Microfluidic device for separating active cells from passive cells

Abstract

The present disclosure describes a sperm separation device (1). The device comprises a sample zone (5) configured to receive semen, a collection zone (15) fluidly connected to the sample zone by a migration zone (10). A cover (20) is disposed over portions of the migration region and the collection region, where the migration region comprises a groove for guiding sperm in the seminal fluid, and where sperm in the seminal fluid migrates to the collection region via the groove.

Description

Technical Field

[0001] This invention relates to the field of assisted reproduction. More specifically, this invention relates to a microfluidic device for separating motile cells from non-motile cells having known predefined characteristics.

[0002] This invention is primarily developed to simulate the natural selection of sperm and to assist in the separation of high-quality sperm from semen. Background Technology

[0003] The following discussion of the prior art is intended to place the invention within a suitable technical context in order to better understand its advantages. However, unless otherwise expressly stated, any prior art mentioned in this specification should not be construed as an express or implied admission that such art is widely known or constitutes part of common knowledge in the field.

[0004] In many IVF applications, it is necessary to separate active organisms such as sperm from other active and passive sperm, as well as from aggregates of passive sperm and the fluids in which these active organisms may be suspended. In IVF, separating active sperm from other organisms and allowing them to flow into a new fluid is essential as part of the process of preparing sperm for assisted reproductive procedures, research, or evaluation.

[0005] Human assisted reproduction and artificial insemination (also known as intrauterine insemination (IUI)) are examples of such procedures that may require the separation of motile sperm. Widely used in livestock and also in humans, IUI is a low-impact and more cost-effective method of infertility treatment. IUI involves using processed sperm and injecting it into a woman's reproductive system.

[0006] Other human reproductive methods include in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). These involve using selected sperm to fertilize an egg outside the body to form an embryo, which is then cultured and implanted into the uterus. IVF involves injecting multiple selected sperm into an egg to promote fertilization, while ICSI involves injecting sperm directly into the egg.

[0007] Sperm selection is a crucial step in the IVF process. Higher sperm quality leads to a higher success rate. Traditional sperm selection methods, such as density gradient centrifugation and upstream methods, can cause DNA fragmentation and cell death, resulting in IVF cycle failure. These methods are also costly, time-consuming, and require highly specialized equipment.

[0008] All of these procedures require some degree of sperm sorting or processing to separate sperm from the semen, which gradually becomes an environment harmful to sperm. Therefore, it is also necessary to remove poor-quality sperm, background cells, and immobile organisms or contaminants. Existing equipment designs and methods of operation have several shortcomings that are crucial for achieving robust and ergonomic sperm separation.

[0009] US Patent 2015 / 0140655A1 describes a device for separating sperm from semen. The device includes an outer chamber for holding a semen sample, a central chamber for collecting the separated sperm, and a network of microchannels between the two. These microchannels are shaped radially, radiating outward from the central chamber. The device operates by utilizing the self-propelled motion of motile sperm and their tendency to swim towards and along surfaces within confined spaces—a behavior known as "wall swimming." The microchannels are designed to allow sperm to swim through them and enter the central chamber, and at least one junction guides the sperm based on their wall swimming behavior. The viscosity of the fluid within the microchannels can be relatively high. The microchannels converge in a cascading fashion from the outer semen chamber, with a smaller number of microchannels emptying into the central chamber.

[0010] The device described in US2015 / 0140655A1 requires sealing the device by covering the central port with tape or a cap after collection. Tape is typically used, but any adhesive that comes into contact with sperm and the sperm-carrying medium poses a toxicity risk. Placing and removing the cap is also a very delicate step, and many clinicians mishandle it, resulting in incomplete sealing, allowing semen to flow into the center of the device during injection. Furthermore, removing the cap creates suction that can inadvertently draw in raw semen, thus disrupting all the separation work performed beforehand. In other words, in US2015 / 0140655A1, raw semen can contaminate the collected sample.

[0011] Furthermore, in US 2015 / 0140655A1, semen was injected into a large channel / reservoir. During injection, the pressure is always highest in the channels closest to the injection port. Therefore, semen is likely to enter the first few channels near the injection port, increasing the risk of contamination.

[0012] Furthermore, the amount of semen collected under US2015 / 0140655A1 is not controlled. For example, if you ask an embryologist to collect 100µL, there is no way to prevent them from collecting 200µL, which means that the extra 100µL must come from the semen sample, which will be aspirated into the channel and contaminate the collected sample.

[0013] US2015 / 0140655A1 requires additional preparation steps to load the semen buffer into the device. Due to the complexity of the ratchet array, loading the semen buffer into the device requires immersing the device in the buffer and then placing it in a vacuum chamber to remove air bubbles. This process is both impractical and time-consuming.

[0014] Furthermore, due to the structure and design of the channel, a large number of sperm in the semen cannot enter the device of US2015 / 0140655A1, thus limiting the semen collection yield.

[0015] Another drawback of US2015 / 0140655A1 is that the channel is located on the bottom plate of the device, so there is a large amount of fluid in the semen reservoir and collection area above the channel. If the fluid is unbalanced, it will create a gravity pump effect, effectively pushing the semen through the device and contaminating the collected semen.

[0016] There is a need for a device that can seal the device without requiring a strap or cover to be placed over the central port after collection.

[0017] There is also a need for a device that can simplify and reduce the number of steps required to collect sperm.

[0018] In addition, a device is needed to limit the maximum amount of fluid that can be collected.

[0019] Additionally, a device is needed that can load sperm buffer without vacuum aspiration.

[0020] A device with inclined grooves and / or channels is also needed to allow sperm to settle and move upwards, thereby increasing sperm collection yield.

[0021] A device is also needed that has grooves and / or channels above the sample area to restrict the flow of any passive material into the channels.

[0022] There is also a need for a device that can separate sperm from semen samples in fewer steps.

[0023] In addition, there is a need for a device that allows for the selection of sperm within the platform in order to further promote fertilization methods.

[0024] Additionally, there is a need for a device that allows for sperm selection within a more stable platform, thereby preventing semen, semen components, passive substances, and contaminants from entering the fluid containing the target sperm.

[0025] Further improvements are needed in equipment and methods that can select sperm from semen samples using methods other than those based on motility separation, thereby obtaining motile sperm with certain (or definable) desired characteristics.

[0026] We also need a platform that can fix the sperm selection device during the selection process to prevent spillover risks and human error, provide environmental control in terms of temperature regulation, and provide visualization and data analysis of the selected sperm for assisted reproductive treatment practices.

[0027] This invention mimics some of the natural selection processes present in the female reproductive tract, aiming to select higher quality individual sperm or sperm populations compared to traditional preparation methods.

