CN112662550B

CN112662550B - Device and method for separating sperms from semen sample

Info

Publication number: CN112662550B
Application number: CN202011107799.6A
Authority: CN
Inventors: S·瓦西列斯库; M·易卜拉欣米·瓦尔基亚尼; R·努斯拉蒂; 樊华; S·R·巴扎兹
Original assignee: Xiwei Suzhou Biomedical Technology Co ltd
Current assignee: Xiwei Suzhou Biomedical Technology Co ltd
Priority date: 2020-10-16
Filing date: 2020-10-16
Publication date: 2023-11-10
Anticipated expiration: 2040-10-16
Also published as: WO2022078144A1; CN112662550A

Abstract

The present invention provides a device and method for separating motile sperm from a semen sample, the device comprising an inlet reservoir for receiving the semen sample; an outlet reservoir for collecting sperm separated from the sample; and at least one but preferably a plurality of microchannels disposed between the inlet and outlet reservoirs to provide fluid communication between the two reservoirs, the microchannels including at least one wall defining a boundary surface along which sperm are directed to the outlet reservoir; and wherein the at least one wall has at least one surface structure selected from the group consisting of, for example, one or more recesses, protrusions, and combinations thereof, extending over at least a portion of the length of the microchannel, thereby providing at least one additional boundary surface along which motile sperm travel toward the outlet reservoir.

Description

Device and method for separating sperms from semen sample

Technical Field

The present invention relates to an apparatus and method for separating sperm cells from a semen sample, and more particularly to an apparatus and method for selecting sperm cells having a predetermined characteristic from the separated sperm cells.

Background

Sperm screening is an important component of Assisted Reproductive Technology (ART) with a great impact on both success rate of reproductive therapy and embryo health (Davies, 2012 and Mcdowell, 2014). Most of the focus of reproductive therapy and biology has been around ovum acquisition and embryo culture, however, since the success of In Vitro Fertilization (IVF) was achieved in 1973, the importance of male gamete health and its isolation strategy has been severely underestimated.

Recent studies (Oseguera-lvopez, 2019 and Sakkas, 2015) have described that sperm have important and complex roles in transferring outside the second set of chromosomes. However, current clinical sperm screening techniques, where Density Gradient Centrifugation (DGC) and upstream methods (SU) are most commonly used, have little innovation since 1978, some of these methods suffer from a number of limitations, such as the introduction of reactive oxygen species by some methods, are time consuming and costly, sperm screening processes also rely largely on embryologist's techniques and non-standardized protocols of operation, which often result in differences in success rates of in vitro fertilization between different operators (Kashaninejad, 2018) which are most pronounced during sperm processing stages, which typically involve three or more gradient centrifugation, cell resuspension and fine aliquoting (world health organization, 2010).

The existing microfluidic sperm screening strategy still only recurs DGC and SU technologies, and has no obvious advantage in terms of flux or selectivity. These techniques currently in use bypass the natural sperm screening process, selecting sperm primarily based on sperm motility or morphology, and ignore other, possibly more important factors such as DNA integrity, degree of sperm apoptosis, and plasma membrane maturity. Indeed, neither DGC nor SU can reduce sperm DNA fragmentation (sDF), even in some samples leading to sperm DNA fragmentation (Robinson, 2012 and oleszczu k, 2013).

The reason for this deficiency in DGC and SU technology is that: they tend to generate reactive oxygen species during centrifugation, which have been shown to impair sperm function by oxidizing and fragmenting DNA adducts (Rappa, 2016 and Aitken, 2013). Since these sperm screening techniques can cause damage to sperm (es, 2015; muradori et al, 2016, 2019), and are largely dependent on the experience of the technician in selecting sperm, this makes the process error-prone, directly affecting fertilization rates. In 2012, the clinical success rate in australia was only between 4% and 30.9% (Wade, 2015). Furthermore, this makes it possible for selected sperm to appear morphologically normal, suitable for In Vitro Fertilization (IVF) but at risk of significant DNA damage. Despite the wide range of applications of these techniques (particularly DGC), in recent years one has turned from recognizing their applicability to recognizing their shortcomings.

