Microfluidic device for separating motile cells from non-motile cells

Abstract

The present disclosure describes a sperm separating 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 lid (20) is disposed over a part of the migration zone and the collection zone, wherein the migration zone includes grooves to guide sperm within the semen, and wherein the sperm in the semen migrate via the grooves to the collection zone.

Description

MICROFLUIDIC DEVICE FOR SEPARATING MOTILE CELLS FROM NON- MOTILE CELLSTechnical Field

[0001] The present invention relates to the field of assisted reproduction. More particularly, the invention relates to a microfluidic device for separating motile bodies from non-motile bodies having known predefined characteristics.

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

[0003] The following discussion of the prior art is intended to present the invention in an appropriate technical context and allow its advantages to be properly appreciated. Unless clearly indicated to the contrary, however, reference to any prior art in this specification should not be construed as an express or implied admission that such art is widely known or forms part of common general knowledge in the field.

[0004] In many IVF applications, there is a need for the separation of motile bodies, such as sperm, from a collection of other motile, non-motile sperm, and non- motile bodies, as well as from the fluid these motile bodies may be suspended in. In IVF, it is desirable to separate motile sperm from other bodies and fluid into new fluids as part of the process of preparing sperm for assisted reproduction procedures, for research, or for assessment.

[0005] Human assisted reproduction and artificial insemination, also known as intrauterine insemination (IUI), are examples of such procedures where the separation of motile sperm may be desirable. IUI is widely used for animal husbandry and is also used for humans as a form of low impact and more cost- effective infertility treatment. IUI involves the use of processed sperm and their injection into a female’s reproductive system.

[0006] Other forms of human reproduction include in vitro fertilisation (IVF) and intracytoplasm ic sperm injection ( ICS I). These involve the use of selected sperm in the fertilisation of an oocyte outside the body to form an embryo, which is then grown and implanted into the uterus. IVF involves the introduction of many selected sperm to an oocyte to facilitate fertilisation, while ICSI involves directly injection a sperm into an oocyte.

[0007] Sperm selection is a crucial step in the IVF process. The higher quality of the sperm, the greater chances the IVF process is successful. Traditional sperm selection methods, such as density gradient centrifugation and swim-up, can cause DNA fragmentation and cell death, leading to unsuccessful IVF cycles. Traditional sperm selection methods are also costly, time consuming and require highly specialised equipment.

[0008] All of these procedures require some level of sperm sorting or preparation where sperm are removed from the semen, which slowly becomes a toxic environment for sperm to remain in and are also removed from less desirable sperm and background cells and non-motile bodies or contaminants. Existing designs for apparatuses and their methods for use are lacking in several aspects, which are crucial for robust and ergonomic sperm separation.

[0009] US 2015 / 0140655 A1 patent describes a device for separating sperm from semen. It consists of an outer chamber containing the semen sample, a central chamber for collecting the separated sperm, and a network of microchannels in between the two. These microchannels are shaped like spokes radiating out from the central chamber. The device works by taking advantage of the self-propelled movement of motile sperm and their tendency to swim towards and along surfaces in confined spaces, a behaviour known as “wall-swimming.” The microchannels are designed such that sperm will swim through them and into the central chamber, with at least one junction directing sperm based on their wall-swimming behaviour. The fluid in the microchannels may be relatively high in viscosity. The microchannels begin at the outer semen chamber and converge in a cascading fashion, with fewer microchannels emptying into the central chamber.

[0010] The device of US 2015 / 0140655 A1 requires the use of tape or a cap to cover the central port after collection to seal the device. An adhesive tape is typically used but any adhesive that is exposed to the sperm and sperm carrying media is a toxicity risk. Placing and removing that cap is also a very delicate step which a lot of clinicians fail to do properly consequently creating an incomplete seal which allows semen to flow into the centre of the device when the semen is injected into the device. Furthermore, the removal of the cap creates a suction force that inadvertently sucks in raw semen which undoes all the separation that has been performed up to that point. In other words, raw semen contaminates the collected sample in US 2015 / 0140655 A1 .

[0011] In addition, in US 2015 / 0140655 A1 , semen is injected into a large channel / reservoir. The pressure during injection is always the highest in the channels closest to the injection port. Accordingly, there is a high chance semen enters into the first several channels near the injection port, increasing the risk of contamination.

[0012] Furthermore, the volume of sperm collected in US 2015 / 0140655 A1 is not moderated. For example, if you tell an embryologist to collect 10OpL, there is nothing stopping them from collecting 200pL meaning an extra 100pL must then come from the semen sample which gets sucked through the channels and contaminates the collected sample.

[0013] US 2015 / 0140655 A1 also requires extra preparation steps to load the device with a sperm buffer. Due to the complex array of ratchets, to load the device with a sperm buffer, the device must be submerged in the buffer and put in a vacuum chamber to suck out the air bubbles. This process is impractical and time consuming.

[0014] In addition, due to the configuration and design of the channels, a lot of sperm in the semen never find their way into the device of US 2015 / 0140655 A1 limiting the yield of collected sperm.

[0015] A further disadvantage of US 2015 / 0140655 A1 is that the channels are on the bottom floor of the device so there is a large volume of fluid in the semenreservoir and collection zone above the channels which, if imbalanced can create a gravity pump, effectively pushing semen through the device and contaminating the collected sperm.

[0016] There is a need for an apparatus that does not require tape or a cap to cover the central port after collection to seal the device.

[0017] There is also a need for an apparatus that simplifies and reduces the number of steps required to collect sperm.

[0018] There is a further need for an apparatus that limits the maximum amount of fluid that can be collected.

[0019] There is an additional need for an apparatus in which no vacuuming is required to load a sperm buffer.

[0020] There is also a need for an apparatus with inclined grooves and / or channels for sperm to settle into and swim up to increase the yield of sperm collected.

[0021] There is also a need for an apparatus with grooves and / or channels above a sample zone to limit any non-motile bodies flowing into the channels.

[0022] There is also a need for an apparatus that separates sperm originating in a semen sample with fewer steps.

[0023] There is a further need for an apparatus that allows the selection of sperm within a platform to be capable of also facilitating methods of fertilisation.

[0024] There is an additional need for an apparatus that allows the selection of sperm within a more stable platform to be able to prevent the ingress of semen, semen components and non-motile bodies and contaminants into fluid containing desirable sperm.

[0025] There is also a need for an improved apparatus and method that allows for the selection of sperm obtained from a semen sample through methods beyond amotility-separation based process, to yield motile sperm having certain (or definable) desired characteristics.