[0028] The object of this invention is to overcome or improve at least one of the disadvantages of the prior art, or to provide a useful alternative. At least one preferred embodiment of the invention aims to provide a microfluidic sperm selection device that provides a more reliable process for selecting high-quality sperm. Summary of the Invention

[0029] According to one aspect of the present invention, a sperm separation apparatus is provided, comprising: The sample area is configured to receive semen. A collection region, configured to be fluidly connected to the sample region via a migration region; and A lid, which is positioned above portions of the migration area and the collection area, The migration zone includes grooves for guiding sperm in the semen; and The sperm in the semen migrates to the collection area via the groove.

[0030] According to another aspect of the invention, a docking device is provided, comprising a container configured to receive an imaging system, a recording system, a temperature control system, a magnifying glass, and a sperm separation device as disclosed herein.

[0031] In some embodiments, the sample area may be shaped to receive and contain a certain volume of semen. In some embodiments, the sample area may be tilted to form a ramp toward the migration zone to increase the flux of the agent toward the migration zone and / or the collection zone.

[0032] In some embodiments, the slope inclination angle can be in the range of 2 degrees to 89 degrees. Preferably, the slope inclination angle is in the range of 7 degrees to 13 degrees.

[0033] In some embodiments, the ramp may be configured to allow passive material to settle and move away from the migration zone to help prevent contamination of the migration and collection zones. In some embodiments, the ramp may include a series of grooves for allowing semen to accumulate in layers and for sperm to settle and migrate upwards to the migration zone. In some embodiments, the series of grooves is linear. In some embodiments, the series of grooves is curved. In some embodiments, the series of grooves is spiral. In some embodiments, the series of grooves is serrated. In some embodiments, the series of grooves is converging. In some embodiments, the series of grooves is diverging. In some embodiments, the series of grooves is inclined. In some embodiments, the series of grooves is downward sloping. In some embodiments, each groove in the series may have a different configuration from the others. Preferably, the grooves are configured in combination to guide sperm toward the migration zone, aiming to increase the yield of sperm involved in separation.

[0034] In some embodiments, the sample area may have a predetermined size and shape, with a depth sufficient to allow semen to deposit into and remain therein. In some embodiments, the sample area may be in the shape of a complete ring (i.e., circular). In some embodiments, the sample area may be in the shape of a semi-circular ring (i.e., semi-circular). In some embodiments, the sample area may be in the shape of a rectangular prism. In some embodiments, the sample area may be in the shape of a serpentine channel. In some embodiments, the sample area may be in the shape of a wavy channel. In some embodiments, the sample area may be in the shape of a bowl. Preferably, the sample area may be in the shape of a complete ring, a semi-circular ring, or a rectangular prism. In other embodiments, the sample area may be other suitable shapes.

[0035] In some embodiments, the depth of the sample area can range from 10 µm to 50,000 µm. In some embodiments, other depths can be used depending on the volume required for the sperm species to be processed.

[0036] In some embodiments, the sample area may be an open area. In some embodiments, the sample area may be a partially closed area. In some embodiments, the sample area may be a closed area. In some embodiments, when the sample area is a closed area, the sample area may include openings for depositing semen into the sample area. In some embodiments, the sample area includes multiple openings for depositing semen into the sample area.

[0037] In some embodiments, the collection area may be configured to concentrate motile sperm in a single location. In some embodiments, the collection area may be located downstream of the sample area. In some embodiments, the collection area may slope downwards from the migration area to encourage sperm migration to the collection area and prevent sperm migration back to the sample area. In some embodiments, the downward slope angle may be in the range of 50 degrees to 90 degrees.

[0038] In some embodiments, the downward slope of the collection area may include one or more blocking members descending along the length of the groove to prevent sperm from migrating upwards and re-entering the migration area. In some embodiments, the downward slope of the collection area may include one or more blocking members descending along a portion of the length of the groove to prevent sperm from migrating upwards and re-entering the migration area. In some embodiments, the blocking member is a protrusion. In some embodiments, the blocking member is a ridge. In some embodiments, the blocking member is a step. In some embodiments, the step may descend in increments between 10µm and 1000µm. Preferably, the step may descend in increments between 30µm and 100µm. In some embodiments, the blocking member may adopt other suitable constructions / shapes to prevent sperm from migrating upwards and re-entering the migration area.

[0039] In some embodiments, the collection area may be in the form of a depression. In some embodiments, the depression may be located below the height of the inlet where the groove enters the collection area to encourage sperm to descend to a level below the end of the migration area and prevent sperm from re-entering the migration area. In some embodiments, the cross-sectional profile of the depression may be cylindrical. In some embodiments, the cross-sectional profile of the depression may be conical. In some embodiments, the cross-sectional profile of the depression may be bowl-shaped. In some embodiments, the depression may have different cross-sectional profiles.

[0040] In some embodiments, the collection area may include a wall. In some embodiments, the collection area may include multiple walls. In some embodiments, the height of the wall may decrease linearly to allow sperm to descend into the collection area. In some embodiments, the height of the wall may decrease gradually to allow sperm to descend into the collection area. In some embodiments, the height of the wall may decrease periodically to allow sperm to descend into the collection area.

[0041] In some embodiments, the wall may be configured to keep sperm in a semi-gliding mode to prevent them from adhering to the device surface and subsequently resisting collection forces. In some embodiments, the wall may include a micropatterned surface to prevent sperm adhesion to the surface and promote overall swimming behavior. In some embodiments, the micropatterned surface may include small protrusions. In some embodiments, the micropatterned surface may include a ridged structure. In some embodiments, the micropatterned surface may include a stepped structure. In some embodiments, the micropatterned surface may include a wavy structure. In some embodiments, the micropatterned surface may include other suitable protrusions or designs to prevent sperm adhesion to the surface and promote overall swimming behavior.

[0042] In some embodiments, the wall may be chamfered. In some embodiments, a portion of the wall may be chamfered. In some embodiments, the top portion of the wall may be chamfered.

[0043] In some embodiments, the collection area may include a selection mechanism within the collection area designed to immobilize sperm with desired characteristics after selection based on motility. In some embodiments, the selection mechanism may be a micropattern of a reagent capable of binding to sperm, the reagent expressing a corresponding mark on the surface of its membrane. In some embodiments, the micropattern may take the form of a zigzag, linear, dotted, or any other arrangement capable of effectively exposing separated sperm to the binding agent.

[0044] In some embodiments, the collection area may include a bottom surface for sperm microscopy. In some embodiments, the bottom surface may be flat. In some embodiments, the bottom surface is aligned with the gap in the cap and the restricted angle prevents direct contact between the suction device and the migration area. In some embodiments, the ratio of the diameter or width of the gap in the cap to the maximum diameter or width of the collection area may be in the range of 1:2 to 1:20. Preferably, this range is between 1:3 and 1:5.

