116] - spermatozoa before selection (ejaculate) compared to spermatozoa obtained after 30 minutes of selection using another sperm selection device available on the market - ZyMbt (paired t-test, p-value = 0.8898, n = 10);

[0117] - spermatozoa obtained after 30 minutes of selection using the device according to the invention compared to spermatozoa obtained after 30 minutes of selection using another sperm selection device available on the market - ZyMbt (Mann- Whitney test, p = 0.04404).

[0118] To assess the characteristics of the data, the Shapiro-Wilk normality test and Levene’s test for the equality of variances were performed.

[0119] Moreover, the reference device did not demonstrate a statistically significant reduction in DFI between the ejaculate and the spermatozoa post-selection. In contrast, the device according to the invention exhibited a significant reduction in DFI, indicating its enhanced efficacy in sperm selection.

Claims

CLAIMS1. A device for in vitro seminological diagnostics and spermatozoa selection, comprising:- a selection system (110) that comprises:- a body (111 ) made of plastic;- an input chamber (112), located in the body (111 ), for receiving an ejaculate sample;- a sorting channel (113), located in the body (111 ), having a first end (114) and a second end (116) and comprising a grid (200) of pillar obstacles (201 ) between the first end (114) and the second end (116);- wherein the first end (114) of the sorting channel (113) is connected to the input chamber (112); and- an output chamber (115), located in the body (111 ), connected to the second end (116) of the sorting channel (113), for receiving spermatozoa of the ejaculate sample that have migrated from the first end (114) to the second end (116) of the sorting channel (113);- and an incubation chamber (120) for generating a temperature gradient in the selection system (110), such that the temperature at the input chamber (112) is lower than the temperature at the output chamber (115).

2. The device according to claim 1 , wherein each of the input chamber (112) and the output chamber (115) has a volume between 20 pl and 200 pl.

3. The device according to any of the preceding claims, wherein the sorting channel (113) has a length (L) between 10 mm and 50 mm, a width (S) between 2 mm and 20 mm, and a height (H) between 40 pm and 1000 pm.

4. The device according to any of the preceding claims, wherein the pillar obstacles (201 ) have a shape of a section of a straight elliptical cylinder whose axis of symmetry is the major axis of the ellipse underlying said elliptical cylinder, the length of the major axis of the ellipse underlying said cylinder is between 500 pm and 1000 pm, the length of the minor axis is between 50 pm and 300 pm, the ratio of the length of the major axis of the ellipse underlying said elliptical cylinder to the length of its minor axis is in therange from 1 to 20, the height (H) of said elliptical cylinder is in the range from 40 pm to 1000 pm, and the internal angle (a1 -3) of said elliptical cylinder slice is in the range from 20° to 75°, with the apex of said cylinder slice facing the output chamber (115).

5. The device according to any of the preceding claims, wherein the pillar obstacles (201 ) are spaced in relation to each other at intervals of 10 pm to 300 pm.

6. The device according to any of the preceding claims, wherein the pillar obstacles (201 ) are arranged in an amount of 3 to 15 per mm2.

7. The device according to any of the preceding claims, wherein the pillar obstacles (201 ) occupy from 60 to 95% of the area of the sorting channel (113).

8. The device according to any of the preceding claims, wherein the grid (200) of pillar obstacles (201 ) has an arrangement selected from: rectangular topology, hexagonal topology, or random topology.

9. The device according to any of the preceding claims, wherein the incubation chamber (120) is configured to generate the temperature gradient in the selection system (110) such that the temperature at the input chamber (112) is lower than the temperature at the output chamber (115) by at least 0.5°C and at most 7°C.

10. The device according to any of the preceding claims, wherein the incubation chamber (120) is configured to generate the temperature gradient in the selection system (110) such that there is a linear temperature gradient between 0.02°C / mm and 0.7°C / mm between the input chamber (112) and the output chamber (115).11 . The device according to any of the preceding claims,- wherein the incubation chamber (120) comprises:- a plate (121 ) of a precise temperature gradient, configured to receive the selection system (110) and made of a thermally conductive material;- temperature control means (122) located on opposite sides of the plate (121 ), perpendicular to a longitudinal axis of the sorting channel (113); and1- wherein the temperature control means (122) is configured to produce a first temperature at the input chamber (112) and a second temperature at the output chamber (115), wherein the first temperature is lower than the second temperature.

12. The device according to claim 11 , wherein the temperature control means (122) is a ceramic heater, a Peltier cell, or a liquid jacket.

13. The device according to any of the preceding claims, wherein the incubation chamber (120) comprises a liquid container (123) configured to maintain the humidity of the air in the incubation chamber (120) at a higher level than the humidity of the air surrounding the incubation chamber (120) such as to reduce evaporation of a liquid from the selection system (110).

14. The device according to any of the preceding claims, configured to observe spermatozoa microscopically during the examination.

15. The device according to any of the preceding claims, wherein the selection system (110) comprises at least two sorting channels (113), each sorting channel (113) having a dedicated input chamber (112) and a dedicated output chamber (115).

16. A method for spermatozoa selection using the device according to any of claims 1 to 15, comprising the steps of:- heating (401 ) the input chamber (112) and the output chamber (115) by the temperature control means (122) to a temperature between 35°C and 45°C, so that the temperature in the input chamber (112) is lower by 0.5°C to 7°C than the temperature in the output chamber (115), and a linear temperature gradient is formed in the sorting channel (113);- filling (402) the selection system (110) with a liquid medium;- introducing (403) the ejaculate sample into the input chamber (112);- incubating (404) the ejaculate sample under the temperature gradient for a time duration of between 10 and 60 minutes; and collecting (405), from the output chamber (115), the liquid medium containing spermatozoa of the ejaculate sample that have passed through the sorting channel (113).

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