[0026] There is also a need for a platform that is able to secure a sperm selection apparatus during the selection process to prevent spill risks, user error, provide environmental control in the form of temperature moderation and provide visualisation and data analysis of selected sperm to treatment practices in assisted reproduction.

[0027] The present invention mimics some of the natural selection processes that exist in the female reproductive tract in an attempt to select a better individual or population of sperm when compared to traditional methods of preparation.

[0028] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. It is an object of the invention in at least one preferred form 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, there is provided a sperm separating device comprising: a sample zone configured to receive semen; a collection zone fluidly connected to the sample zone by a migration zone; and a lid disposed over a part of the migration zone and the collection zone, wherein the migration zone includes grooves to guide sperm within the semen; and wherein the sperm in the semen migrate via the grooves to the collection zone.

[0030] According to another aspect of the present invention, there is provided a dock comprising receptacles configured for receiving an imaging system, a recordingsystem, a temperature control system, a magnification lens, and the sperm separating device, as herein disclosed.

[0031] In some embodiments, the sample zone may be shaped to receive and hold a volume of semen. In some embodiments, the sample zone may be inclined to form a ramp toward the migration zone to increase the throughput of motile bodies towards the migration and / or collection zone.

[0032] In some embodiments, the angle of the incline of the ramp may be in the range of 2 degrees and 89 degrees. Preferably, the angle of the incline of the ramp is in the range of 7 degrees and 13 degrees.

[0033] In some embodiments, the ramp may be configured to allow non-motile bodies to settle down and away from the migration zone to help prevent contamination in the migration and collection zones. In some embodiments, the ramp may include a series of grooves for the semen to be layered on top of and for sperm to settle into and migrate up towards the migrations zone. In some embodiments, the series of grooves are in a straight configuration. In some embodiments, the series of grooves are in a curved configuration. In some embodiments, the series of grooves are in a spiral configuration. In some embodiments, the series of grooves are in a zig-zag configuration. In some embodiments, the series of grooves are in a converging configuration. In some embodiments, the series of grooves are in a diverging configuration. In some embodiments, the series of grooves are in an inclining configuration. In some embodiments, the series of grooves are in a declining configuration. In some embodiments, each of the grooves in the series of grooves may be in a different configuration from one another. Preferably, the grooves are in a combination of configurations to guide sperm towards the migration zone with the intent to amplify the yield of sperm engaged for separation.

[0034] In some embodiments, the sample zone may have a predetermined size and shape that is deep enough to allow for semen to be deposited into the sample zone and remain there. In some embodiments, the sample zone may be in the shape of a full ring (i.e. circular in shape). In some embodiments, the sample zone may be in the shape of a semi-circle ring (i.e. semi-circular in shape). In someembodiments, the sample zone may be in the shape of a rectangular prism. In some embodiments, the sample zone may be in the shape of a serpentine channel. In some embodiments, the sample zone may be in the shape of wave-like channel. In some embodiments, the sample zone may be in the shape of a bowl. Preferably, the sample zone is in the shape of a full ring, a semi-circle ring, or a rectangular prism. In other embodiments, the sample zone may be another suitable shape.

[0035] In some embodiments, the sample zone may have a depth within the range of 10pm and 50,000pm. In some embodiments, other depths may be used depending on the volume required for the species of sperm being processed.

[0036] In some embodiments, the sample zone may be an open area. In some embodiments, the sample zone may be a partially enclosed area. In some embodiments, the sample zone may be an enclosed area. In some embodiments, where the sample zone is an enclosed area, the sample zone may include an opening for depositing semen into the sample zone. In some embodiments, the sample zone includes a plurality of openings for depositing semen into the sample zone.

[0037] In some embodiments, the collection zone may be configured to concentrate motile sperm into a single location. In some embodiments, the collection zone may be located at a position downstream of the sample zone. In some embodiments, the collection zone may be declining from the migration zone to encourage sperm to migrate to the collection zone and to prevent the sperm from migrating back to the sample zone. In some embodiments, the angle of the decline may be in the range of 90 degrees and 50 degrees.

[0038] In some embodiments, the decline of the collection zone may comprise one or more blocking members descending along the length of the groove to prevent the sperm from migrating back up and re-entering the migration zone. In some embodiments, the decline of the collection zone may comprise one or more blocking members descending along a part of the length of the groove to prevent the sperm from migrating back up and re-entering the migration zone. In some embodiments, the blocking members are bumps. In some embodiments, the blocking members are ridges. In some embodiments, the blocking members are steps. In someembodiments, the steps may drop in increments in the range of 10pm and 1000pm. Preferably, the steps drop in increments in the range of 30pm and 100pm. In some embodiments, the blocking members may be another suitable configuration / shape to prevent the sperm from migrating back up and re-entering the migration zone.

[0039] In some embodiments, the collection zone may be in the form of a well. In some embodiments, the well may be positioned below the height of entry of the grooves into the collection zone to encourage sperm to descend to a lower level than the end of the migration zone and prevent sperm from re-entering the migration zone. In some embodiments, the cross-sectional profile of the well may be cylindrical in shape. In some embodiments, the cross-sectional profile of the well may be conical in shape. In some embodiments, the cross-sectional profile of the well may be a bowl shape. In some embodiments, the well may have a different cross-sectional profile.

[0040] In some embodiments, the collection zone may include a wall. In some embodiments, the collection zone may include a plurality of walls. In some embodiments, the wall may have a sheer decline in height for sperm to descend into the collection zone. In some embodiments, the wall may have a gradual decline in height for sperm to descend into the collection zone. In some embodiments, the wall may have a periodic decline in height for sperm to descend into the collection zone.

[0041] In some embodiments, the wall may be configured to keep the sperm in a semi-slither mode to prevent their adhesion to the surfaces of the device and subsequent resistance to collection forces. In some embodiments, the wall may include a micropatterned surface to prevent sperm from adhering to surfaces and promote bulk swimming behaviour. In some embodiments, the micropatterned surface may include small bumps. In some embodiments, the micropatterned surface may include ridges. In some embodiments, the micropatterned surface may include stairs. In some embodiments, the micropatterned surface may include waves. In some embodiments, the micropatterned surface may include other suitable projections or designs to prevent sperm from adhering to surfaces and promote bulk swimming behaviour.

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

[0043] In some embodiments, the collection zone may include a selection mechanism within the collection zone designed to immobilise sperm with desirable features following motility-based selection. In some embodiments, the selection mechanism may be a micropattern of an agent able to bind to sperm externally expressing a corresponding marker on this surface of its membrane. In some embodiments, the micropattern may take the form of a zig-zag, line, dots or any other arrangement that will efficiently expose separated sperm to the binding agent.