[0045] In some embodiments, the collection area may include a bottom section configured to concentrate sperm in a specific local area of ​​the collection area so that the sperm are kept near the collection force, i.e., near the suction force generated by the tool used to aspirate fluid within the collection area.

[0046] In some embodiments, the bottom section may be in the shape of a convex cone. In some embodiments, the bottom section may be in the shape of a concave cone. In some embodiments, the bottom section may be in other suitable shapes.

[0047] In some embodiments, the collection area may be covered with a surface coating to prevent sperm from adhering to any surface within the collection area. In some embodiments, the surface coating may include, but is not limited to, surfactant additives, polymer coatings (e.g., polyvinyl alcohol or PVA), proteins (e.g., BSA or HAS), polyethylene glycol (PEG) polymers, and any other hydrophilic treatment agents.

[0048] In some embodiments, the volume of the collection zone can range from 50 µL to 1,000 µL. Preferably, the volume of the collection zone can range from 150 µL to 500 µL. In other embodiments, other volumes may be used.

[0049] In some embodiments, the depth of the collection area can range from 100µm to 10,000µm. Preferably, the depth of the collection area can range from 2,000µm to 4,000µm. In other embodiments, other depths may be used depending on the geometry of the pit.

[0050] In some embodiments, the collection area may be loaded with a chemical reagent and diffused into the migration area to induce sperm sensitive to the chemical reagent to move toward the collection area. In some embodiments, the chemical reagent may be a chemical attractant. In some embodiments, the chemical attractant may include, but is not limited to, progesterone, follicular fluid, fallopian tube fluid, lactic acid, prostaglandins, and chemokines.

[0051] In some embodiments, the migration zone may include the confluence of two sub-grooves to provide sperm selection during sperm migration, one sub-grooves being a dead end and the other sub-grooves leading to the collection zone and containing a chemical attractant.

[0052] In some embodiments, the sub-groove with a dead end may include a decrease in groove height to prevent unwanted sperm from returning to the path connected to the collection area. In some embodiments, the sub-groove with a dead end may be closed at its end or open to air so as to allow the migration area to be fully filled with air when in an open configuration, or to generate air bubbles to seal the end of the groove when in a closed configuration, thereby providing an edge for sperm penetration and helping to prevent sperm backflow.

[0053] In some embodiments, the groove not connected to the collection area may have a dead end, and the end may form air bubbles due to the loading of the device buffer solution, such that the bends created between the air bubbles, the passage surface, and the fluid are deep enough to prevent sperm from migrating back once they enter, forming a trap for sperm to enter and hindering their return. In some embodiments, the height of the groove with the dead end at the inlet of the dead end may vary to prevent backflow of non-chemisensitive sperm.

[0054] In some embodiments, when the collection area is loaded with chemical reagents, the groove may have an appropriate length and geometry to provide a stable chemical reagent gradient throughout the duration of sperm selection. In some embodiments, the length of the groove may range from 1 mm to 15 mm. Preferably, the length of the groove is in the range of 6 mm to 8 mm.

[0055] In some embodiments, the cap may include a slit configured to receive a suction device configured to collect sperm within a collection area. In some embodiments, the slit may be configured to remove some or all of the fluid from the collection area without collecting unwanted passive material that may be located further upstream in the device. In some embodiments, the slit may be configured as a buffer loading port for filling the device with a selected buffer solution, thereby utilizing capillary forces to deliver the buffer solution into the recess. In some embodiments, the slit may be configured as a buffer loading port for filling the device with a selected buffer solution, thereby causing the force of the buffer solution ejected from the buffer dispensing device to push the buffer solution into the recess.

[0056] In some embodiments, the sperm buffer may be dispensed to the inlets on either side of any groove, thereby allowing capillary forces to move the buffer into the groove. In some embodiments, the sperm buffer may be dispensed into the migration zone.

[0057] In some embodiments, the cap includes an inward-facing cavity such that when suction is applied to the collection area (i.e., when the pit is emptied during sperm collection), an air bubble forms between the cap and the collection area. This air bubble blocks the fluid connection between the migration area and the collection area, thereby limiting the amount of sperm collected from the collection area and preventing unwanted material from being collected from the sample area or migration area. In some embodiments, the inward-facing cavity may be chamfered towards the gap to help narrow the collection area inlet at the intersection of the collection area and the migration area, and to promote air bubble formation by buffering the surface tension of the fluid there. In some embodiments, the collection area with the cap on top may have a generally rhomboid cross-sectional profile.

[0058] In some embodiments, the migration region and the cap are connected, thereby forming a fluid boundary between the sample region and the migration region.

[0059] In some embodiments, the lid may include a lip located beneath the lid to allow air exchange.

[0060] In some embodiments, the collection area may not be covered by a lid, but may be an open fluid reservoir that may or may not be covered with an oil coating to allow for the pickup of individual sperm using a micromanipulator.

[0061] In some embodiments, the migration region may be an open region. In some embodiments, the migration region may be a partially enclosed region. In some embodiments, the migration region may be a closed region. Preferably, the migration region is a partially enclosed region to help form a fluid boundary.

[0062] In some embodiments, the total length of the migration region may be greater than 0.5 mm. Preferably, the total length of the migration region is in the range of 3 mm to 5 mm.

[0063] In some embodiments, the height of the groove may be sufficient to generate adequate fluid resistance to prevent semen with a viscosity higher than water from flowing into the collection area and to form a fluid boundary between the semen and the migration area in the sample area. In some embodiments, the height of the groove may range from 5 µm to 400 µm. Preferably, the height of the groove is in the range of 40 µm to 120 µm.

[0064] In some embodiments, the migration zone may be located downstream of the sample area. In some embodiments, the migration zone may slope downwards from the sample area to facilitate sperm migration to the collection area and prevent sperm from migrating back to the sample area. In some embodiments, the downward slope angle may be in the range of 50 to 90 degrees.

[0065] In some embodiments, the migration zone may include at least one abrupt drop in vertical height to a depth, preventing sperm traveling along the base of the migration zone from (easily) returning to that depth, thereby increasing the yield of sperm collected in the collection zone and preventing the loss of desired sperm due to backward migration at any stage of the selection process. In some embodiments, the height drop may take the form of a cut in the base of the migration zone. In some embodiments, the height drop may take the form of a new groove. In some embodiments, the height drop may take the form of an existing groove with an extended depth. In some embodiments, the height drop may take the form of a semi-circular recess configured to guide and facilitate the redirection of sperm toward the collection zone, rather than forming a sharp corner, in which sperm would burrow in and become stuck and unable to turn away.

