System for selecting spermatozoa

Abstract

The present invention relates to a system for selecting spermatozoa that comprises a dish having at least one recessed microfluidic circuit (6), where each microfluidic circuit (6) is formed by three cavities (1,2,3) and two channels (4,5). The two channels form a capital "L" shape (L), with the length of the first channel (4) being greater than the length of the second channel (5). The first cavity (1) is the cavity in which the semen sample is deposited and said cavity is arranged at the free end of the first channel (4); the third cavity (3) is arranged at the free end of the second channel (5); and the second cavity (2) is arranged at the intersection between the first channel (4) and the second channel (5) and has a greater diameter than the first cavity (1) and the third cavity (3), the first channel (4) corresponding to a sector for dispersion and thermotaxis and the second channel (5) corresponding to a sector for sperm selection using rheotaxis.

Description

[0001] SPERM SELECTION SYSTEM

[0002] Technical sector

[0003] The present invention relates to a microfluidic sperm selection system consisting of two channels with opposing flow currents, forming an angle between them, which can be integrated into an intracytoplasmic sperm injection (ICSI) plate, without the need for an external system to create these flows.

[0004] State of the art

[0005] Sperm preparation and sperm selection are essential steps during assisted reproduction treatments (ART), as any manipulation can impact sperm viability, especially the integrity of their genetic information, which can influence reproductive success and the health of the newborn.

[0006] Sperm selection methods were developed with two fundamental objectives: the elimination of seminal plasma and the selection of the best sperm, essential steps to preserve their fertilization capacity and the proper development of the embryo.

[0007] The most commonly used sperm selection methods currently are Wash and Swim-Up (WSU) and Density Gradient Centrifugation (DGC). In WSU, the semen sample, diluted in culture medium, is initially centrifuged to concentrate the cellular fraction into a sediment, or pellet, and separate it from the seminal plasma. Culture medium is then carefully added to the pellet and incubated for a specified time to allow motile sperm to migrate to the upper fraction. After a final wash of this fraction, the final sample is obtained, which will be used for in vitro fertilization or ICSI. The total procedure takes 60 to 70 minutes. In DGC, the entire semen sample is deposited on top of two layers of density gradients of a colloidal medium prepared at different concentrations and then centrifuged.The result is a pellet containing the most motile sperm. After additional washing of this pellet by centrifugation, the final sample of motile sperm is obtained in approximately 40 minutes. Both methods have widely demonstrated their effectiveness in removing seminal plasma and selecting motile sperm, but they have several limitations. First, centrifugation (WSU and CGD) and incubation temperature (WSU) generate oxygen-free radicals (ROS) that induce sperm DNA (FADN) fragmentation. This fact is relevant due to the association between FADN and reproductive failure, particularly at the level of fertilization and embryonic development, especially in couples with poor gamete quality.Furthermore, both methods require the semen sample to be handled and re-contained (tubes or culture plates) several times, each of which poses a potential risk of sperm confusion or loss. Third, they require a long preparation time and expensive equipment to carry out the entire process.

[0008] For all these reasons, one of the most important lines of research currently focuses on the search for new methodologies that allow for safer sperm selection, reducing the iatrogenic impact on sperm related to both the generation of ROS and the number of steps required for sample preparation. Furthermore, it is important to achieve greater efficiency of the procedure by reducing time and costs.

[0009] The use of microfluidics has become a promising trend that meets these premises. Several commercial devices designed to select motile sperm are currently available. All of them are external platforms, in the form of slides, most of them with predefined circuits carved into the surface and often using polydimethylsiloxane (PDMS). Several authors have described the usefulness of microfluidic systems for selecting motile sperm, which also contain their intact DNA. The main difference between microfluidics and traditional sperm selection methods, such as WSU or CGG, is that sample centrifugation is not required for processing. Furthermore, they allow obtaining the definitive fraction of motile sperm in approximately 20 minutes.However, the application of microfluidics in ART, as currently known, requires the use of devices external to those routinely used in assisted reproduction laboratories. This is a factor that adds complexity and cost to the procedure and does not entirely eliminate the risk associated with sample handling, since sperm transfer from the microfluidic device to the ICSI plate is still required. Furthermore, there are cases, especially those with very severe dyspermia (very low sperm count, very low motility, or very few sperm with normal morphology), in which it is possible that no sperm will reach the end of the microfluidic channel, so there is a risk of running out of sample to perform ICSI when sperm become trapped in the solid-walled and -ceilinged tunnels of current microfluidic chips.

[0010] On the other hand, it should be noted that not all microfluidic systems utilize sperm rheotaxis, as they do not feature a current flow. The fluid remains static, and it is the sperm that move, but not against the current.

[0011] In search of an improvement in the sperm selection process, several groups have proposed microfluidic systems made by hand in the ICSI plate itself, based on the union of drops of culture medium, so that a drop of the semen sample is placed at one point of the system and the sperm are subsequently selected at another.

[0012] These systems have the advantage of simplicity and efficiency, but also several disadvantages, such as the fact that the system is manually created, depending on the user's personal skill and preventing absolute reproducibility of the system, and above all, the failure to take advantage of all sperm selection tools.

[0013] Most of the aforementioned systems do not have any current flow, as they connect droplets of identical volume. Others connect two droplets of different volumes, but because they only have one microfluidic channel, it is impossible to generate two opposing currents.