[0044] In some embodiments, the collection zone may include a bottom face for microscopic imaging of the sperm. In some embodiments, the bottom face may be flat. In some embodiments, the bottom face aligns with the void of the lid and prohibits, through a limited angle, a suction device from coming into direct contact with the migration zone. In some embodiments, the ratio of the diameter or width of the void of the lid to the maximum diameter or width of the collection zone may be in the range of 1 :2 and 1 :20. Preferably, the range is between 1 :3 and 1 :5.

[0045] In some embodiments, the collection zone may include a bottom section configured to concentrate sperm in a particular localised area of the collection zone to keep sperm near the force of collection, i.e. the suction force created by the tool used to suck up the fluid in the collection zone.

[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 another suitable shape.

[0047] In some embodiments, the collection zone may be covered in a surface coating to prevent sperm from sticking to any surfaces in the collection zone. In some embodiments, the surface coating may consist of but are not limited to surfactant additives, polymer coatings (for example, polyvinyl alcohol or PVA),proteins (for example, BSA and HAS), polyethylene glycol (PEG)-like polymers, and any other hydrophilic treatment.

[0048] In some embodiments, the collection zone may have a volume in the range of 50pL and 1 ,000pL. Preferably, the collection zone has a volume in the range of 150pL and 500pL. In other embodiments, other volumes may be used.

[0049] In some embodiments, the collection zone may have a depth in the range of 100pm and 10,000pm. Preferably, the collection zone has a depth in the range of 2,000pm and 4,000pm. In other embodiments, other depths may be used depending on the geometry of the well.

[0050] In some embodiments, the collection zone may be loaded with a chemical agent and allowed to diffuse into the migration zone to encourage sperm receptive to the chemical agent towards the collection zone. In some embodiments, the chemical agent may be a chemoattractant. In some embodiments, the chemoattractive agent may consist of but are not limited to progesterone, follicular fluid, oviductal fluid, lactate, prostaglandins, and chemokines.

[0051] In some embodiments, the migration zone may include an intersection of two sub-grooves for providing a choice for sperm during their migration, wherein one sub-groove has a dead end, and the other sub-groove leads to the collection zone and has the chemoattractive.

[0052] In some embodiments, the sub-groove with a dead end may include a drop in groove height to prevent the return of undesirable sperm to paths that connect with the collection zone. In some embodiments, the sub-groove with a dead end may be close or open to the air at their termination to either allow complete filling of the migration zone, when in an open configuration, or result in the generation of a bubble to cap the end of the groove, thereby providing an edge for sperm to burrow into and assist in the prevention of returning sperm, when in a closed configuration.

[0053] In some embodiments, the grooves that are not connected to the collection zone may have dead ends and may end with an air bubble from device buffer loading such that the comers generated between the air bubble, the surfacesof the pathway and the fluid are both deep enough into the dead end to prevent sperm migrating back once they’ve committed and create a trap for sperm to burrow into and discourage them from turning back. In some embodiments, the grooves with dead ends may have a change in height at the entrance to the dead end to prevent the return of non-chemoreceptive sperm.

[0054] In some embodiments, when the collection zone is loaded with a chemical agent, the grooves may be of suitable lengths and geometries to provide a stable gradient of the chemical agent for the duration of the sperm selection. In some embodiments, the grooves may have a length in the range of 1 mm and 15 mm. Preferably, the grooves have a length in the range of 6mm and 8mm.

[0055] In some embodiments, the lid may include a void configured to receive a suction device that is configured to collect sperm within the collection zone. In some embodiments, the void may be configured for removing some or all of the fluid in the collection zone without collecting undesired non-motile bodies that may reside further upstream in the device. In some embodiments, the void may be configured as a buffer loading port for filling the device with a buffer of choice whereby capillary forces move the buffer into the grooves. In some embodiments, the void may be configured as a buffer loading port for filling the device with a buffer of choice whereby the force of the buffer being ejected from the buffer dispensing device pushes the buffer into the grooves.

[0056] In some embodiments, a sperm buffer may be dispensed at the entrance of either side of any grooves whereby capillary forces move the buffer into the grooves. In some embodiments, a sperm buffer may be dispensed in the migration zone.

[0057] In some embodiments, the lid includes an inwardly facing cavity such that when suction is applied to the collection zone, i.e. when the well is emptied during sperm collection, an air bubble is created between the lid and collection zone, wherein the air bubble terminates the fluid connection between the migration zone and collection zone to limit the amount of sperm collected from the collection zone and prevent the collection of undesired bodies from the sample or migration zones. In some embodiments, the inwardly facing cavity may be chamfered towards thevoid to help pinch the entrance of the collection zone where it meets the migration zone and promote the formation of an air bubble through the surface tension of the buffer fluid at that point. In some embodiments, the collection zone with the lid on top may have a substantially diamond shaped cross section profile.

[0058] In some embodiments, the migration zone and the lid communicate to create a fluid boundary between the sample zone and the migration zone.

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

[0060] In some embodiments, the collection zone may not be covered by the lid and may be instead an open fluid reservoir which may or may not be covered by an oil overlay to enable individual sperms to be picked up with a micromanipulator.

[0061] In some embodiments, the migration zone may be an open area. In some embodiments, the migration zone may be a partially enclosed area. In some embodiments, the migration zone may be an enclosed area. Preferably, the migration zone is a partially enclosed area to assist in the creation of a fluid boundary.

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

[0063] In some embodiments, the grooves may be a height that facilitates enough fluid resistance to prevent an influx of semen, which is of higher viscosity than water, into the collection zone and creates a fluid boundary between semen in the sample zone and the migration zone. In some embodiments, the height of the grooves may be in the range of 5um and 400pm. Preferably, the height of the grooves is in the range of 40pm and 120pm.

[0064] In some embodiments, the migration zone may be located at a position downstream of the sample zone. In some embodiments, the migration zone may be declining from the sample zone to encourage sperm to migrate through the migration zone to the collection zone and to prevent the sperm from migrating back to thesample zone. In some embodiments, the angle of the decline may be in the range of 90 degrees and 50 degrees.

[0065] In some embodiments, the migration zone may comprise at least one sharp drop in vertical height to a depth which sperm travelling along the floor of the migration zone may not (easily) return from to improve the yield of collected sperm in the collection zone and to prevent the loss of desirable sperm through backwards migration at any point during the selection process. In some embodiments, the drop in height may be in the form of a cut into the floor of the migration zone. In some embodiments, the drop in height may be in the form of a new groove. In some embodiments, the drop in height may be in the form of an existing groove with an extended depth. In some embodiments, the drop in height may be in the form of a semi-circular indent that is configured to promote the redirection of sperm towards the collection zone rather than a sharp corner where sperm can burrow into and become stuck and unable to turn away from.