In other current techniques, due to time constraints on the sample (e.g., obtained by testicular surgery), actual sperm separation is not possible, but only sperm purification is performed. Sperm purification processes are generally more time consuming; screening sperm from testicular samples is a manual operation, typically requiring 2-3 hours, and only removes seminal plasma from sperm (world health organization, 2010).

This means that this and other current methods of sperm production may not be able to pick the most viable sperm from the sample. DGC typically selects up to about 36% of the sperm population from 0.5mL of raw semen in 30 minutes, whereas upstream experiments typically select about 12% of the sperm population from 1mL of semen in 1 hour, resulting in an increase in sperm motility of 18-19% and 5%, respectively (world health organization, 2010).

U.S. patent No. 2015/0140655 A1 describes a method and apparatus for separating sperm from a semen sample. The device comprises an outer annular chamber for semen samples, a plurality of spoke-like micro-channels (i.e. radial arrays or networks of such channels), and a central chamber that collects sperm that have passed through the array of micro-channels. The function of such devices is achieved by the migration of autonomously swimming sperm (motile sperm) through effectively stagnant fluid (which may be a relatively high viscosity fluid) in a microchannel and exploiting the natural swimming behavior of sperm in confined spaces, such as the surface accumulation behavior of sperm and the swimming behavior along the boundary. This action of the adherent swimming is hereinafter referred to simply as "the wall swimming action of sperm in the microfluidic structure". The microchannels starting from the annular semen sample chamber are connected together in cascade, taking into account the geometry involved, so that the number of microchannels draining into the central chamber is reduced. That is, there is at least one sperm-based wall play between the inlet and outlet of the microchannel path as a junction for directing sperm.

There is therefore a need for an improved device and method for separating sperm from a semen sample that provides acceptable yields as disclosed in U.S. patent document' 655.

There is also a need for an apparatus and method that can separate sperm cells from a semen sample while reducing the potential for sperm cell damage during the processing stage.

There is also a need for an improved apparatus and method that is capable of selecting sperm cells from a semen sample by a motion-based separation process (as described in the aforementioned U.S. patent document' 655) to yield motile sperm cells having certain (or definable) desired characteristics. The present invention addresses one or more of these unmet needs.

In this specification, the reference to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as, an acknowledgment or admission or any form of suggestion that the prior publication (or information derived from it) or known matter forms part of the common general knowledge in the technical field in which this specification relates.

Disclosure of Invention

According to a first aspect of the present invention there is provided a device for separating motile sperm from a semen sample, the device comprising an inlet reservoir for receiving a semen sample; an outlet reservoir for collecting sperm separated from the sample; and at least one but preferably a plurality of microchannels disposed between the inlet and outlet reservoirs to provide fluid communication between the two reservoirs, the microchannels including at least one wall defining a boundary surface along which sperm are directed to the outlet reservoir; and wherein the at least one wall has at least one surface structure selected from the group consisting of, for example, one or more recesses, protrusions, and combinations thereof, extending over at least a portion of the length of the microchannel, thereby providing at least one additional boundary surface along which motile sperm travel toward the outlet reservoir.

In some embodiments, the device comprises a housing, preferably made of a polymeric material, such as Cyclic Olefin Copolymer (COC), polycarbonate (PCA), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), or photopolymer resin used in additive manufacturing techniques that may advantageously be used to manufacture the device of the invention (but do not exclude other manufacturing techniques such as casting, molding and machining), the housing being designed to integrally form or completely encase an inlet reservoir, the outlet reservoir and one or more microchannels extending between the inlet reservoir and the outlet reservoir, at least one port in the housing being arranged to supply a semen sample into the inlet reservoir, and at least one extraction port for removing sperm separated from semen from the outlet chamber.

In the following, reference will be made mainly to embodiments with a plurality of said micro-channels, it being understood that embodiments with a single or a small number of micro-channels are also within the scope of the inventive idea.

The cross-section of each microchannel may be constant or variable, such as square, rectangular, circular, oval, etc. It will be appreciated that when the cross-section of the microchannel is circular or elliptical, there will be one annular wall defining one contiguous boundary surface, while in the case of a rectangular cross-section there are four major boundary surfaces defining sharp corners (or rounded corners) at the plane where the contiguous walls meet. Sperm that exhibit wall-swimming behavior will tend to move toward and along the boundary surface.