[0014] The present invention proposes a system that is equally or even simpler to use, but much more sophisticated, as it takes advantage of the peculiarities that the best spermatozoa have been shown to have when moving, such as the tendency to swim towards the edges following a curvilinear and not a rectilinear movement, the ability to swim against the current due to the rheotaxis phenomenon, and the tendency to swim from colder areas to warmer areas due to the thermotaxis phenomenon. To take advantage of these characteristics, it is necessary to generate current flows and temperature gradients, but all while taking into account the importance of maintaining intact the characteristics of the ICSI plate, especially the temperature and pH of the drops where the oocytes will be placed, so that the treatment is as effective as possible.Therefore, if a microfluidic system with these characteristics is to be integrated into an ICSI plate, a fundamental parameter must be taken into account to ensure the system's viability in everyday clinical practice. This parameter is the time it takes for the system to provide sufficient high-quality sperm to perform ICSI. The proposed system achieves this in between ten and fifteen minutes, less than half the time of most commercially available systems. This unique feature is what allows this system to be integrated into an ICSI plate.

[0015] There is an ICSI plate on the market with a built-in microfluidic system (Fertdish, published in Lab on a Chip 21 (775) 2021). This system presents several clear differences with the system object of the present invention. Firstly, there is no current flow in the path and therefore no system of opposing currents, so the sperm swim through the system, but not against the current, so the rheotaxis phenomenon is not used to select the best sperm. There is also no temperature difference, so the thermotaxis phenomenon is also wasted to select sperm. Thirdly, the path that the sperm follow is straight, so the possibility of selecting those with curvilinear progression and that may have more complete DNA than those with rectilinear progression is not taken advantage of.Furthermore, the recommended culture medium for the microchannels is polyvinylpyrrolidone (PVP), a dense medium known to affect sperm movement, slowing their velocity and reducing the tendency of the best sperm to swim toward the edges. The pathway is covered by a solid material, which, unlike the proposed system, makes it impossible to retrieve sperm if none reach the endpoint, as they become trapped within the system. Furthermore, the system incorporates PDMS into the microinjection plate, a material used to construct the microfluidic system and not routinely used to configure the ICSI plate. Finally, the system requires an incubation time of one to two hours at 37°C, which could affect the microenvironment of the droplets intended to contain the oocytes.

[0016] Document WO2022184960A1 also describes an ICSI plate incorporating a sperm selection system based on the interaction of the sperm with the oocyte's cumulus oophorus cells. It consists of a straight track with a cavity in the center for the cumulus cells to be placed there. At both ends of the channel are two other cavities: one at one end for the sperm sample, and the other at the other end for collecting the sperm that have reached the channel and using them for ICSI. This system does not select sperm by rheotaxis, as no premeditated current flow is generated in the channels that causes the best sperm to swim against the current, and only these reach the final drop. Selection is based on the interaction of the sperm with the cumulus oophorus cells. It has the following disadvantages:

[0017] A different material, cumulus oophorus cells, is incorporated into the ICSI plate than that used on a daily basis to configure the ICSI plate.

[0018] It is necessary to manipulate the oocytes to remove these cumulus cells.

[0019] The patient's oocytes may have low-quality cumulus cells.

[0020] Obtaining these cumulus cells and placing them in the sperm selection system is done manually, which reduces the reproducibility of the system and makes it more user-dependent.

[0021] - Some sperm may swim along the edge of the canal without interacting with these cumulus cells.

[0022] The plate must be incubated at 37°C, which could generate oxygen-free radicals (ROS) that could affect the integrity of the sperm DNA. A prolonged incubation time of the entire plate is required to select sperm, possibly because there is no swimming effect. Furthermore, they must swim through the cumulus cells, which can prolong the recovery time. This can have several negative consequences for the pregnancy prognosis when performing ICSI in this system, since the droplets placed on the ICSI plate will have been prepared for a long time by the time the oocytes are transferred to them, and their pH may have changed, which could affect the proper function of the oocytes.

[0023] If the channels are very deep or protrude from the bottom of the plate, it will be difficult to recover sperm from the final drop with the ICSI pipette.

[0024] The present invention provides a microfluidic sperm selection system formed by one or more microfluidic circuits having two channels with opposing flow currents, forming an angle between them, which can be integrated into an ICSI plate, the objectives of which are: to improve the effectiveness of sperm selection, simplify it, make it safer and more efficient.The system takes advantage of the natural characteristics of the movement of the best spermatozoa, reduces the manipulation of gametes and therefore the iatrogenic damage to them, simplifies the selection process making it faster and less expensive, thus improving efficiency, and, in the preferred option in which the system is integrated into the ICSI plate, reduces the risk of confusion when identifying the sample by not having to pass it from one container to another, since all that needs to be done is to place a drop of semen on the ICSI plate and wait to be able to recover the spermatozoa that reach the end of the journey, which will be selected to microinject the oocytes, in the same plate.

[0025] To achieve this, the microfluidic systems in the plate are not static, but have a current flow—in fact, two opposing current flows—that prevent seminal plasma, dead cells, or poor-quality sperm from reaching the end of the path. Furthermore, the circuit layout is not rectilinear; rather, both currents form a right angle to improve the selection of the best sperm and ensure that no unwanted elements are carried to the end. It has the advantage of using the same elements, in terms of culture media and consumables, that are routinely used when configuring the droplets in an ICSI plate to carry out the treatment, without introducing media, materials, or cells foreign to the procedure, making it simpler.It also doesn't require incubating the semen sample at a high temperature, which reduces ROS production and thus damage to the sperm, and shortens recovery time. By shortening the preparation time, the culture medium droplets that will contain the oocytes are not affected. The entire microfluidic circuit is minimally recessed into the surface of the plate and does not protrude beyond it. Since it is not covered by any solid material, it allows for easy recovery of sperm from any point along the path.