[0066] In some embodiments, the downward slope of the migration zone may include one or more blocking members descending along the length of the groove to prevent sperm from migrating upwards back and re-entering the sample area. In some embodiments, the downward slope of the migration zone may include one or more blocking members descending a portion of the length of the groove to prevent sperm from migrating upwards back and re-entering the sample area. In some embodiments, the blocking member is a protrusion. In some embodiments, the blocking member is a ridge. In some embodiments, the blocking member is a step. In some embodiments, the step may descend in increments ranging from 10 µm to 1000 µm. Preferably, the step may descend in increments ranging from 30 µm to 100 µm. In some embodiments, the blocking member may be other suitable constructions / shapes to prevent sperm from migrating upwards back and re-entering the sample area.

[0067] In some embodiments, the migration region includes a single layer of grooves. In some embodiments, the migration region includes multiple layers of grooves. In some embodiments, the multiple layers of grooves may intersect each other.

[0068] In some embodiments, each groove may be linearly constructed. In some embodiments, each groove may be curved. In some embodiments, each groove may be labyrinthine. In some embodiments, each groove may be spirally constructed. In some embodiments, each groove may be converging. In some embodiments, each groove may be diverging. In some embodiments, each groove may be inclined. In some embodiments, each groove may be downwardly inclined. In some embodiments, each groove may have other suitable constructions. In some embodiments, each groove may have a different construction from each other. Preferably, the grooves are constructed in combination to maximize fluid resistance within the migration zone while selecting sperm.

[0069] In some embodiments, the width of each groove along its length may remain constant. In some embodiments, the width of each groove along its length may vary. Preferably, each groove narrows near the collection area to increase fluid resistance and reduce the potential opening for sperm to return in the direction of the sample area.

[0070] In some embodiments, each groove may extend into the sample area to facilitate the guidance of more sperm from the semen and into the migration zone. Thus, these grooves may contain both semen and buffer solution simultaneously, while still maintaining the fluid boundary between the two zones.

[0071] In some embodiments, each groove may extend the entire length of the migration region. In some embodiments, each groove may extend a portion of the length of the migration region. In some embodiments, each groove may extend from the migration region to the collection region. In some embodiments, each groove may extend from the sample region to the collection region.

[0072] In some embodiments, the length of each groove can range from 10µm to 30,000µm. In some embodiments, the length of each groove can depend on the area in which the groove is located.

[0073] In some embodiments, the height of each groove along its length may remain constant. In some embodiments, the height of each groove along its length may vary. In some embodiments, the top of each groove along its entire length is open. In some embodiments, the top of each groove along its entire length is closed. In some embodiments, the top of each groove along its entire length is partially closed. In some embodiments, the height of each groove may be in the range of 5µm to 400µm. Preferably, the height of each groove is in the range of 20µm to 12µm.

[0074] In some embodiments, the width of each groove along its length may remain constant. In some embodiments, the width of each groove along its length may vary. In some embodiments, the width of each groove may range from 5 µm to 10,000 µm. In some embodiments, the width of each groove connecting the sample area and the migration area may range from 20 µm to 100 µm. In some embodiments, the width of each groove connecting the migration area and the collection area may range from 10 µm to 40 µm.

[0075] In some embodiments, the cross-sectional profile of each groove along its length may remain constant to achieve optimal sperm persistence, depending on the type of sperm being processed in the device—persistence refers to the ability of sperm to migrate through the groove to the end of the migration zone with maximum displacement from its sperm initiation position at the beginning of the migration zone in the shortest possible time, thereby conserving energy. In some embodiments, the cross-sectional profile of each groove along its length may vary.

[0076] In some embodiments, each groove may include a guide member for guiding sperm toward the collection area. In some embodiments, each groove may include multiple guide members for guiding sperm toward the collection area. In some embodiments, each groove may include a combination of different guide devices to provide multiple guide paths for sperm to reach the collection area. In some embodiments, the guide member may be in the form of a angular edge. Preferably, each groove includes two angular edges. In some embodiments, the guide member may be a wavy wall protrusion for guiding sperm forward and preventing sperm from migrating backward. In some embodiments, other suitable guide members may be used. Preferably, the groove will include a combination of corners and grooves to provide multiple guide paths for sperm to reach the collection area.

[0077] In some embodiments, each groove may have a wall profile designed to provide additional edges for sperm to follow.

[0078] In some embodiments, the migration zone may include outwardly extending protrusions to prevent semen from passing through the migration zone and to prevent semen from being injected into the migration zone.

[0079] In some embodiments, a junction may exist between the migration zone and the sample zone. In some embodiments, the junction may include a series of protrusions. In some embodiments, the protrusions may be in the form of pillars. In some embodiments, these pillars may extend from the base plate of the ramp or the base plate of the sample zone to the height of the migration zone or higher, and are configured to act as an additional initial barrier against viscous semen, but allow sperm to pass through between them into the migration zone. In some embodiments, these pillars may be cylindrical in shape. In some embodiments, these pillars may be prismatic in shape. In some embodiments, these pillars may be mound-shaped. In some embodiments, these pillars may be of different shapes suitable for assisting in preventing seminal fluid from flowing into downstream areas. In some embodiments, the distance between the pillars may be in the range of 10 µm to 300 µm. Preferably, the distance between the pillars is in the range of 40 µm to 80 µm.

[0080] In some embodiments, the column can be configured to prevent the user from injecting semen directly into the migration area by guiding the pipette tip and syringe away from the migration area and toward the base of the sample area.

[0081] In some embodiments, the sperm separation device may include a hydrophilic surface. In some embodiments, the grooves in the migration region may have a hydrophilic surface to prevent bubble formation and promote sperm migration by preventing sperm from adhering to the surface within the device. In some embodiments, a section of the grooves in the migration region may have a hydrophilic surface.

[0082] In some embodiments, the sperm separation device may include proteins that bind to a hydrophilic surface, allowing the proteins to bind naturally to the device.

[0083] In some embodiments, the sperm separation apparatus may include an oocyte region for receiving oocytes and sperm collected from a collection region, wherein the oocyte region is configured for intracytoplasmic sperm injection (ICSI) of the oocytes. In some embodiments, the oocyte region may include mechanisms for containing selected sperm and oocytes. In some embodiments, the oocyte region may include spatial droplets for containing selected sperm and oocytes.

[0084] In some embodiments, a sperm separation device including an oocyte region can be shaped to be mounted on a microscope stage.