[0066] In some embodiments, the decline of the migration zone may comprise one or more blocking members descending along the length of the groove to prevent the sperm from migrating back up and re-entering the sample zone. In some embodiments, the decline of the migration zone may comprise one or more blocking members descending along a part of the length of the groove to prevent the sperm from migrating back up and re-entering the sample zone. In some embodiments, the blocking members are bumps. In some embodiments, the blocking members are ridges. In some embodiments, the blocking members are steps. In some embodiments, the steps may drop in increments in the range of 10pm and 1000pm. Preferably, the steps drop in increments in the range of 30pm and 100pm. In some embodiments, the blocking members may be another suitable configuration / shape to prevent the sperm from migrating back up and re-entering the sample zone.

[0067] In some embodiments, the migration zone includes one layer of grooves. In some embodiments, the migration zone includes a plurality of layers of grooves. In some embodiments, the plurality of layers of grooves may intersect with one another.

[0068] In some embodiments, each of the grooves may be in a straight configuration. In some embodiments, each of the grooves may be in a curved configuration. In some embodiments, each of the grooves may be in a maze-like configuration. In some embodiments, each of the grooves may be in a spiral configuration. In some embodiments, each of the grooves may be in a converging configuration. In some embodiments, each of the grooves may be in a diverging configuration. In some embodiments, each of the grooves may be in an inclining configuration. In some embodiments, each of the grooves may be in a declining configuration. In some embodiments, each of the grooves may be in another suitable configuration. In some embodiments, each of the grooves may be in a different configuration from one another. Preferably, the grooves are in a combination of configurations to select the sperm whilst maximising the fluid resistance within the migration zone.

[0069] In some embodiments, each of the grooves may have a constant width along their length. In some embodiments, each of the grooves may have a varying width along their length. Preferably, each of the grooves constrict near the collection zone to increase fluid resistance and decrease the potential opening for sperm to return in the direction of the sample zone.

[0070] In some embodiments, each of the grooves may extend to the sample zone to facilitate the guidance of additional sperm out of the semen and into the migration zone. As a result, these grooves may contain both semen and buffer at the same time but still retain the fluid boundary between the two zones.

[0071] In some embodiments, each of the grooves may extend the entire length of the migration zone. In some embodiments, each of the grooves may extend a part of the length of the migration zone. In some embodiments, each of the grooves may extend from the migration zone to the collection zone. In some embodiments, each of the grooves may extend from the sample zone to the collection zone.

[0072] In some embodiments, each of the grooves may have a length in the range of 10pm and 30,000pm long. In some embodiments, each of the grooves may have a length that is dependent on the zone the groove is located in.

[0073] In some embodiments, each of the grooves may have a constant height along their length. In some embodiments, each of the grooves may have a varying height along their length. In some embodiments, each of the grooves may have an open top along their entire length. In some embodiments, each of the grooves may have a closed top along their entire length. In some embodiments, each of the grooves may have a partially closed top along their entire length. In some embodiments, each of the grooves may have a height in the range of 5pm and 400pm. Preferably, each of the grooves have a height in the range of 20pm and 12 pm.

[0074] In some embodiments, each of the grooves may have a constant width along their length. In some embodiments, each of the grooves may have a varying width along their length. In some embodiments, each of the grooves may have a width in the range of 5pm and 10,000pm. In some embodiments, each of the grooves connecting the sample zone to the migration zone may have a width in the range of 20pm and 100pm. In some embodiments, each of the grooves connecting the migration zone to the collection zone may have a width in the range of 10pm and 40pm.

[0075] In some embodiments, each of the grooves may have a constant crosssection profile along their length for optimal sperm persistence, depending on the species of sperm being processed in the device - persistence being the ability of sperm to migrate through the grooves with maximum displacement from their starting position at the beginning of the migration zone to the end of the migration zone within the least amount of time to conserve energy. In some embodiments, each of the grooves may have a varying cross-section profile along their length.

[0076] In some embodiments, each of the grooves may include a guiding member to guide sperm towards the collection zone. In some embodiments, each of the grooves may include a plurality of guiding members to guide sperm towards the collection zone. In some embodiments, each of the grooves may include a combination of different guiding means to provide multiple guided avenues for sperm to reach the collection zone. In some embodiments, the guiding member may be in the form of a corner edge. Preferably, each of the grooves includes two corneredges. In some embodiments, the guiding member may be an undulating wall intrusion to guide sperm forward and prevent the backwards migration of sperm. In some embodiments, other suitable guiding members may be used. Preferably, the grooves will contain a combination of comers and grooves to provide multiple guided avenues for sperm to reach the collection zone.

[0077] In some embodiments, each of the grooves may have walls that are contoured to provide additional edges for the sperm to follow.

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

[0079] In some embodiments, there may be a junction 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, the pillars may extend from the floor of the ramp or the floor of the sample zone up to or beyond the height of the migration zone and are configured to act as an added initial barrier to the viscous semen but to allow sperm to pass between them to the migration zone. In some embodiments, the pillars may be cylindrical in shape. In some embodiments, the pillars may be a prism shape. In some embodiments, the pillars may be a mound shape. In some embodiments, the pillars may be a different shape suitable for assisting in preventing an influx of semen plasma into downstream areas. In some embodiments, the distance between the pillars may be in the range of 10pm and 300pm. Preferably, the distance between the pillars is in the range 40pm and 80pm.

[0080] In some embodiments, the pillars may be configured to prevent users from injecting semen directly in the migration zone by guiding pipette tips and syringes away from the migration zone and towards the base of the sample zone.

[0081] In some embodiments, the sperm separating devices may include hydrophilic surfaces. In some embodiments, the grooves in the migration zone may have hydrophilic surfaces to both prevent bubble generation and promote sperm migration by preventing their sticking to surfaces within the device. In someembodiments, a section of the grooves in the migration zone may have hydrophilic surfaces.

[0082] In some embodiments, the sperm separating device may include proteins bound to the hydrophilic surfaces, which allow the protein to bind naturally to the device.

[0083] In some embodiments, the sperm separating device may include an oocyte zone for receiving sperm collected from the collection zone and an oocyte, wherein the oocyte zone is configured for intracytoplasm ic sperm injection of the oocyte. In some embodiments, the oocyte zone may include mechanisms to hold selected sperm and oocytes. In some embodiments, the oocyte zone may include space droplets to hold selected sperm and oocytes.

[0084] In some embodiments, the sperm separating device, which includes an oocyte zone, may be shaped such that it is able to fit onto a microscope stage.