[0026] In this way, the system allows the following:

[0027] 1- Select the sperm to be microinjected on the ICSI plate itself, thus avoiding successive transfers of the sample and thus reducing the risk of confusion when identifying it, as well as reducing the risk of sample contamination.

[0028] 2- Making this sample selection from whole semen, that is, without prior processing of the sample, reducing the risk of iatrogenic damage to the sperm produced, especially but not exclusively, by centrifugation and thus improving the effectiveness of the treatment.

[0029] 3- Selecting the best sperm by taking advantage of their specific characteristics of swimming towards the edges, swimming against the current (rheotaxis) and swimming towards warmer areas (thermotaxis), thus improving the effectiveness of the treatment.

[0030] 4- Simplify the sperm selection technique for ICSI, reducing the time required to obtain the sperm for microinjection and the expense of inventory and consumables, which overall improves efficiency compared to conventional sperm selection techniques for ICSI.

[0031] 5- Treat most semen samples, unlike the limitations that other microfluidic systems have in samples with severe dispermia.

[0032] 6- Being able to recover sperm that have not reached the final zone of the journey.

[0033] 8- Scaling the system, allowing its use in different types of treatment other than ICSI (in vitro fertilization or artificial insemination) Explanation of the invention

[0034] The invented system combines the use of physical and biological principles that allow for the selection of the best sperm, such as:

[0035] 1- Tendency of sperm to swim against the current (rheotaxis).

[0036] 2- Tendency of sperm to swim towards the edges.

[0037] 3- Tendency of sperm to swim towards warmer areas (thermotaxis).

[0038] 4- Laplace's Law.

[0039] 5- Laws of Hydrostatics.

[0040] To improve the prognosis of ICSI assisted reproduction treatment, it is essential to select the best sperm, defined as those with the greatest potential to fertilize the egg and develop a healthy, developing embryo until delivery. It is known that sperm with poorer morphology, and especially those with DNA integrity defects, are associated with a lower fertilization rate via ICSI, a higher incidence of embryonic development arrest, a lower embryo implantation rate, a higher miscarriage rate, and a lower live birth rate per embryo transfer. The morphology or motility of a sperm does not indicate whether its DNA is intact or not.

[0041] It has been shown that the best sperm, those with the highest DNA integrity, tend to swim against the current. This phenomenon is known as rheotaxis. In contrast, sperm with DNA defects have a diminished ability to swim against the current, advancing less rapidly or being swept away by the current. This tendency to swim against the current appears to be a passive effect. The best sperm beat their tails more and with greater amplitude, becoming a kind of virtual sail that causes the effect of the current to steer the sperm head against the current. It has been shown that applying a current flow in a microfluidic system (systems with very small amounts of fluid in sub-centimeter dimensions) selects sperm with a higher probability of having intact DNA.It has also been shown that the best sperm tend to swim toward the edges, not in a straight line, but rather, moving progressively to one side or the other. It has been shown that when sperm were made to swim through a microfluidic channel with right and left detours, those that continued swimming in a straight line to the end of the channel, without deviating through the detours, had a higher incidence of DNA fragmentation than those that swam close to the channel wall and therefore exited through the detour. This improvement in DNA integrity is independent of sperm motility or morphology, as all sperm exhibited good motility, allowing them to move progressively. It has also been observed that more sperm with intact DNA are recovered from the detours when they are at a 45° angle than when the angle is 90°.

[0042] It has been shown that the best sperm tend to swim from colder to warmer regions. This phenomenon is known as thermotaxis, and taking advantage of it can reduce sperm recovery time and select those with the highest DNA integrity.

[0043] It has been proven (Laplace's Law) that droplets of the same liquid, but with different radii, present different pressures, with the pressure being greater in the droplet with the smaller radius. This means that when two droplets of different radii are connected, a flow or current is created that goes from the droplet with the smaller radius to the one with the larger radius. This principle makes it possible to create a microfluidic system with a current flow without the need for an external system, such as a pressure pump, to create these flows.

[0044] It has also been demonstrated (laws of hydrostatics) that the pressure at a given point within a fluid is proportional to the distance from that point to the surface. Therefore, when a droplet of a given volume and another of a larger volume are placed in a Petri dish, and both are covered with oil, the pressure of the oil on the surface of each droplet will be different, being greater in the droplet of smaller volume, due to the greater distance between its surface and the oil surface, than in the droplet of larger volume, whose surface will be closer to the oil surface. This concept can also be leveraged to create a pressure difference between two drops in a microfluidic system and thus, a current flow that persists over time and without the need for an external system, which would be unfeasible in the ICSI dish.Thus, the sperm selection system of the present invention consists of one or more microfluidic circuits (or microfluidic and rheotaxis sperm selection circuits) excavated in a plate, in which controlled flows of opposing currents are created, without the need for any external system, by joining droplets with different pressures obtained by the specific dimensions of each drop, in a layout with specific deviations that allow the best sperm to be selected. A specific temperature gradient is also added to speed up this sperm selection and make the selection even more effective.

[0045] Preferably, the sperm selection system object of the present invention is incorporated into a conventional polystyrene ICSI plate in which the microfluidic circuit has been excavated.