[0085] In some embodiments, a sperm separation device including an oocyte region may include a collection area having a surface coated with a reagent micropattern for selectively binding and immobilizing sperm having desired biological characteristics expressed externally on their membranes.

[0086] In some embodiments, the micropattern may be a zigzag shape. In some embodiments, the micropattern may be in the form of dots. In some embodiments, the micropattern may be in the form of strips. In some embodiments, the micropattern may be in the form of other suitable shapes covering part or all of the width / diameter of the collection area. In some embodiments, the micropattern may include multi-row / layered micropatterns. In some embodiments, the sperm separation device may include proteins bound to the device surface through a hydrophilic coating that allows the proteins to bind naturally.

[0087] In some embodiments, the micropattern may include a bonding layer for attaching an active sperm binding agent to the device. In some embodiments, the bonding layer may vary depending on the material used to fabricate the device and may include PEG chains, polydopamine, agarose, heparin, or any other layer that mediates sperm selection and binding agent attachment.

[0088] In some embodiments, the sperm binding agent can be attached to the device via a connecting layer to promote positive sperm binding, thereby facilitating examination of sperm morphology and micromanipulation pickup. In some embodiments, the sperm binding agent may be hyaluronic acid (HA), anti-Juno antibodies, integrin α-9, zona pellucida protein 3 (ZP3), antibodies against externally expressed sperm markers (e.g., anti-SP17 and anti-TEX101 antibodies), or sex-specific antigens (e.g., requimod, anti-CD52, imiquimod, and gadiquimod). In some embodiments, other suitable sperm binding agents may be used.

[0089] In some embodiments, active selection can be performed in the same apparatus, followed by hyaluronic acid binding. Unlike conventional sperm separation methods (e.g., density gradient centrifugation (DCG)), hyaluronic acid binding in DCG is performed in a separate culture dish after sperm washing. Advantageously, 91.1% of motile sperm (i.e., all motile sperm in the claimed apparatus, since only motile sperm can reach the concentration zone) bind to the micropattern, while only 79.75% of motile sperm (some sperm are immobile after DGC) bind to the micropattern after DGC. The claimed invention is superior to conventional sperm separation methods (e.g., DGC) because centrifugation-based damage can occur during sperm selection, and reactive oxygen species generated by DGC can inhibit sperm binding ability.

[0090] In some embodiments, the sperm separation device containing the oocyte region may be configured to allow a mineral oil coating to ensure the sterility of the device contents, the mineral oil coating being shallow enough that a micromanipulator can access the device contents. In some embodiments, the depth of the sperm separation device may be in the range of 3 mm to 40 mm. Preferably, the depth of the sperm separation device is in the range of 10 mm to 20 mm.

[0091] In some embodiments, the sperm separation device may be made of a polymeric material, such as cyclic olefin copolymer (COC), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), polycarbonate (PCA), or any other gamete biocompatible polymer that may be advantageously used to manufacture the device of the present invention.

[0092] In some embodiments, the sperm separation apparatus may include a protective device for reducing the risk of spillage, maintaining the sterility of the apparatus, and preventing the medium from evaporating from the apparatus. In some embodiments, the protective device is in the form of a cover. In other embodiments, other suitable protective devices may be used. Attached Figure Description

[0093] Preferred embodiments of this disclosure will now be described by way of example only, with reference to the accompanying drawings, in which: Figure 1 This is a top-view perspective view of the sperm separation device; Figure 2 yes Figure 1 A cross-sectional view of the device shown; Figure 3 yes Figure 2 A close-up view of the cross-section shows the slope, stepped migration zone, and collection zone of the sample area; Figure 4 yes Figure 1 A close-up view of the inclined groove and columnar structure of the device shown; Figure 5 yes Figure 1 A close-up view of the stepped descending groove of the device shown; Figure 6 This is a top perspective view of an alternative embodiment of the device with a lid; Figure 7 yes Figure 1 A cross-sectional view of the device with a lid shown; Figure 8 yes Figure 1 A close-up view of the sub-grooves of the device shown, one of which is a dead end, while the other has a chemical attractant leading to the collection area; Figure 9 yes Figure 1 A close-up view of the corner of the groove in the device shown; Figures 10a to 10f This is a cross-sectional view of an alternative embodiment of the recessed area in the collection zone; Figure 11 yes Figure 1 A top perspective view of the device with a cover and lid shown; Figure 12 yes Figure 11 Exploded top perspective view of the device shown; Figure 13 This is a top perspective view of an alternative embodiment of a sperm separation device with a lid; Figure 14 yes Figure 13 Exploded top perspective view of the device shown; Figure 15 This is a cross-sectional view of an alternative embodiment of a device having multiple grooves; Figure 16 yes Figure 15 A close-up cross-sectional view of the device shown; Figure 17 yes Figure 15 A top-down perspective close-up view of the collection area of ​​the device shown; Figure 18 This is an exploded view of an alternative embodiment of the device, which includes an oocyte region, a lid, and a cover; Figure 19 This is an exploded view of an alternative embodiment of the device, which includes an oocyte region, a lid, and a cover; Figures 20a to 20c This is a close-up view of an alternative embodiment of the step in the groove; Figures 21a to 21c This is a close-up view of an alternative embodiment of the downward groove; Figure 22 This is an exploded view of an alternative embodiment of a sperm separation device, which includes an oocyte region and micropatterns; Figure 23This is a diagram illustrating sperm DNA fragmentation through sperm selection methods; Figure 24 It is a chart showing the comparison of sperm motility through sperm selection methods; Figure 25 It is a chart showing the binding efficiency according to sperm selection methods; Figure 26 This is a flowchart illustrating the operation of an embodiment of the device; and Figure 27 This is a flowchart of an alternative embodiment of the device. Detailed Implementation

[0094] The invention will now be described with reference to the following examples, which should be considered exemplary and not limiting in all respects. In the figures, corresponding features in the same embodiment are given the same reference numerals.

[0095] Overview and first reference Figure 1 The sperm separation device 1 is configured to mimic natural selection of sperm and assist in separating high-quality sperm from semen. In the illustrated embodiment, the device is generally circular in shape. However, in other embodiments, the shape of the device may differ. Figure 13 and Figure 14 An alternative embodiment of the device 1, which is rectangular in shape, is shown. Figure 18 and Figure 19 Alternative embodiments of the device 1 in different shapes are shown, having an additional oocyte region 85 (hereinafter referred to as...). Figure 18 and Figure 19 (Description to follow). The sperm separation device 1 includes a sample area 5 configured to receive semen (not shown), a collection area 15 fluidly connected to the sample area 5 via a migration area 10, and a cover 20 disposed above the migration area 10 and a portion of the collection area 15 (as described above). Figure 6 (As shown). The migration zone 10 includes a groove 35 for guiding sperm (not shown) in the semen from the sample zone 5 into the collection zone 15.