Additional surface structures are provided in the other planar walls of the microchannel, such as one or more recesses, one or more protrusions, and a combination of one or more recesses and protrusions, which extend over at least a portion of the length of the microchannel, with the aim of increasing the number of boundary surfaces of the microchannel path along which sperm can travel. As mentioned above, the movement of sperm is affected by the surface with which it interacts and sperm typically exhibits wall-swimming behavior, or when in a restricted channel geometry such as a microfluidic channel, sperm tends to move closer to the boundary surface. The profile of the microchannel walls, which can provide or form additional edges and boundary surfaces, is more conducive to motility-based sperm separation than is the case when the microchannel walls are smooth or flat profiles (as described in U.S. patent' 655).

It should be appreciated that the device of U.S. patent' 655 employs the concept of: the initially larger number of microchannels in the radially outer region of the radial microchannel network are connected in sequence such that their number decreases in the radially inner discharge region, seeking to maintain the width and height (i.e., cross-sectional geometry) of the substantially rectangular cross-sectional channels substantially constant in order to sort sperm based entirely on the directionality of the wall-flow behavior.

In contrast, the device according to the first aspect of the invention does not seek to distinguish sperm based on the direction of travel within the microchannel, but rather alters the geometry of the microchannel to include additional boundary surfaces along which sperm will swim indiscriminately based on its wall-swimming behaviour. Sperm cells having the desired characteristics for fertilization typically travel a greater distance than sperm cells having reduced motility travel, and the increase in boundary surface allows more sperm cells having the desired characteristics to reach the outlet reservoir where they can then be collected or further processed.

Furthermore, by providing additional surface structures in the microchannels as described above, it is possible to increase the maximum cross-sectional dimensions of the individual microchannels compared to smooth-walled channels without compromising the microfluidic properties of the channels seeking to meet the wall-swimming behavior of motile sperm, as the increased surface structures provide additional boundary surfaces within the channel boundaries for motile sperm to travel based on their wall-swimming behavior.

The configuration or shape of the surface structure is not limited to any particular geometry, as long as the structure can form an additional boundary surface for guiding sperm, and many geometries can achieve this objective, as described below. In addition, the number of sperm motility boundaries can be controlled to regulate sperm throughput.

From a manufacturing perspective, the structure may include one or more grooves, such as rectangular, semi-circular, or stepped cross-sections, ribs, and combinations thereof, extending the entire length of the microchannel from the inlet to the outlet, or along only a portion thereof.

The grooves/ribs define additional surface paths within the microchannel from the inlet reservoir to the outlet reservoir along which sperm may travel based on wall-travel behavior. These structures may extend along part or all of the length of the microchannel along one or more paths that are axially straight, serrated, wavy, or a combination thereof. The depth of the grooves/the height of the ribs may be constant or variable. The cross-sectional shape or profile of the groove/rib may be selected as appropriate, provided that there is at least one sharp or rounded edge with channel walls along its extension that exceeds the edge defined by the smooth-walled channel.

Depth or height discontinuities in the surface structure may be provided by stepped sidewalls of the microchannel. This is to add more boundary surfaces in the microchannel side walls to allow sperm to travel along the microchannel as it passes through the microchannel.

In a preferred embodiment, the device is designed such that the plurality of micro-channels extend in spokes within the housing from an inlet reservoir in the form of an annular radially outer chamber (which may be subdivided into segments with individual semen sample inlets) to an outlet reservoir in the form of a circular radially inner outlet chamber.

Adjacent microchannels may share a common separation sidewall and surface structures providing additional boundary surfaces may be present in one or both sidewalls of a microchannel having a rectangular cross-section. Of course, alternatively or additionally, there may also be surface structures in one or both of the bottom and top boundary walls of such rectangular-section microchannels.

Other microchannel arrangements may be employed without excluding other microchannel arrangements, for example, a plurality of microchannels extending parallel to each other (as opposed to a radial network) between inlet and outlet chambers at opposite ends of a microchannel, or a plurality of microchannels may be arranged in a spiral from a radially outer annular inlet chamber toward a radially inner cylindrical outlet chamber.