[0046] Although many microfluidic systems are constructed from polydimethylsiloxane (PDMS), this one is preferably constructed from polystyrene or a similar material, as it is the material commonly used in this type of ICSI plate and is generally a preferred material for cell culture.

[0047] Optionally, the sperm selection system is placed on one side of the plate, so that the rest of it can be used for conventional ICSI.

[0048] Alternatively, the sperm selection system object of the present invention consists of one or more microfluidic circuits excavated in a plate independent of the ICSI plate, where sperm selection is carried out on said plate for subsequent use in a conventional ICSI plate.

[0049] In general, when the sperm selection system is incorporated into an ICSI plate, there will be three distinct zones. An area dedicated to sperm selection, where the microfluidic systems and the droplets where the selected sperm will be transferred will be located; a microinjection zone where the drops in which the oocytes to be microinjected will be placed can be placed and which will generally occupy the center of the plate as it is the area where the temperature is best controlled; and a third zone where drops of culture medium can be placed to wash the oocytes when transferring them from one culture medium to another. A single plate can include several microfluidic circuits so that sperm selection can be performed in more than one of them in order to recover more sperm from a larger initial sample volume (for example, 4 drops of semen instead of a single drop).

[0050] Each circuit consists of two sectors. A first sector for semen sample dispersal and sperm selection by thermotaxis, and a second sector for sperm selection by rheotaxis.

[0051] The final appearance of the complete pathway, viewed from above, is that of a capital L, with a longer arm (first channel) and a shorter arm (second channel), in which there is an alveolus at each end and an alveolus at the intersection of both sides. Preferably, and without limitation, the cross-section of the channels is rounded, rather than square, to allow for better aspiration of sperm with the ICSI pipette, should this be necessary.

[0052] The long arm of the L would correspond to the dispersal and thermotaxis sector and the short arm to the sperm selection sector by rheotaxis.

[0053] The length of each sector or arm of the L responds to the specific needs of the circuit. In general, it is important to make the route as short as possible, so that the sperm have to swim a shorter distance and can be selected more quickly. However, an excessively short distance from the first sector could cause seminal plasma and dead cells to reach the intermediate drop. Indeed, in this first channel, the current flows from the first drop, where the semen is deposited, to the second, to disperse the coagulum of the ejaculate sample, encourage the sperm to exit the drop, with the best sperm swimming toward the edges and passively bringing them closer to the rheotaxis zone. However, it is possible that some may turn around and begin swimming against the current.In order for the best sperm to continue moving from drop 1 to drop 2, a temperature gradient is established between them, so that the best sperm will tend to head towards the second drop.

[0054] There, those that continue swimming along the edge of the channel will encounter a countercurrent from the second channel. The best sperm, due to their rheotaxis, will continue swimming against the current toward the final third drop, while the poorest sperm will be retained in the second drop. It is in this second channel, therefore, that the best sperm are selected. This second route can be shortened compared to the first, but not excessively, because if the drops were too close together, the channel connecting them would lose its proper morphology.

[0055] Each circuit is excavated at the bottom of the slab, and from the top it looks like an L-shaped structure formed by two perpendicular channels and three cells. The two end cells (the first and third cells, the first cell being the cell at the beginning of the path and the third cell being the cell at the end of the path) are preferably the same size, while the cell at the intersection (the second cell) is larger, preferably twice as large. The first channel is also longer than the second (preferably twice as long).

[0056] Optionally, both canals and three alveoli are equally deep.

[0057] Preferably, the first channel has a gradual slope, such that it is deeper at the beginning (at the end in contact with the first alveolus) and shallower at the end (at the end in contact with the second alveolus). This configuration facilitates the deposit of non-motile cells at the bottom of the channel, while motile sperm can swim above the deposit of non-motile cells.

[0058] Preferably, the first channel has a progressive widening at its end, near the end in contact with the second alveolus. This widening at the end of the first channel serves two purposes. On the one hand, as the channel widens, the current velocity is reduced, making it easier for the best sperm, which are swimming close to the edge of the channel, to continue swimming and divert toward the second channel, since a faster current could drag them straight ahead and not allow them to enter the second channel. Furthermore, it has been proven that this inverted funnel morphology produces a divergent flow, which means that the flow direction remains the same at the edges of the channel, but a turbulence effect is produced in the central area of ​​the channel, which would hinder the advance of sperm that do not swim close to the edge of the channel and which theoretically are of poorer quality.Preferably, the second channel tapers gradually at its beginning, such that it is wider at the end in contact with the second alveolus. This gradual narrowing at the beginning of the second channel produces the same divergent flow effect, which facilitates the countercurrent movement of sperm that continue to swim along the edge of the droplet and makes it difficult for sperm swimming in the center to enter the second channel. Furthermore, the flow velocity will be greater at the end of this second channel, which is where the best sperm are selected.

[0059] Preferably, the intersections between the alveoli and the channels are curved, avoiding sharp angles. These smoothed junctions between the channels and the alveoli help maintain contact between the best sperm and the channel rim. If the area where the channel meets the alveolus forms a right angle, the sperm is more likely to separate from the rim, while a smoothed intersection makes it more likely that the sperm will stay attached to the rim of the microfluidic circuit.

[0060] Preferably, the alveolus at the end of the path has sperm recovery zones formed by progressively shallower protrusions, which cause the sperm to enter and remain trapped there, preventing them from continuing to swim back and returning. Instead, they remain in those zones, ready to be aspirated and used for ICSI.