[0096] In the illustrated embodiment, sample area 5 has an open top surface and a groove 35. However, in other embodiments, the top surface of sample area 5 may be partially open or partially closed, and may not have any groove.

[0097] refer to Figure 3 The sample area 5 is inclined to form a ramp 40 facing the migration area 10. This advantageously allows the semen sample to remain on the ramp 40, causing the passive components in the semen to settle under gravity and move away from the migration area 10, while actively promoting sperm to enter the migration area 10 along the groove 35 and cross the fluid interface formed between the semen and the sperm buffer (not shown). The inclination angle of the ramp 40 is between 2 degrees and 89 degrees.

[0098] Sperm buffer can be any medium that can maintain sperm motility. The viscosity of the medium can also be adjusted to create an environment more similar to the female reproductive tract, thereby increasing the yield of collected sperm. Medium can be buffered with bicarbonate, MOPS (3-(N-morpholino)propanesulfonic acid), or HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), and typically contains human serum albumin, some antioxidants, antibiotics (such as gentamicin), glucose, and lactate. This list is not exhaustive, and other types of media may also be used.

[0099] like Figure 2 and Figure 3 As shown in the optimal configuration, the collection area 15 slopes downward relative to the migration area 10. The angle of downward slope is between 90 and 50 degrees. Specifically, the collection area 15 exists on a plane above the bottom of the sample area 5. This configuration facilitates the creation of a zero-fluid-pressure environment because there are no enclosed grooves that would push the semen sample through and fill the device, thus eliminating the risk of semen flowing into the downstream area (i.e., the collection area).

[0100] refer to Figure 2 The collection area 15 is in the form of a well 45, positioned below the entrance height of the collection area 15, allowing sperm from the migration area 10 to fall into the collection area 15. The collection area 15 is configured to accommodate a sperm volume of 50 µL to 2000 µL. The collection area 15 also has a flat bottom surface 50 for sperm microscopy. In the illustrated embodiment, the well 45 is generally inverted trapezoidal in shape. In alternative embodiments, the shape of the well 45 may be as follows: Figures 10a to 10f Other shapes shown.

[0101] refer to Figure 5 The downward slope of the collection zone 15 includes one or more steps 55 designed to prevent sperm from easily returning towards the migration zone 10 and to promote bulk-swimming behavior, thereby increasing collection yield. The one or more steps 55 descend in increments between 10 µm and 1000 µm. In the illustrated embodiment, the steps 55 are generally semi-circular in shape. In alternative embodiments, the steps may be shaped as follows: Figures 20a to 20c Other shapes shown.

[0102] The migration zone 10 is configured such that sperm can be confined within the traversable geometry of the migration zone 10 by fluid surface tension or by the arrangement of surfaces extending from the device itself.

[0103] In such Figure 8In the alternative arrangement shown, the migration zone 10 also includes the confluence of two sub-grooves, one of which has a dead end 60, while the other sub-grooves 65 leads to the collection zone 15 and contains a chemical reagent, which is present in the form of a chemical attractant (not shown). Advantageously, the use of a chemical attractant increases sperm production in the collection zone 15 because the chemical attractant induces sperm to move towards it unless the sperm can sense a change in the concentration of the chemical attractant, in which case the sperm will turn 135 degrees and follow the gradient of increasing concentration towards the collection zone 15. This alternative embodiment allows for the selective selection of specific types of sperm to the collection zone 15. Examples of chemical attractants include progesterone, RANTES, lysine, bortezidine, atrial natriuretic peptide, hyaluronic acid, or female follicular fluid. This list is not exhaustive, and other types of chemical attractants may also be used.

[0104] like Figure 4 As shown in the optimal configuration, the junction of the sample area 5 and the migration area 10 also includes an outwardly extending protrusion in the form of a columnar protrusion 70. The columnar protrusion 70 is located at the inlet of the migration area 10 and serves to prevent semen from passing through and being injected into the migration area 10. The columnar protrusion 70 effectively prevents the possibility of viscous semen flowing into downstream areas by increasing the fluid resistance within the space through which the fluid needs to pass. Furthermore, the columnar protrusion 70 also helps prevent the user from directly depositing semen at the inlet of the migration area 10, thereby preventing semen from being injected directly into subsequent areas at high speed.

[0105] In the illustrated embodiment, the migration region 10 includes a single-layered groove 35. However, in alternative arrangements, such as... Figures 15 to 17 As shown, the migration zone may include multiple layers of grooves 35. The multiple layers of grooves 35 can improve the sperm yield migrating from the sample zone 5 to the collection zone 15.

[0106] In the illustrated embodiment, the migration area 10 is partially covered by the cover 20, meaning the migration area 10 has a partially open top surface, such as... Figure 6 As best shown. However, in other embodiments, the top surface of the migration region 10 may be open or closed.

[0107] The lid 20 also covers the collection area 15 and includes a gap 25 configured to receive a suction device for collecting sperm within the collection area 15. Figure 7As best shown, the cap 20 has an inwardly chamfered cavity 30. Advantageously, the combination of the recess 45 and the chamfered cavity 30 in the cap 20 causes the fluid at the junction of the migration zone 10 and the collection zone 15 to be compressed. This creates a fluid meniscus at the junction between the migration zones 10, causing air bubbles to form between the cap 20 and the collection zone 15 when suction is applied to the collection zone 15. These air bubbles inhibit the fluid connection between the migration zone 10 and the collection zone 15, thereby limiting the amount of sperm collected from the collection zone 15.

[0108] In the illustrated embodiment, the groove 35 extends into the sample area 5 to facilitate the guidance of more sperm out of the semen and into the migration area 10. However, in an alternative arrangement, the groove 35 in the migration area 10 extends at least a portion of the length of the migration area 10 and may continue to extend from or into the sample area 5 or collection area 15, with the aim of separating sperm capable of passing through the geometry within the migration area 10.

[0109] In the illustrated embodiment, as Figure 9 As shown, each groove 35 also includes a pair of corners 75 for guiding sperm, as sperm motility is influenced by the surfaces with which they interact, and sperm typically exhibit wall-swimming behavior, or tend to travel closer to the boundary surface within confined groove geometry (e.g., in microfluidic channels). However, in alternative embodiments, the groove may have one or more corners. Compared to those present in microchannels with smooth / planar walls, the walls of the groove 35 are profiled to provide / form additional edges and boundary surfaces, thereby enabling more advantageous improvement in sperm separation according to motility criteria. Furthermore, the corners 75 advantageously provide additional edges for guiding sperm.