Furthermore, the microchannels need not extend along a straight line, but may be arranged in a serpentine or meandering manner between the inlet and outlet reservoirs.

The number of microchannels of the device is 400 to 700, more preferably 100 to 500, for producing motile sperm within an acceptable (shorter) collection time range, but the number of microchannels can range from a minimum of 5 to a maximum of 5000.

Depending on the number of existing micro-channels and the semen species to be collected or separated from the semen sample, the width of the plurality of micro-channels may be between about 10 μm and about 5000 μm, preferably between 50 μm and 250 μm, and the height between about 10 μm and about 5000 μm, preferably between 100 μm and 1000 μm. Preferably, one or both of the width and height of all of the microchannels are the same.

Typically, the length of the microchannels is between about 0.1mm and 15mm, preferably between about 5mm and 9 mm.

Furthermore, particularly in embodiments where the microchannels are in a radially converging arrangement, two or more microchannels starting at the inlet reservoir end of the device may be joined along their path to form a single microchannel at the outlet reservoir.

It should also be appreciated that the microchannels may be arranged in a single or multi-layered stack within the device housing, with the start points of the microchannels being in a common or separate inlet chamber and discharging into a common outlet chamber.

According to a second aspect of the present invention there is provided an apparatus for screening sperm cells having a defined characteristic from a semen sample, the apparatus comprising: an inlet reservoir for receiving a semen sample; a binding chamber arranged in downstream fluid communication with the inlet chamber by a guiding structure designed to guide motile sperm from the semen sample to the binding chamber, preferably based on the wall-play of motile sperm; and an outlet reservoir in downstream fluid communication with the binding chamber, the outlet reservoir being designed to receive motile sperm that have passed through the binding chamber; wherein the binding chamber is configured to receive a selective particle having a selectivity for binding to motile sperm of: (i) Having passed through the guide structure, and (ii) having a predetermined, undesired marking, the binding chamber and/or the selection particles are designed to substantially confine sperm bound to the particles in the binding chamber and to prevent further travel of sperm toward the outlet reservoir without impeding passage of motile sperm not bound to the selection particles.

In some embodiments, the guiding structure will advantageously take the form described with reference to the first aspect of the invention, i.e. comprising a plurality of micro-channels extending between the inlet reservoir and the binding chamber (rather than the outlet chamber).

In one embodiment, the inlet side of the binding chamber is bounded by the downstream ends of the walls of the plurality of microchannels (thus providing a grid-like inlet structure) and the outlet side is defined by a series of spaced apart columns or posts arranged in a configuration defining a grid-like outlet structure, whereby the binding chamber is a "cage-like structure" into which motile sperm can enter from the plurality of microchannels and from which outflow of sperm bound by the particles is prevented by a plurality of blocking posts. In some embodiments, the end portions of the walls separating adjacent microchannels adjacent to the binding chamber are advantageously configured such that (i) the microchannels narrow toward their respective discharge ends, and (ii) collectively define a grid-like boundary structure that inhibits motile sperm that have exited the microchannels from re-entering the microchannels. In other words, the end cross-section of the microchannel narrows and provides an inner surface along which motile sperm can travel, along which the motile sperm travels from the microchannel into the binding chamber in a preferential converging path, such that the motile sperm is substantially (but not absolutely) impossible to re-enter the microchannel due to the narrowing of the cross-section. For example, assuming that the microchannel side walls are substantially rectangular in vertical cross section and the ends widen in thickness, then they are substantially triangular in plan view of the wall ends.

In a preferred embodiment, a plurality of blocking posts are shaped and/or arranged with respect to one another to limit the return of motile sperm that have exited the binding chamber to the binding chamber in a manner functionally similar to the shaping of the microchannel defining sidewall ends. The gap between the blocking posts needs to be large enough to allow unbound motile sperm to flow through the binding chamber to the outlet reservoir. At the same time, the gap between the blocking posts needs to be small enough to accommodate the selected particles within the binding chamber. In a preferred embodiment, the gap between adjacent blocking posts is between 10 and 100 μm. Also, the cross-section of the microchannel at its ends needs to be as small to accommodate the selected particles in the binding chamber.