[0061] Optionally, along the second canal there is also a set of sperm recovery zones formed by progressively shallower protrusions, arranged on one side of the canal or on both.

[0062] Preferably, the sperm selection system is incorporated into an ICSI plate and consists of at least two microfluidic systems to be able to perform the procedure in duplicate and at the same time to be able to recover a greater quantity of sperm.

[0063] The sperm selection system is prepared by placing a drop of the same volume of culture medium in each alveolus and then a fourth drop of the same volume on the drop in the middle alveolus, so that it will have twice the volume of the others. Depending on the plastic finish of the plate (hydrophobic or hydrophilic), the drops may connect to each other automatically as the channels fill by capillary action, or a sterile pipette tip may need to be passed over the channels, from one drop to the next, to connect them. In any case, as soon as the three drops connect, a pressure difference will automatically be created between them.

[0064] Once the microfluidic circuit has been primed with the culture medium, if the sperm selection system is incorporated into an ICSI plate, the rest of the ICSI plate is set up by arranging the droplets into which the sperm selected for ICSI and the oocytes (eggs) will later be transferred and covering everything with mineral oil.

[0065] Once this is done, a drop of semen, half the volume of the previous ones, is added to the initial alveolus of the system, which is the one located at the end of the longest channel.

[0066] Next, a temperature gradient is generated between the first and second droplets of the system, so that the first droplet is cooler. To achieve this, the plate that incorporates the system (preferably an ICSI plate, although it can be a separate plate) is placed on a plate made of a material with high thermal conductivity (for example, a 2 mm aluminum plate) with a specific configuration with a series of cutouts, such that the plate is in contact with the entire plate except for the cells where the semen sample will be placed.

[0067] Finally, the plate-and-sheet assembly is placed on a heated flat surface. The aluminum will transmit the heat to the entire plate, ensuring that the temperature of all the drops is preferably 37°C, except for the drops containing the semen sample, which will remain cooler. This creates a temperature gradient between the first and second drops necessary for the thermotaxis process to occur.

[0068] Heating the system, except for the drop containing the semen sample, serves two purposes. First, it reduces the temperature of the drop containing the whole semen, since an increased temperature causes cells with a larger cytoplasm, such as white blood cells, germline cells, and immature sperm, to generate oxygen-free radicals (ROS), which can damage the DNA of healthy sperm. Second, it creates a temperature gradient between the first and second drop, which results in more sperm being recovered in less time due to the tendency of the best sperm to swim toward warmer areas due to a thermotaxis effect.

[0069] Preferably, there is a lid to cover the plate during the sperm selection process, reducing contact of the mineral oil with the environment. This will promote stable plate conditions with respect to temperature, pH, and osmolarity, while preventing the risk of contamination.

[0070] According to the results, in most semen samples with at least one million sperm with good rectilinear progression per milliliter, in less than fifteen minutes there will be enough sperm to microinject at least 20 oocytes in the final drop of the circuit and in cases of normozoospermia (semen samples with all parameters within the QMS normal reference limits), in less than ten minutes.

[0071] The droplets vary in diameter and volume, so, according to Laplace's Law, the pressure of each droplet is inversely proportional to its diameter. Thus, a droplet of a given diameter will have more pressure than a droplet of the same culture medium with a larger diameter, and when the two drops merge, a flow will be created, i.e., a displacement of the medium from the droplet with higher pressure to the droplet with lower pressure.

[0072] Furthermore, as is commonly done in the ICSI technique, the droplets are covered with mineral oil to prevent changes in pH and osmolarity in the culture medium due to evaporation. This oil covering the droplets, in accordance with the laws of hydrostatics, also exerts a different pressure on smaller droplets than on larger droplets, so that this pressure is also inversely proportional to the droplet volume.

[0073] Overall, this means that when two droplets of different sizes join a channel in the same culture medium, the medium will progressively move from the smaller droplet to the larger droplet. As the small droplet becomes smaller and the larger the droplet becomes, the pressure difference between them will increase, so this current flow will persist for a long time. This time and the flow rate can be regulated by modifying the volume and diameter of the droplets, their distance from each other, and the dimensions of the connecting channel.

[0074] The system object of the present invention is further characterized in that it comprises the following operating method:

[0075] Place a drop of the same volume of culture medium in each alveolus and then a fourth drop of the same volume on the drop in the intermediate alveolus, so that it will have twice the volume of the others.

[0076] Pass a sterile pipette tip over the channels, from one drop to another, so that they are connected.

[0077] In the preferred embodiment in which the system is incorporated into an ICSI plate, configuration of the rest of the ICSI plate by arranging the drops that will contain the selected sperm and oocytes (eggs)

[0078] Covering the entire plate with mineral oil.

[0079] - Addition of a drop of semen, half the volume of the previous ones, in the first alveolus, which is the one located at the end of the longest canal.

[0080] Lid arrangement on the plate.

[0081] Arrangement of the plate on a sheet of a material with high thermal conductivity.

[0082] Arrangement of the plate-and-sheet assembly made of a highly thermally conductive material on a flat surface heated to 37°C. The plate will transmit the temperature to the entire ICSI plate except for the drop containing the semen sample, creating a temperature gradient between the first and second drop, which is necessary for the thermotaxis process to occur.