[0085] In some embodiments, the sperm separating device, which includes an oocyte zone, may include a collection zone with a micropattern on the surface of the collection zone coated with agents to selectively bind and immobilise sperm with desired biological features externally expressed on their membrane.

[0086] In some embodiments, the micropattern may be zig-zag in 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 that cover part or all of the width / diameter of the collection zone. In some embodiments, the micropattern may include numerous rows / layers of micropatterns. In some embodiments, the sperm separating device may include proteins bound to surfaces of the device via hydrophilic coatings on the device surface, which allow the protein to bind naturally.

[0087] In some embodiments, the micropattern may include a connective layer to bind the active sperm binding agent to the device. In some embodiments, the connective layer may be dependent on the material the device is made of and mayinclude PEG chains, polydopamine, agarose, heparin, or any other layer mediating the connection of the sperm selection and binding agent.

[0088] In some embodiments, the sperm binding agent may be bound to the device via the connective layer to facilitate positive sperm binding for easy examination of sperm morphology and micromanipulator pickup. In some embodiments, the sperm binding agent may be hyaluronic acid (HA), anti-Juno antibodies, integrin alpha-9, zona pelucida protein 3 (ZP3), antibodies for externally expressed sperm markers such as anti-SP17 and anti-TEX101 antibodies, or sex specific antigens such as resiquimod, anti-CD52, imiquimod, and gardiquimod. In some embodiments, other suitable sperm binding agents may be used.

[0089] In some embodiments, motile selection may be followed by hyaluronic acid binding in the same device. In contrast to traditional methods of sperm separation such as Density Gradient Centrifugation (DCG), where hyaluronic acid binding is done in a separate dish after washing the sperm. Advantageously, 91.1 % of motile sperm (which is all of the motile sperm in the claimed device as only motile sperm can make it to the concentration zone) bind to the micropattern, while only 79.75% of motile sperm (some sperm post DGC are immotile) after DGC bind. The claimed invention is advantageous over traditional methods of sperm separation, such as DGC, as during the sperm selection process there is the potential for centrifugation-based damage and reactive oxygen from DGC which inhibit binding potential of sperm.

[0090] In some embodiments, the sperm separating device, which includes an oocyte zone, may be configured to allow for a mineral oil overlay to ensure sterility of the device’s contents, the mineral oil overlay being shallow enough to allow a micromanipulator access to the content of the device. In some embodiments, the sperm separating device may have a depth in the range of 3 mm and 40 mm. Preferably, the sperm separating device has a depth in the range of 10 mm and 20 mm.

[0091] In some embodiments, the sperm separating device may be made from a polymer material such as a cyclic olefin copolymer (COC), polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), polycarbonate (PCA) or anyother gamete biocompatible polymer which may be advantageously employed in the manufacture of the device of the present invention.

[0092] In some embodiments, the sperm separating device may include a protective means for reducing spill risk, maintaining sterility of the device, and preventing evaporation of media from the device. In some embodiments, the protective means is in the form of a cover. In other embodiments, other suitable protective means may be used.Brief description of the drawings

[0093] A preferred embodiment of the disclosure will now be described, by way of example only, with reference to the accompanying drawings in which:

[0094] Figure 1 is a top perspective view of the sperm separating device;

[0095] Figure 2 is a cross-sectional view of the device of Figure 1 ;

[0096] Figure 3 is a close-up cross-sectional view of Figure 2 showing the ramp of the sample zone, the migration zone which steps down, and the collection zone;

[0097] Figure 4 is a close-up view of the inclining grooves and pillars of the device of Figure 1 ;

[0098] Figure 5 is a close-up view of the declining grooves with steps of the device of Figure 1 ;

[0099] Figure 6 is a top perspective view of alternative embodiment of the device with a lid;

[0100] Figure 7 is a cross-sectional view of the device of Figure 1 with a lid;

[0101] Figure 8 is a close-up view of sub-grooves of the device of Figure 1 where one sub-groove has a dead end, and the other sub-groove has a chemoattractive leading to the collection zone;

[0102] Figure 9 is a close-up view of comers of the grooves of the device of Figure 1 ;

[0103] Figures 10a to 10f are cross-sectional views of alternative embodiments of the well of the collection zone;

[0104] Figure 11 is a top perspective view of the device of Figure 1 with a cover and a lid;

[0105] Figure 12 is an exploded top perspective view of the device of Figure 11 ;

[0106] Figure 13 is a top perspective view of an alternative embodiment of the sperm separating device with a lid;

[0107] Figure 14 is an exploded top perspective view of the device of Figure 13;

[0108] Figure 15 is a cross-sectional view of an alternative embodiment of the device with multiple layers of grooves;

[0109] Figure 16 is a close-up cross-sectional view of the device of Figure 15;

[0110] Figure 17 is a close-up top perspective view of the collection zone of the device of Figure 15;

[0111] Figure 18 is an exploded view of an alternative embodiment of the device with an oocyte zone, a lid and a cover;

[0112] Figure 19 is an exploded view of an alternative embodiment of the device with an oocyte zone, a lid and a cover;

[0113] Figures 20a to 20c are close up views of alternative embodiments of the steps in the grooves;

[0114] Figures 21a to 21c are close up views of alternative embodiments of the declining grooves;

[0115] Figure 22 is an exploded view of an alternative embodiment of the sperm separating device with an oocyte zone and a micropattern;

[0116] Figure 23 are graphs showing sperm DNA fragmentation by method of sperm selection;

[0117] Figure 24 are graphs showing motility comparison by method of sperm selection;

[0118] Figure 25 is a graph showing binding efficiency by method of sperm selection;

[0119] Figure 26 is a workflow of an embodiment of the device; and

[0120] Figure 27 is a workflow of an alternative embodiment of the device.Detailed description

[0121] The present invention will now be described with reference to the following example which should be considered in all respects as illustrative and non- restrictive. In the Figures, corresponding features within the same embodiment have been given the same reference numerals.

[0122] In overview and referring initially to Figure 1 , the sperm separating device 1 is configured to simulate the natural selection of sperm and to assist in the separation of high quality sperm from semen. In the illustrated embodiment, the device is substantially round in shape. However, in other embodiments, the device may be a different shape. Figures 13 and 14 show an alternative embodiment of the device 1 in a rectangular shape. Figures 18 and 19 show alternative embodiments of the device 1 in different shapes with an additional oocyte zone 85 (described hereinafter in relation to Figures 18 and 19). The sperm separating device 1 includes a sample zone 5 configured to receive semen (not shown), a collection zone 15 fluidly connected to the sample zone 5 by a migration zone 10, and a lid 20 (shown in Figure 6) disposed over a part of the migration zone 10 and the collection zone 15. The migration zone 10 includes grooves 35 to guide sperm (not shown) within the semen from the sample zone 5 to the collection zone 15.