In some embodiments, the cross-sectional shape of the posts is chevron, semi-annular, or crescent shaped, and the posts are arranged so as not to obstruct the passage between the posts toward the outlet chamber, but so as to inhibit motile sperm from returning from the outlet chamber into the binding chamber.

Selected particles of suitable shape, size and bulk density are selected to be incorporated into the binding chamber so as not to substantially impede the passage of motile sperm which do not exhibit undesirable indicia upon exiting the binding chamber, but are sufficiently shaped, sized and bulk density to adequately contain motile sperm exhibiting undesirable characteristics within the binding chamber.

According to various embodiments of the invention, the selection particles contained in the binding chamber comprise beads, in particular microspheres or microbeads, conjugated with a ligand comprising a protein capable of attaching to a label selected from the group consisting of lectin, anti-CRISP (cytosine-rich secreted protein), annexin A5, anti-chemokine receptor antibody and anti-CD 63, anti-CD 9, ALIX or TSG101m, or the ligand may be a drug comprising resiquimod (resiquimod), imiquimod (imiquimod) and gardimmod (gardimquimod). Preferably, the ligand is annexin A5, which chemically binds to a marker expressed by motile sperm.

The term "ligand" as used in the examples of the present invention refers to a substance that forms a complex with a biomolecule to function in a biochemical pathway. For example, a ligand may be a molecule that when bound to a particular cellular site will trigger a cellular response. In other examples, the ligand may be an ion or protein or any other suitable molecule having a chemical structure capable of interacting with the binding site.

In a preferred embodiment, the ligand is a protein, preferably annexin A5, by which sperm expressing phosphatidylserine at the outer surface of sperm can be bound and captured in the binding chamber. Thus preventing these phosphatidylserine expressing sperm from traveling further toward the outlet reservoir, while those that do not can travel substantially unimpeded through the binding chamber toward the outlet reservoir where they can then be collected or further processed.

According to another aspect of the invention, the device further comprises an oocyte holding chamber in fluid communication with or as part of the outlet reservoir. Optionally, a structure may be selected for regulating the passage of screened (i.e., non-particle-bound) motile sperm from the outlet reservoir into the oocyte holding compartment. The oocyte may be introduced directly into the oocyte holding compartment so that motile sperm, which has been isolated from the sample and "screened" during passage through the binding chamber, may be combined with the oocyte to effect fertilization. In some embodiments, a chemoattractant may be applied to the outlet reservoir and/or oocyte holding chamber. The chemoattractant helps direct the separated and screened sperm to the oocyte.

In one form of the device of the invention, different functional units or structures are divided within the device housing. For example, the device may have a first region comprising an inlet reservoir, a second region comprising a microchannel or guide structure, a third region comprising a binding chamber, a fourth region comprising an outlet reservoir, and a fifth region comprising an oocyte holding chamber in its housing. The third zone and the fourth zone may preferably be juxtaposed, which means that the oocyte holding chamber will be located within the range of the outlet reservoir. For example, when the outlet reservoir is designed as a substantially cylindrical disc chamber, there may be one or more separate wall structures therein, each for accommodating an oocyte, but at the same time motile sperm can pass through the wall structures to contact the oocyte.

In other embodiments, some of the regions are at different heights relative to a datum or reference plane (e.g. the support surface on which the device is placed in use), the first region (e.g. the inlet reservoir in the form of an annular chamber) may be located at the same height as the second region (microchannel) or above or below the second region (microchannel), e.g. an annular outer inlet chamber having radially inwardly directed channels at the same height, with an annular junction chamber or outlet chamber at the same height at the end. In another embodiment, the second region (microchannel) may be located at the same level as the third region (i.e. also the binding chamber in the form of a ring chamber). However, in embodiments where the first region and the third region are located at different heights, the micro-channel (i.e., the second region) is located between these two heights, and thus the channel itself will extend obliquely relative to the horizontal plane.