[0083] - Aspiration of sperm accumulated in the sperm retrieval areas, preferably with an ICSI micropipette. Brief description of the drawings

[0084] To complement the description being made and in order to help better understand the characteristics of the invention, in accordance with a preferred example of practical embodiment thereof, a set of figures is attached as an integral part of said description, in which the following has been represented for illustrative and non-limiting purposes:

[0085] Figure 1 shows a schematic view of the microfluidic circuit7 according to an embodiment of the present invention.

[0086] Figure 2 shows a top view of a round ICSI plate with two microfluidic sperm selection circuits and rheotaxis, according to an option of the present invention.

[0087] Figure 3 shows a top view of a round ICSI plate with four microfluidic sperm selection circuits and rheotaxis, according to an option of the present invention.

[0088] Figure 4 shows a diagram of the possible general dimensions of two sperm selection circuits, according to an embodiment option of the present invention.

[0089] Figure 5 shows a section of the system shown in Figure 4.

[0090] Figure 6 shows a section of the system shown in Figure 4.

[0091] Figure 7 shows a diagram of the possible general dimensions of two sperm selection circuits, according to an embodiment option of the present invention.

[0092] Figure 8 shows a detailed view showing the rounded junctions between the canals and the alveoli.

[0093] Figure 9 shows the example configuration of the ICSI plate in Figure 2 with the plate made of a material with high thermal conductivity underneath it to achieve a temperature difference between the first and second drop of each system, according to an embodiment of the present invention. Figure 10 shows a top view of a rectangular ICSI plate with a possible arrangement of two microfluidic and rheotaxis sperm selection circuits, according to an embodiment of the present invention.

[0094] Figure 11 shows the same plate as in Figure 10 with a plate of a material with high thermal conductivity under it to achieve a temperature difference between the first and second drop of each circuit.

[0095] Figure 12 shows a top view of a rectangular ICSI plate with another possible arrangement of two microfluidic and rheotaxis sperm selection circuits, according to an option of the present invention.

[0096] Figure 13 shows the same plate as in Figure 12 with the plate system made of a material with high thermal conductivity underneath it to achieve a temperature difference between the first and second drop of each system.

[0097] Figure 14 shows an example of preferred locations of the sperm accumulation and recovery zones according to the relationship between the circuit layout and the entry direction into the ICSI micropipette plate, according to an embodiment option of the present invention.

[0098] Figure 15 shows an example of preferred locations of the sperm accumulation and recovery zones according to the relationship between the circuit layout and the entry direction into the ICSI micropipette plate, according to an embodiment option of the present invention.

[0099] Figure 16 shows an exemplary embodiment of the invention on a board independent of the ICSI board comprising a set of excavated microfluidic circuits.

[0100] Figure 17 shows the plate of Figure 16 with the sheet of a material with high thermal conductivity underneath it to achieve a temperature difference between the first and second drop of each system, according to an embodiment of the present invention. Description of the preferred embodiments of the invention

[0101] In view of the aforementioned figures, and in accordance with the numbering adopted, one can observe in them an example of a preferred embodiment of the invention, which comprises the parts and elements indicated and described in detail below.

[0102] Thus, the system object of the present invention comprises an ICSI plate (7) that has at least one microfluidic circuit (6) excavated.

[0103] As shown in Figure 1 , the microfluidic circuit (6) is formed by three alveoli (1,2,3) and two channels (4,5), where the two channels form a capital L (L), the length of the first channel (4) being twice the length of the second channel (5). The first alveolus (1) is the alveolus in which the semen sample is deposited and is arranged at the free end of the first channel (4). The third alveolus (3) is the alveolus from which the selected sperm are recovered, and is arranged at the free end of the second channel (5), and has a diameter equal to the first alveolus. The second alveolus (2) is arranged at the intersection between the first channel (4) and the second channel (5), and has twice the diameter of the first alveolus (1) and the third alveolus (3), the first channel (4) corresponding to a sector of dispersion and thermotaxis and the second channel (5) to a sector of sperm selection by rheotaxis.

[0104] In this way, the system is formed by joining three alveoli of different volume, so that the second alveolus (2) has twice the volume of the first and second alveoli (1 and 3) so that, according to the Law of Laplace and Hydrostatics, the first and third alveoli have more pressure than the second alveolus when they contain fluid inside. By joining the alveoli with the channels (4 and 5) a current flow is created from the first and third alveoli at the ends (1, 3), which have more pressure, towards the second central alveolus (2).

[0105] When a semen sample (6) is placed in the first alveolus (1), as there is pressure towards the second alveolus (2), the seminal plasma is dispersed through the first channel (4) and the sperm with the best curvilinear mobility move towards the edges of the channels and advance with the current. When they reach the second alveolus (2) they encounter a current flow coming from the third alveolus (3). The best sperm will continue swimming along the edges and will manage to overcome the countercurrent and reach the third alveolus (3). To prevent them from continuing to swim, going back, and accumulating in the third alveolus (3) as they arrive, it has small, progressively shallower protrusions that cause the sperm to enter and remain trapped there, ready to be aspirated and used for ICSI.

[0106] The right angle formed between the channels and the opposing current flow of each channel prevents seminal plasma, immobile cells, or low-quality sperm from reaching the third drop. Only the best sperm will be able to swim against the current and reach the final drop. Figures 2 and 3 show two examples of ICSI plates (7) with two and four microfluidic sperm selection systems and rheotaxis.