[0110] The sperm separation features within the migration zone 10 also prevent sperm from returning to the sample area 5, which is beneficial for improving sperm yield and quality, as the first sperm capable of navigating the path to the collection area 15 typically possesses the most desirable characteristics. For example, incorporating a height difference (i.e., a step) during sperm journey can prevent sperm from returning to the migration zone 10. Preferably, this height difference is a sharp, curved incision to prevent sperm from getting stuck at the corner of the groove. The length of the migration zone 10 is also configured such that it reduces the chance of sperm deflection, yet is long enough to prevent sperm from being accidentally carried away from the sample area 5. Preferably, the length of the migration zone 10 is between 3 mm and 5 mm.

[0111] In the illustrated embodiment, device 1 has an open top. However, in an alternative embodiment, the device may include a cover 80 to reduce the risk of spillage, maintain the sterility of the device, and prevent the medium from evaporating from the device; see [link to previous embodiment]. Figure 11 and Figure 12 . Figure 28 A cross-section of the device 1 with the cover 80 is shown.

[0112] like Figure 11 , Figure 12 and Figure 28 As shown, the cover 80 has a gap 82 for receiving the cover 20. Figure 11 As shown, the void 82 has a vertical wall 122. Figure 12 As shown, the edge of the lid 20 has a vertical wall 120 that abuts against the vertical wall 122 of the gap 82 when the lid 20 is inserted into the gap 82. Both the vertical walls 120 and 122 of the lid 20 and the gap 82 include attachment devices, such as snaps, threads, etc., to ensure a tight fit between the lid 20 and the gap 82. When the cover 80 is engaged with the lid 20, the gap 82 allows the gap 25 to still receive the suction device.

[0113] The edge of the lid 20 also has a horizontal section perpendicular to the vertical wall of the lid 20. This horizontal section can be used to set the maximum level of the lid 20 insertion gap 82.

[0114] The cover 80 also engages with the device 1 to cover the sample area 5 and the migration area 10. In one arrangement, the outer lip of the cover 80 is threaded to engage with a complementary thread on the outer lip of the device 1. Other attachment devices can also be used to attach the cover 80 to the device 1.

[0115] When the cover 80 is positioned above the device 1, the sample area 5 and the migration area 10 are protected by the cover 80. As previously described, the collection area 15 is covered by the lid 20. The gap 82 also allows the user to enter the gap 25 to collect semen from the collection area 15. Furthermore, the cover 80 and the lid 20 prevent sample spillage and contamination.

[0116] As previously described, the migration zone 10 may include multiple layers of grooves 35 to improve sperm yield at the collection zone 15. In one arrangement, a portion of the migration zone 10 having multiple layers of grooves 35 is integrated with a cap 20. This arrangement provides the migration zone 10 with multiple layers of grooves 35 when the cap 20 is engaged with the device 1.

[0117] Figures 29 to 32A cover 20 is shown with recesses 35 having multiple layers 2900A to 2900C. Layers 2900A to 2900C can be attached to the cover 20, along with other layers 2900A to 2900C. In another arrangement, layers 2900A to 2900C are integrated into the cover 20. In yet another arrangement, one of layers 2900A is integrated into the cover 20, while the other two layers 2900B and 2900C can be attached to layers 2900A and 2900C, respectively. Layers 2900A to 2900C can be attached using attachment devices such as snaps, threads, or adhesive. Although... Figures 29 to 32 Only three layers of 2900A to 2900C are shown, but one or more layers of 2900 can be attached to / integrated into the cover 20.

[0118] In another embodiment, such as Figure 18 and Figure 19 As shown, the sperm separation device 1 also includes an oocyte region 85 for receiving oocytes (not shown) and sperm collected from the collection region 15. The oocyte region 85 is configured for intracytoplasmic sperm injection (ICSI) of oocytes and also includes an oil coating layer (not shown) to ensure no fluid contact between the sperm, oocytes, and sperm buffer.

[0119] refer to Figure 26 To use the sperm separation device 1, the device first needs to be filled with the required buffer solution (step 1). The device is filled by filling the collection area 15 with buffer fluid through the gap 25 in the cap at the collection area 15, or by placing the fluid directly into the open collection area 15, where the force generated when the fluid is ejected from the buffer fluid dispensing device pushes the fluid through the groove 35. In an alternative embodiment, time can be allowed for the buffer fluid dispensed into the collection area 15 to enter the groove 35 via hydrophilic capillary action. After the device is filled, sperm are deposited into the sample area 5 (step 2). In the illustrated embodiment, a cover 80 is placed on the device to reduce the risk of spillage, maintain the sterility of the device, and prevent the medium from evaporating from the device (step 3). The sperm then migrate upwards along the groove 35 in the ramp 40 to the migration area 10, while inactive sperm settle at the bottom of the ramp 40 of the sample area 5. The sperm continue to migrate through the groove 35 until they reach the collection area 15 (in the form of a pit 45), where they migrate downwards along the downward-sloping groove 35 with steps 55 to the bottom of the pit 45 for collection. The steps 55 advantageously prevent the sperm from migrating upwards back and re-entering the migration area 10.

[0120] To collect sperm from the collection area (step 4), the suction device is placed in the gap 20 of the lid 25 and activated. Upon activation, air bubbles form between the lid 20 and the collection area 15. Specifically, as fluid is removed from the collection area 15, air enters the device 1 to fill the space occupied by the fluid; the air interacts with the geometry of the device, generating air bubbles. Advantageously, these air bubbles can block the fluid connection between the migration area 10 and the collection area 15, thereby limiting the amount of sperm collected from the collection area 15 and preventing unwanted substances from being collected from the sample area 5 or the migration area 10. Advantageously, depending on the number of motile sperm in the semen sample and the user's needs, sperm can be collected from the collection area 15 within 2 minutes after deposition in the sample area 5. All desired sperm must be collected within 1 hour after deposition in the sample area 5. Specifically, the device enables the migration of high-quality sperm to the collection area 15 while suppressing unwanted sperm and substances in the sample area 5 and the migration area 10.