[0123] In the illustrated embodiment, the sample zone 5 has an open top face and grooves 35. However, in other embodiments, the sample zone 5 may have a partially open or closed top face and may not have any grooves.

[0124] Referring to Figure 3, the sample zone 5 is inclined to form a ramp 40 toward the migration zone 10 which advantageously allows a semen sample to sit on top of the ramp 40 and allow gravity to let non-motile bodies in the semen settle down and away from the migration zone 10 whilst also actively encouraging sperm to follow the grooves 35 up and into the migration zone 10 and to cross the fluid boundary created between the semen and a sperm buffer (not shown). The angle of the incline of the ramp 40 is between 2 degrees and 89 degrees.

[0125] The sperm buffer may be considered as any media that keeps sperm viable. The viscosity of the media may also be moderated to make the environment more similar to the female reproductive tract advantageously increasing the yield of sperm collected. The media could be bicarbonate, MOPS (3-(N- morpholino)propanesulfonic acid) or HEPES (4-(2-hydroxyethyl)-1 - piperazineethanesulfonic acid) buffered and typically contains human serum albumin, some antioxidants, antibiotics like gentamicin, glucose, and lactate. This list is not exhaustive, and different types of media may be used.

[0126] The collection zone 15 is declined from the migration zone 10, as best shown in Figures 2 and 3. The angle of the decline is between 90 degrees and 50 degrees. Specifically, the collection zone 15 exists in a plane above the bottom of the sample zone 5. This configuration advantageously results in a zero fluid pressure environment since there are no closed grooves to push the semen sample through to fill the device and, consequently, there is no risk of a semen influx into downstream zones, i.e. the collection zone.

[0127] Referring to Figure 2, the collection zone 15 is in the form of a well 45 and is positioned below the height of the entry into the collection zone 15 such that sperm from the migration zone 10 falls into the collection zone 15. The collection zone 15 is configured to hold a volume of sperm between 50pL and 2000pL. The collection zone 15 also has a flat bottom face 50 for microscopic imaging of the sperm. In the illustrated embodiment, the well 45 is substantially shaped as an inverted trapezium. In the alternative embodiments, the well 45 may be other shapes as shown in Figures 10a to 10f .

[0128] Referring to Figure 5, the decline of the collection zone 15 comprises one or more steps 55 to both prevent sperm from easily returning towards the migration zone 10 and promote bulk-swimming behaviour to improve the yield upon collection. The one or more steps 55 drops in increments of between 10pm and 1000pm. In the illustrated embodiment, the steps 55 are substantially semi-circular. In the alternative embodiments, the steps may be other shapes as shown in Figures 20a to 20c.

[0129] The migration zone 10 is configured such that sperm are confined in the traversable geometry of the migration zone 10 either by the fluid surface tension or by the provision of surfaces extending from the device itself.

[0130] In an alternative arrangement as shown in Figure 8, the migration zone 10 further comprises an intersection of two sub-grooves, wherein one sub-groove has a dead end 60, and the other sub-groove 65 leads to the collection zone 15 and has a chemical agent in the form of a chemoattractive agent (not shown). Advantageously, the use of a chemoattractive agent increases the yield of sperm in the collection zone 15 as the chemoattractive agent encourages sperm to travel towards the chemoattractive agent unless the sperm are able to sense a change in chemoattractive agent concentration in which the sperm then turn 135 degrees and pursue increasing concentrations of the gradient towards the collection zone 15.This alternative embodiment enables a selection of a particular type of sperm toward the collection zone 15. Examples of a chemoattractive agent include progesterone, RANTES, lyral, bourgeonal, atrial natriuretic peptides, hyaluronic acid, or follicular fluid from a woman. This list is not exhaustive, and other types of chemoattractive agents may be used.

[0131] As best shown in Figure 4, the junction of the sample zone 5 and migration zone 10 further comprises outwardly extending protrusions, in the form of pillars 70. The pillars 70 are located at the entrance of the migration zone 10, to inhibit the semen from passing through the migration zone 10 and to prevent the semen from being injected into the migration zone 10. The pillars 70 advantageously prevent the potential for the viscous semen to flow into downstream zones by increasing the fluid resistance in spaces that the fluid needs to squeezethrough. The pillars 70 also help to prevent the user from depositing semen directly at the entrance to the migration zone 10 and thereby prevent the injection of semen at a high velocity directly into subsequent zones.

[0132] In the illustrated embodiment, the migration zone 10 comprises a single layer of the grooves 35. However, in an alternative arrangement, as shown in Figures 15 to 17, the migration zone may comprise a plurality of layers of the grooves 35. The plurality of layers of the grooves 35 increases the yield of sperm migrating from the sample zone 5 to the collection zone 15.

[0133] In the illustrated embodiment, the migration zone 10 is partially covered by a lid 20, i.e. the migration zone 10 has a partially open top face, as best shown in Figure 6. However, in other embodiments, the migration zone 10 may have an open or closed top face.

[0134] The lid 20 also covers the collection zone 15 and comprises a void 25 configured to receive a suction device which is configured to collect sperm within the collection zone 15. The lid 20 has an inwardly facing chamfered cavity 30, as best shown in Figure 7. Advantageously, the combination of a well 45 with a chamfered cavity 30 in the lid 20 results in the pinching of the fluid at the junction between the migration zone 10 and collection zone 15. This results in the generation of a fluid meniscus at the junction between the migration zone 10 such that when suction is applied to the collection zone 15, an air bubble is created between the lid 20 and collection zone 15, wherein the air bubble inhibits the fluid connection between the migration zone 10 and collection zone 15 to limit the amount of sperm collected from the collection zone 15.

[0135] In the illustrated embodiment, the grooves 35 extend into the sample zone 5 to facilitate the guidance of additional sperm out of the semen and into the migration zone 10. However, in an alternative arrangement, the grooves 35 in the migration zone 10 extend for at least part of the length of the migration zone 10 and may continue from or into either the sample zone 5 or collection zone 15 and are intended to separate sperm able to traverse the geometry within the migration zone 10.

[0136] In the illustrated embodiment, each of the grooves 35 also comprises a pair of corners 75, as shown in Figure 9, to guide the sperm as sperm movement is influenced by the surfaces they interact with, and sperm typically exhibit wallswimming behaviour, or the tendency to travel closer to boundary surfaces when within a confined groove geometry, such as in microfluidic channels. However, in an alternative embodiment, the grooves may have one or a plurality of corners. The walls of the grooves 35 are contoured to provide / form additional edges and boundary surfaces compared to those present in microchannels with smooth / planar walls, to advantageously improve the separation of sperm based on motility criteria. The corners 75 also advantageously provide an additional edge to guide the sperm.