[0107] The rest of the plate is free to freely arrange the rest of the drops in that area to carry out the ICSI procedure. The examples shown in Figures 2 and 3 indicate a possible sperm selection area (18), which has the excavated microfluidic circuits (6) and other areas (8) to optionally arrange drops of polyvinylpyrrolidone (PVP) or other dense medium where the aspirated sperm from the recovery areas can be placed to be immobilized and subsequently microinjected. A washing area (10) with areas where drops of culture medium can be placed where the oocytes can be progressively washed. A microinjection area (9) with areas where drops of culture medium can be placed in which the oocytes will be placed to be microinjected.

[0108] A possible embodiment of the microfluidic circuits (6) is shown in Figures 4 and 5. In the embodiment shown, the first cell (1) and the third cell (3) have a diameter of 3 mm and a depth of 0.1 mm, while the second cell (2) has a diameter of 4 mm and a depth of 0.1 mm. The first channel (4) has a length of 10 mm and a width of 0.7 mm, while the second channel (5) has a length of 5 mm and a width of 0.7 mm.

[0109] Another possible embodiment of microfluidic circuits is shown in Figures 7 and 8. The width of both the first channel (4) and the second channel (5) is in the range of 0.5 to 0.7 mm. The first channel (4) has a section of increasing width (13) in the last 2 mm of its length near the second alveolus (2), its width increasing from 0.5-0.7 mm to 1 mm. The second channel (5) has a section of decreasing width (14) in the first 2 mm of its length near the second alveolus (2), its width decreasing from 1 mm to 0.5-0.7 mm. The circuit also comprises a sperm accumulation and recovery zone (11) of progressively smaller depth, from 0.1 mm to 0 mm over a distance of 1 mm.

[0110] Figure 9 shows the configuration example of Figure 2 with the plate system (15) made of a material with high thermal conductivity underneath it to achieve a temperature difference between the first and second drop of each system, where it can be seen that the plate (15) has some cutouts in the area of ​​the alveoli where the semen is deposited. This could also be achieved instead of through cutouts in the plate, by interposing some non-thermal conducting element between the plate and the area of ​​the plate where the alveoli are located where the semen is deposited.

[0111] Figures 10 to 13 show examples of rectangular ICSI plates (7) that have the excavated microfluidic circuits (6) with zones (8) to optionally place drops of polyvinylpyrrolidone (PVP) or other dense medium where the aspirated spermatozoa from the recovery zones are placed to be immobilized and subsequently microinjected. A washing zone (10) with zones where drops of culture medium are placed where a progressive washing of the oocytes can be done. A microinjection zone (9) with zones where drops of culture medium are placed in which the oocytes will be placed to be microinjected. The arrangement of the plates of a material with high thermal conductivity (15) in these ICSI plates (7) is also shown.

[0112] Figures 14 and 15 show examples of different arrangements of the sperm accumulation and recovery zones (11) of the third alveolus (3), as well as the sperm recovery zones (19) of the second channel (5). There are also pillars (16) in the third alveolus (3) for redirecting the sperm towards the recovery zone (11).

[0113] Figures 16 and 17 show an alternative embodiment of the invention in which the plate on which the microfluidic circuits (6) of the sperm selection system are excavated is an independent plate (20) of an ICSI plate, comprising a set of excavated microfluidic circuits (6) and optionally an area (8) for placing the recovered sperm.