In one embodiment, the microchannels are arranged in a radial array, the inlet reservoir is an annular chamber in the region above the annular junction chamber or outlet chamber, the 3D channel array will be defined by a frustoconical (through channel) envelope or a truncated parabolic (or similar) envelope, and the channels have a curvature in a plane perpendicular to the horizontal plane. The third zone (binding chamber) may be located at the same height as the fourth zone (outlet reservoir) and the fourth zone (outlet reservoir) may be located at the same height as or above the fifth zone (oocyte holding chamber).

More closely, these regions (whether at the same or different elevations) are in fluid communication with each other, the buffer solution fills the various structures such that (a) motile sperm can be separated from the semen sample within the microchannel or guide structure, (b) motile sperm that have entered the binding chamber and have predetermined characteristics can be separated from motile sperm that have bound to selected particles and thus remain in the binding chamber, and (c) motile sperm that have not bound to particles can reach the outlet reservoir. The so-screened sperm (not bound to particles) may then be removed from the outlet reservoir and/or "swim" into the oocyte holding chamber where they may be combined with one or more oocytes contained in the oocyte holding chamber to effect fertilization.

In some embodiments, ports into each chamber may also be provided to enable removal of particle-bound sperm from the binding chamber, removal of non-particle-bound sperm from the outlet reservoir and/or oocyte holding chamber, for example using a pipette or other known in vitro fertilization output/removal instrument.

According to another aspect of the present invention, there is provided a method of screening and/or separating motile sperm from a semen sample, comprising the steps of: providing a device according to the first or second aspect of the invention as the case may be (i.e. separating motile sperm from a semen sample alone, or separating and screening motile sperm); filling the microchannel or guide structure (as the case may be) and the inlet and outlet reservoirs of the device with a sperm buffer solution to produce a substantially non-flowing liquid in which motile sperm can travel; introducing the semen sample into the inlet reservoir in a manner that does not cause or minimizes the flow of liquid in the device, e.g. while removing a portion of the buffer solution or prefilling the associated chambers and microchannels only to some extent; waiting a predetermined period of time during which motile sperm will pass through the various regions of the device into the outlet chamber; and recovering the accumulated sperm in the outlet reservoir.

According to another aspect of the present invention there is provided a method of fertilising an oocyte in vitro using motile sperm isolated and screened from a semen sample, the method including the steps of: providing an apparatus according to the second aspect of the invention; filling the microchannel or guide structure, and the inlet and outlet reservoirs of the device, with sperm buffer solution as appropriate; filling the binding chamber with a selection particle having a selectivity for binding motile sperm having a predetermined undesired characteristic associated with fertilization of the oocyte, such as expression of an apoptosis-related marker, expression of a sex-selective related marker, or having a permeable membrane, thereby confining the motile sperm bound to the particle in the binding chamber without impeding passage of motile sperm not bound to the selection particle; introducing one or more oocytes into an oocyte holding chamber; introducing a semen sample into an inlet reservoir; waiting for a period of time to allow the screened motile sperm (i.e., motile sperm not bound to the selected particles) to accumulate in the outlet reservoir; allowing motile sperm to enter the oocyte holding chamber from the outlet reservoir; and allowing the selected motile sperm sufficient time to fertilize in combination with an oocyte contained in the oocyte holding compartment.

Aspects related to the "surface profile" of the microchannel walls defined in the present invention, as well as the binding chamber containing selective particles capable of binding to "undesired" sperm, may be added to various sperm separation apparatus described in the patent document US 2015/0140655A1 to Nosrati et al, which discloses the basic principle of providing multiple microchannel paths for guiding sperm based on the wall-walk behavior of the sperm in a confined space, as well as the typical dimensions of such microchannels, the type of anti-reflux chamber and reservoir defined within the microchannel network. Accordingly, the content of U.S. patent document' 655 is incorporated by reference in its entirety for short, cross-reference, but does not therefore suggest that virtually all of the theories presented in this document are supported by evidence.

Other aspects and further features of the various aspects of the invention will become apparent from the description of various non-limiting embodiments of the invention provided herein below with reference to the accompanying drawings.