[0137] Sperm separation features within the migration zone 10 also prevent sperm from returning towards the sample zone 5, which is beneficial for both yield and quality since the first sperm able to navigate a path to the collection zone 15 often have the most desirable characteristics. For example, the inclusion of a drop in height, i.e. a step, as the sperm progress prevents sperm from returning to the migration zone 10. Preferably, the drop is sharp and is a curved cut to prevent sperm from getting stuck in the corners of the grooves. The length of the migration zone 10 is also configured so that there is less opportunity for sperm to turn around but long enough to prevent sperm from accidentally being picked up from the sample zone 5. Preferably the length of the migration zone 10 is between 3mm and 5 mm.

[0138] In the illustrated embodiment, the device 1 has an open top. However, in alternative embodiments, the device may include a cover 80 for reducing spill risk, maintaining sterility of the device, and preventing evaporation of media from the device, see Figures 11 and 12. Figure 28 shows a cross-section of the device 1 with the cover 80.

[0139] As shown in Figures 11 , 12, and 28, the cover 80 has a void 82 configured for receiving the lid 20. As shown in Figure 11 , the void 82 has a vertical wall 122. As shown in Figure 12, the edge of the lid 20 has a vertical wall 120 that abuts the vertical wall 122 of the void 82, when the lid 20 is inserted into the void 82. The vertical walls 120 and 122 of both the lid 20 and void 82 include attachment means, such as snap lock, threads, and the like, that enable the lid 20 to securelyengage the void 82. When the cover 80 is engaged with the lid 20, the void 82 allows the void 25 to still receive the suction device.

[0140] The edge of the lid 20 also has a horizontal section perpendicular to the vertical wall of the lid 20. The horizontal section can be used to set the maximum level of insertion of the lid 20 into the void 82.

[0141] The cover 80 also engages the device 1 , to cover the sample zone 5 and the migration zone 10. In one arrangement, the outer lip of the cover 80 includes threads that engage complementary threads disposed on the outer lip of the device 1 . Other attachment means may also be used so that the cover 80 is attached to the device 1 .

[0142] When the cover 80 is disposed above the device 1 , the sample zone 5 and the migration zone 10 are protected by the cover 80. As discussed hereinbefore, the collection zone 15 is covered by the lid 20. The void 82 also enables a user to access the void 25 to collect sperm from the collection zone 15. Additionally, the cover 80 and the lid 20 prevent spillage and contamination of the sample.

[0143] As discussed hereinbefore, the migration zone 10 may include multiple layers of grooves 35 to increase the sperm yield at the collection zone 15. In one arrangement, part of the migration zone 10 having the multiple layers of grooves 35 are integrated to the lid 20. This arrangement provides the migration zone 10 with multiple layers of grooves 35 when the lid 20 is engaged with the device 1 .

[0144] Figures 29 to 32 show the lid 20 having multiple layers 2900Ato 2900C with grooves 35. The layers 2900Ato 2900C may be attachable to the lid 20 and to other layers 2900A to 2900C. In another arrangement, the layers 2900A to 2900C are integrated to the lid 20. In another arrangement, one of the layers 2900A is integrated to the lid 20 while the other layers 2900B and 2900C are attachable to the layer 2900A and 2900B respectively. The layers 2900A to 2900C may be attached using attachment means such as snap lock, thread, glue, and the like. Although only 3 layers 2900Ato 2900C are shown in Figures 29 to 32, one or more layers 2900 may be attachable / integrated to the lid 20.

[0145] In another embodiment, as shown in Figures 18 and 19, the sperm separating device 1 further comprises an oocyte zone 85 for receiving sperm collected from the collection zone 15 and an oocyte (not shown). The oocyte zone 85 is configured for intracytoplasm ic sperm injection of the oocyte and further comprises an oil overlay (not shown) to ensure there is no fluid contact between the sperm, oocyte and the sperm buffer.

[0146] Referring to Figure 26, to use the sperm separating device 1 , the device first needs to be primed with a desired buffer (step 1 ). Priming the device occurs by filling the collection zone 15 with a buffer fluid either through the void in the lid 25 at the collection zone 15 or by placing fluid in the open collection zone 15, the force of the fluid being ejected from a buffer fluid dispensing device pushes the fluid through the grooves 35. In an alternative embodiment, time is allowed for the buffer fluid dispensed in the collection zone 15 to enter the grooves 35through hydrophilic capillary forces. Once the device is primed, sperm is deposited into the sample zone 5 (step 2). In the illustrated embodiment, a cover 80 is placed over the device for reducing spill risk, maintaining sterility of the device, and preventing evaporation of media from the device (step 3). The sperm then migrates up the grooves 35 in the ramp 40 to the migration zone 10 while the non-motile bodies settle at the bottom of the ramp 40 of the sample zone 5. The sperm continue to migrate through the grooves 35 until they reach the collection zone 15, in the form of a well 45, where the sperm migrates down declining grooves 35 with steps 55 to reach the bottom of the well 45 for collection. The steps 55 advantageously prevent the sperm from migrating back up and re-entering the migration zone 10.

[0147] To collect the sperm from the collection zone (step 4), a suction device is placed in the void 20 of the lid 25, and the suction device is activated. Once the suction device is activated, an air bubble is created between the lid 20 and collection zone 15. Specifically, when fluid is removed from the collection zone 15, air enters the device 1 to replace the space the fluid was occupying, which interacts with the geometry of the device and generates the air bubble. Advantageously, the air bubble terminates the fluid connection between the migration zone 10 and collection zone 15 to limit the amount of sperm collected from the collection zone 15 and prevent the collection of undesired bodies from the sample zone 5 or migration zone 10.Advantageously, sperm can be collected from the collection zone X within 2 minutes of sperm being deposited into the sample zone 5 depending on the number of motile sperm present in the semen sample and the needs of the user. All desired sperm must be collected within an hour of sperm being deposited into the sample zone 5. This device specifically enables the migration of high quality sperm to the collection zone 15 while inhibiting undesirable sperm and bodies within the sample zone 5 and migration zone 10.