[0121] refer to Figure 27 In order to perform intracytoplasmic sperm injection (ICSI) on oocytes, this device (such as...) is used. Figure 18 and Figure 19 An alternative embodiment of the device (shown) includes an oocyte region 85 and a collection region 15 with a micropattern 90, best shown in Figure 21. The micropattern 90 contains a sperm binding agent capable of selectively binding and advantageously immobilizing sperm with desired biological characteristics expressed externally on their membranes. The desired characteristics that the binding agent may have include, but are not limited to, sperm maturity, sperm-egg identification markers, and sperm sex markers. As described above, the device is first filled with a buffer solution (step 1). In this embodiment, capillary action draws the buffer fluid into the recess 35. An oocyte is then placed in the oocyte region 85 (step 2), and oil is applied to the collection region 15 and the oocyte region 85 (step 3) to ensure no fluid contact between the sperm, the oocyte, and the sperm buffer solution. Sperm is then injected into the sample region 5 (step 4), where it migrates through the migration region 10 to the micropattern 90 in the collection region 15, where it binds to the binding agent. In this embodiment, the micropattern 90 is in a serrated shape. However, in alternative embodiments, other shapes may be used. Finally, sperm are collected from the micropattern 90 and injected into oocytes in the oocyte region 85 (step 5). In alternative embodiments, sperm may be collected without the use of a binding agent, and / or sperm may be transferred to an intermediate droplet typically made of polyvinylpyrrolidone (PVP) before being injected into the oocytes in the oocyte region 85.

[0122] Figure 23The percentage of sperm DNA fragmentation (SDF) obtained by sperm chromatin dispersion (SCD) analysis is shown, comparing the results of raw semen, density gradient centrifugation (DGC, a conventional semen processing method), and device 1 (referred to as MFD in the figure) in aliquoted semen samples. It also shows a comparison of the percentage of sperm DFI distribution after device 1 and DGC treatment. Figure 23 A semen sample before processing in device 1 and sperm collected from the device after processing the semen sample are shown.

[0123] Figure 24 and Figure 25 A graph showing the results of comparing device 1 with DGC is presented. Figure 24 This demonstrates a comparison of raw semen, DGC-treated semen, and semen treated with device 1, based on World Health Organization (WHO) standards, using conventional manual assessment of sperm motility. Figure 24 The semen obtained (referred to as MFD in China). Figure 24 A comparison of the percentage of sperm DFI distribution in device 1 and DGC-treated sperm is also shown. Figure 25 The comparison of hyaluronic acid binding scores of sperm treated with DGC (i.e., the conventional device) with those of a variant of device 1 with a hyaluronic acid coating after motility-based selection is shown.

[0124] The sperm separation device described in this article is particularly suitable for separating high-quality motile sperm from unwanted non-motile sperm and other substances in semen, representing a practical and commercially significant improvement over existing technologies. Another advantage is that sperm separation from semen does not damage the sperm.

[0125] Although the invention has been described with reference to specific embodiments, those skilled in the art will understand that the invention can be implemented in many other forms.

[0126] Reference Table

Claims

1. A sperm separation device, comprising: The sample area is configured to receive semen. A collection area, which is configured to be fluidly connected to the sample area via a migration area; as well as A lid, which is positioned above portions of the migration area and the collection area, The migration zone includes grooves for guiding sperm in the semen; and The sperm in the semen migrates to the collection area via the groove.

2. The sperm separation device according to claim 1, wherein, Each of the grooves extends into the sample area.

3. The sperm separation apparatus according to claim 1 or claim 2, wherein, Each of the grooves includes at least one corner.

4. The sperm separation apparatus according to any one of the preceding claims, wherein, The sample area is tilted to form a slope toward the migration area.

5. The sperm separation apparatus according to any one of the preceding claims, wherein, The collection area slopes downward from the migration area.

6. The sperm separation apparatus according to claim 5, wherein, The downward tilt angle is between 90 degrees and 50 degrees.

7. The sperm separation apparatus according to claim 5 or claim 6, wherein, The downward slope of the collection area includes one or more blocking members to prevent the sperm from migrating upward back into the collection area.

8. The sperm separation apparatus according to any one of the preceding claims, wherein, The migration zone slopes downward from the sample area, and the downward slope of the migration zone includes one or more blocking members to prevent the sperm from migrating upward back to the migration zone.

9. The sperm separation apparatus according to claim 7 or claim 8, wherein, The blocking member includes steps that descend in increments of 10µm to 1000µm.

10. The sperm separation apparatus according to any one of the preceding claims, wherein, The migration zone is in communication with the lid to form a fluid boundary between the sample area and the migration zone.

11. The sperm separation apparatus according to any one of the preceding claims, wherein, The height and width of the groove are not constant along its length.

12. The sperm separation apparatus according to any one of the preceding claims, wherein, The migration zone includes the intersection of two sub-grooves, one of which has a dead end, while the other sub-grooves leads to the collection zone and contains chemical reagents.

13. The sperm separation apparatus according to any one of the preceding claims, wherein, The migration zone includes multiple layers of the aforementioned grooves.

14. The sperm separation apparatus according to any one of the preceding claims, wherein, The collection area is in the form of a pit and is configured to accommodate sperm volumes ranging from 50 µL to 2000 µL.

15. The sperm separation apparatus according to any one of the preceding claims, wherein, The collection area has a flat bottom surface for microscopic imaging of the sperm.

16. The sperm separation apparatus according to any one of the preceding claims, wherein, The lid also includes a gap configured to receive a suction device, which is configured to collect the sperm within the collection area.

17. The sperm separation apparatus according to claim 15 or 16, wherein, The cap also includes an inward cavity that forms an air bubble between the cap and the collection area when suction is applied to the collection area. The air bubble prevents fluid connection between the migration area and the collection area, thereby limiting the amount of sperm collected from the collection area.

18. The sperm separation apparatus according to any one of the preceding claims, wherein, The junction between the sample area and the migration area also includes outwardly extending protrusions to prevent the semen from passing through the migration area and to prevent the semen from being injected into the migration area.

19. The sperm separation apparatus according to any one of the preceding claims further includes an oocyte region for receiving an oocyte and sperm collected from the collection region, wherein the oocyte region is configured for intracytoplasmic sperm injection (ICSI) of the oocyte.

20. The sperm separation apparatus according to claim 18, wherein, The surface of the collection area also includes micropatterns for receiving sperm binding agents to selectively bind and fix sperm with desired properties on the surface.

21. The sperm separation apparatus according to claim 19, wherein, The micropattern also includes at least one connecting layer for binding the sperm binding agent to the device.

22. The sperm separation apparatus according to any one of the preceding claims further includes a cover configured to engage with the apparatus such that the sample area and the migration area are covered.

23. The sperm separation apparatus according to claim 22, wherein, The cover has a gap configured to receive the lid.

24. The sperm separation apparatus according to claim 23, wherein, The cover is attached to the lid.

25. The sperm separation apparatus according to any one of claims 22 to 24, which is dependent on claim 16, wherein, The gaps in the cover allow the lid to receive the suction device.

26. The sperm separation apparatus according to any one of claims 22 to 25, which is dependent on claim 13, wherein, The multi-layered grooves are integrated with the cover.

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