Claims

MODIFIED CLAIMS received by the International Bureau on 29 October 2024 (29.10.2024) 1. Sperm selection system, characterized in that it comprises an ICSI plate (7) with at least one excavated microfluidic circuit (6), where each microfluidic circuit (6) is formed by three alveoli (1, 2, 3) and two channels (4, 5); where the two channels form a capital L (L), the length of the first channel (4) being greater than the length of the second channel (5); where the first alveolus (1) is the alveolus in which the semen sample is deposited and is arranged at the free end of the first channel (4); where the third alveolus (3) is arranged at the free end of the second channel (5);where the second alveolus (2) is arranged at the intersection between the first channel (4) and the second channel (5), and has a larger diameter than the first alveolus (1) and the third alveolus (3) in order to be able to place therein a drop of culture medium of greater radius and volume than those arranged in the drops of the alveoli at the ends, so that when these drops communicate with each other with a culture medium through the channels and cover the entire plate with mineral oil, a current flow is generated from the smallest drops of the alveoli at the ends (1 and 3) towards the largest drop of the intermediate alveolus (2); the first channel (4) corresponding to a dispersion and thermotaxis sector and the second channel (5) to a sperm selection sector by rheotaxis;and characterized in that the system comprises a plate (15) of a material with high thermal conductivity that can be placed under the plate and that has a specific configuration with a series of cutouts, such that said plate is in contact with the entire plate except with the alveoli where the semen sample will be placed; 2.- Sperm selection system, according to claim 1, characterized in that the depth of the three alveoli (1, 2, 3) and the two channels (4, 5) is the same. 3.- Sperm selection system, according to claim 2, characterized in that the depth of the three alveoli (1, 2, 3) and the two channels (4, 5) is 0.1 mm. 4.- Sperm selection system, according to claim 1, characterized in that the first channel (4) has a progressive slope so that it is deeper at the beginning (at the end in contact with the first alveolus (1)) and less deep at the end (at the end in contact with the second alveolus (2)). 27 MODIFIED SHEET (ARTICLE 19) 5.- Sperm selection system, according to any of claims 1 to 4, characterized in that the first channel (4) has a progressive widening in its final section, near the end in contact with the second alveolus (2). 6.- Sperm selection system, according to claim 4, characterized in that the first channel (4) has a section of increasing width (13) in the last 2 mm of its length close to the second alveolus (2), its width increasing from 0.5-0.7 mm to 1 mm. 7.- Sperm selection system, according to any of claims 1 to 6, characterized in that the second channel (5) has a progressive narrowing, at its beginning, so that it is wider at its end in contact with the second alveolus (2). 8.- Sperm selection system, according to claim 7, characterized in that the second channel (5) has a section of decreasing width (13) in the first 2 mm of its length close to the second alveolus (2), its width decreasing from 1 mm to 0.5-0.7 mm. 9.- Sperm selection system, according to any of claims 1 to 8, characterized in that the intersections between the alveoli and the channels are curved. 10.- Sperm selection system, according to any of claims 1 to 9, characterized in that the radius of the first alveolus (1) and the third alveolus (3) is equal. 11.- Sperm selection system, according to claim 10, characterized in that the diameter of the first alveolus (1) and the third alveolus (3) is 3 mm. 12.- Sperm selection system, according to claim 11, characterized in that the radius of the second alveolus (2) is 4 mm. 13.- Sperm selection system, according to any of claims 1 to 12, characterized in that the length of the first channel (4) is 10 mm. 14.- Sperm selection system, according to any of the claims 1 to 13, characterized in that the length of the second channel (5) is 5 mm. 28 MODIFIED SHEET (ARTICLE 19) 15.- Sperm selection system, according to any of claims 1 to 14, characterized in that the plate is preferably a polystyrene plate. 16.- Sperm selection system, according to any of claims 1 to 15, characterized in that the plate comprises more than one excavated microfluidic circuit (6). 17.- Sperm selection system, according to any of claims 1 to 10, characterized in that the third alveolus (3) has sperm recovery zones (11) formed by protrusions, progressively less deep, which cause the sperm to enter and remain trapped there, ready to be aspirated and used for ICSI. 18.- Sperm selection system, according to claim 17, characterized in that the sperm accumulation and recovery zone (11) has a progressively smaller depth, from 0.1 mm to 1 mm over a distance of 1 mm. 19.- Sperm selection system, according to any of claims 17 to 18, characterized in that there are pillars (16) in the third alveolus (3) for redirecting the sperm towards the recovery zone (11). 20.- Sperm selection system, according to any of claims 1 to 19, characterized in that along the second channel there is a set of sperm recovery zones (19) formed by progressively less deep projections, arranged on one side of the channel or on both. 21.- Sperm selection system, according to any of claims 1 to 20, characterized in that the plate (15) is made of aluminum. 22.- Sperm selection system, according to any of the claims 1 to 21, characterized in that it comprises a lid. 24.- Sperm selection system, according to any of claims 1 to 22, characterized in that the ICSI plate (7) comprises a sperm selection zone (18), which has the excavated microfluidic circuits (6) and with zones (8) where the aspirated spermatozoa are placed from the recovery zones to be immobilized and subsequently microinjected; a washing zone (10) with zones 29 MODIFIED SHEET (ARTICLE 19) where drops of culture medium can be placed where the oocytes can be progressively washed; and a microinjection zone (9) with zones where drops of culture medium can be placed in which the oocytes will be placed to be microinjected. 25.- Sperm selection system, according to any of claims 1 to 26, characterized in that the section of the channels (4,5) of the circuit (6) is rounded. 26.- Sperm selection method that uses the sperm selection system of any of claims 1 to 25, characterized in that it comprises the following steps: • Place a drop of the same volume of culture medium in each alveolus (1, 2, 3) and then a fourth drop of the same volume on the drop of the second alveolus (2), so that it will have twice the volume of the others. • Pass a sterile pipette tip over the channels, from one alveolus to another, so that the drops remain connected if they are not connected by capillarity. • Covering the entire plate with mineral oil. • Disposition of a drop of semen in the first alveolus (1). • Place a lid on the plate. • Placement of the plate on a plate made of a material with high thermal conductivity (15) that has cutouts to be in contact with the entire plate except for the alveolus in which the semen sample is located. • Placement of the plate-sheet assembly on a heated flat surface where the sheet of material with high thermal conductivity will transmit the temperature to the entire plate except for the alveolus in which the semen sample has been placed, generating a temperature gradient between the first alveolus and the second alveolus. • Aspiration of sperm from the sperm retrieval areas. 27.- Method of sperm selection, claim 26, characterized in that it is used when the plate is an ICSI plate (7), and comprises a third step 30 MODIFIED SHEET (ARTICLE 19) consisting of configuring the rest of the ICSI plate by arranging the drops that will contain the selected sperm and oocytes (eggs). MODIFIED SHEET (ARTICLE 19) DECLARATION ACCORDING TO ARTICLE 19 (1) Amendments are made pursuant to Art. 19 PCT. The modifications are as follows: A new set of claims is presented in which claim 1 has been modified, incorporating the content of the previous claims 21 and 23, thus adding the characteristics that the plate is an ICSI plate, and that the system also comprises a plate of a material with a high thermal conductivity that has a set of cuts to generate a temperature gradient between the first and second alveolus and thus provoke the response of the spermatozoa to the thermal stimulus and therefore swim from the first alveolus in which the semen sample is deposited towards the second alveolus. Claims 25 and 26 have also been eliminated because they are incompatible with the new claim 1.

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