[0148] Referring to Figure 27, to conduct intracytoplasm ic sperm injection of an oocyte, an alternative embodiment of the device (as shown in Figures 18 and 19) is used which contains an oocyte zone 85 and a collection zone 15 with a micropattern 90, as best shown in Figure 21 . The micropattern 90 contains a sperm binding agent which selectively binds to and advantageously immobilises sperm with desired biological features externally expressed on their membrane. Desirable features for which a binding agent may include but are not limited to are sperm maturity, spermoocyte recognition markers, and sperm sex markers. As detailed above, the device is first primed with a buffer (step 1). In this embodiment, capillary forces suck the buffer fluid into the grooves 35. An oocyte is then placed in the oocyte zone 85 (step 2), and the collection zone 15 and the oocyte zone 85 are covered in oil (step 3) to ensure there is no fluid contact between the sperm, oocyte and the sperm buffer. Sperm is then injected into the sample zone 5 (step 4) where it migrates through the migration zone 10 to the micropattern 90 in the collection zone 15 where the sperm binds to a binding agent. In this embodiment, the micropattern 90 is in the form of a zig-zag. However, in alternative embodiments, other shapes may be used. The sperm is then collected from the micropattern 90 and injected into an oocyte in the oocyte zone 85 (step 5). In an alternative embodiment, the sperm may be collected without the binding agent and / or the sperm may be transferred to an intermediate droplet, normally made of polyvinylpyrrolidone (PVP), before being injected into an oocyte in the oocyte zone 85.

[0149] Figure 23 shows percentage sperm DNA fragmentation (SDF) analysed by sperm chromatin dispersion (SCD) assay comparing the result of raw semen, density gradient centrifugation (DGC), which is a conventional semen processing method, and the device 1 (referred as MFD in the figure) in split semen samples. Italso shows a comparison of percentage DFI distribution of sperm from the device 1 versus DGC. Figure 23 shows a semen sample before processing in the device 1 and sperm collected from the device after processing said semen sample.

[0150] Figures 24 and 25 show graphs showing the result of the device 1 compared to DGC. Figure 24 shows sperm motility analysis by conventional manual assessment according to World Health Organisation (WHO) criteria comparing raw semen, semen obtained via DGC, and semen obtained via the device 1 (referred to as MFD in Figure 24) in split semen samples. Figure 24 also shows a comparison of percentage DFI distribution of sperm from the device 1 versus DGC. Figure 25 shows a comparison of the binding score for hyaluronic acid binding of sperm post- DGC (i.e. , a conventional device) and on the variant of the device 1 possessing a hyaluronic acid coating after motility-based selection.

[0151] The sperm separation device described herein is particularly useful for the separation of high quality motile sperm from undesirable non-motile sperm and bodies in semen and represents a practical and commercially significant improvement over the prior art. Another advantage is that the separation of sperm from semen does not damage the sperm.

[0152] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms.Reference table

Claims

Claims1 . A sperm separating device comprising: a sample zone configured to receive semen; a collection zone fluidly connected to the sample zone by a migration zone; and a lid disposed over a part of the migration zone and the collection zone, wherein the migration zone includes grooves to guide sperm within the semen; and wherein the sperm in the semen migrate via the grooves to the collection zone.

2. A sperm separating device according to claim 1 , wherein each of the grooves extend to the sample zone.

3. A sperm separating device according to claim 1 or claim 2, wherein each of the grooves comprises at least one corner.

4. A sperm separating device according to any one of the previous claims, wherein the sample zone is inclined to form a ramp toward the migration zone.

5. A sperm separating device according to any one of the previous claims, wherein the collection zone is declined from the migration zone.

6. A sperm separating device according to claim 5, wherein the angle of the decline is between 90 degrees and 50 degrees.

7. A sperm separating device according to claim 5 or claim 6, wherein the decline of the collection zone comprises one or more blocking members to prevent the sperm from migrating back up the collection zone.

8. A sperm separating device according to any one of the previous claims, wherein the migration zone is declined from the sample zone and wherein the decline of the migration zone comprises one or more blocking members to prevent the sperm from migrating back up the migration zone.

9. A sperm separating device according to claim 7 or claim 8, wherein the blocking members comprise steps that drop in increments of between 10pm and 1000pm.

10. A sperm separating device according to any one of the previous claims, wherein the migration zone and the lid communicate to create a fluid boundary between the sample zone and the migration zone.

11. A sperm separating device according to any one of the previous claims, wherein the height and width of the grooves is not constant along their length.

12. A sperm separating device according to any one of the previous claims, wherein the migration zone comprises an intersection of two sub-grooves, wherein one sub-groove has a dead end, and the other sub-groove leads to the collection zone and has a chemical agent.

13. A sperm separating device according to any one of the previous claims, wherein the migration zone comprises a plurality of layers of the grooves.

14. A sperm separating device according to any one of the previous claims, wherein the collection zone is in the form of a well and is configured to hold a volume of sperm between 50pL and 2000pL.

15. A sperm separating device according to any one of the previous claims, wherein the collection zone has a flat bottom face for microscopic imaging of the sperm.

16. A sperm separating device according to any one of the previous claims, wherein the lid further comprises a void configured to receive a suction device which is configured to collect sperm within the collection zone.

17. A sperm separating device according to claim 15 or 16, wherein the lid further comprises an inwardly facing cavity such when suction is applied to the collection zone, an air bubble is created between the lid and collection zone, wherein the air bubble inhibits the fluid connection between the migration zone and collection zone to limit the amount of sperm collected from the collection zone.

18. A sperm separating device according to any one of the previous claims, wherein the junction between the sample zone and migration zone further comprises outwardly extending protrusions to inhibit the semen from passing through the migration zone and to prevent the semen from being injected into the migration zone.

19. A sperm separating device according to any one of the previous claims, further comprising an oocyte zone for receiving sperm collected from the collection zone and an oocyte, wherein the oocyte zone is configured for intracytoplasm ic sperm injection of the oocyte.

20. A sperm separating device according to claim 18, wherein a surface of the collection zone further comprises a micropattern for receiving a sperm binding agent to selectively bind and immobilise sperm with desirable traits on the surface.21 . A sperm separating device according to claim 19, wherein the micropattern further comprises at least one cnnective layer to bind the sperm binding agent to the device.

22. A sperm separating device according to any one of the preceding claims, further comprising a cover configured to engage the device such that the sample zone and the migration zone are covered.

23. A sperm separating device according to claim 22, wherein the cover has a void configured for receiving the lid.

24. A sperm separating device according to claim 23, wherein the cover attaches to the lid.

25. A sperm separating device according to any one of claims 22 to 24, when dependent on claim 16, wherein the void of the cover allows the void of the lid to receive the suction device.

26. A sperm separating device according to any one of claims 22 to 25, when dependent on claim 13, wherein the plurality of layers of the grooves is integrated to the lid.

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