Drawings

FIG. 1 shows in a highly schematic simplified diagram a sperm screening apparatus in accordance with the present invention and in a generalized diagram a sperm screening step performed during operation of the apparatus when performing a sperm screening method in accordance with another aspect of the present invention;

FIGS. 2A through 2F schematically and in part detail illustrate a first embodiment of a sperm separation and screening apparatus in accordance with the present invention; wherein,

FIG. 2A is a top perspective view of the top housing portion of the device;

FIG. 2B is a bottom perspective view of the top housing portion showing a plurality of spoke-like microchannel sperm conduits extending from a radially outer annular sperm receiving reservoir or chamber to a radially inner annular sperm screening (or binding) chamber, which in turn is contiguous with a substantially cylindrical radially inner motile sperm outlet (or collection) reservoir or chamber;

FIG. 2C is a top perspective view of a lower bottom housing portion joined to a top housing portion containing nine individual oocyte holding structures in a radially central region according to a raised detail view, with the sperm collecting chamber of the top housing portion receiving and in fluid communication with the oocyte holding chamber in a radially centermost position when the two housing portions are brought together as schematically shown in FIG. 2E;

FIG. 2D is an enlarged, fragmentary detail view of the top housing portion showing (i) the radially inner end of the microchannel providing a circular grill boundary structure for the screening (or binding) chamber, (ii) the screening chamber and (iii) the radially inner circular grill boundary structure in the screening chamber formed by a plurality of closely spaced blocking posts, and (iv) the radially inner circular sperm outlet reservoir;

FIG. 2E is a cross-section along the diameter of the device with the upper and lower halves separated from each other;

FIG. 2F is a schematic microscopic illustration depicting the device of FIG. 2B or 2D (an enlarged view) from left to right with (i) a plurality of microchannel sperm conduits in spoke form extending from a radially outer annular sperm receiving reservoir or chamber, (ii) a radially inner annular sperm screening (or binding) chamber, (iii) a radially inner circular grid boundary structure in the screening chamber formed of a plurality of closely spaced blocking posts, and (iv) a radially inner circular sperm outlet reservoir;

figures 3A to 3I show schematically and in part detail another embodiment of a device for sperm separation and screening according to the present invention, the device having 100 or more micro-channels;

FIG. 3A is a bottom perspective view of a top housing portion similar to FIG. 2B, but showing another embodiment of the apparatus according to the present invention showing a plurality of microchannel sperm tubes extending in a radial pattern from a radially outer annular sperm receiving reservoir or chamber to a radially inner annular sperm screening (or binding) chamber, which in turn is contiguous with a radially inner movable sperm outlet (or collection) reservoir or chamber that is also substantially annular;

FIGS. 3B and 3C are different perspective views of the detail highlighted in FIG. 3A, showing an embodiment of a microchannel according to another aspect of the invention, the microchannel including structures in the form of ribs and grooves in the opposing sidewalls, the structures extending over a portion of the length of the microchannel and increasing the number of boundary surfaces;

FIG. 3D illustrates three of the many other configurations of facing surfaces of opposing sidewalls of the microchannel shown in FIGS. 3B and 3C, with an additional increase in the number of boundary surfaces along which motile sperm can travel based on wall-walking by forming a surface structure at each sidewall;

FIG. 3E is a view similar to FIG. 3B, but further enlarged to better illustrate how the microchannel side walls are shaped in the radially outer end region as compared to the adjoining radially inner region, thereby providing additional boundary surface for motile sperm to travel based on wall-swimming behavior;

FIGS. 3F through 3I are schematic top and detailed perspective views of four different embodiments of a radially inner multi-post grid barrier structure forming one of the grid boundaries of an annular bonding (or screening) chamber;

figures 3J to 3L schematically and in part detail show another embodiment of a device for sperm separation and screening according to the present invention having 45 microchannels, wherein the features shown are further (but not to scale) exaggerated to better illustrate the wall structure and wall inserts of this embodiment;

FIG. 4 schematically illustrates an embodiment of a manufacturing process of a device using additive manufacturing techniques, i.e., 3D printing, in accordance with various embodiments of one aspect of the present invention;

FIG. 5A schematically illustrates a preparation step of a selection (binding) particle according to one embodiment of this aspect of the invention, which will be received in the form of protein-coated microbeads and contained in the annular binding chamber of the device when the device of FIGS. 1, 2, 3 and 4 is used;

FIG. 5B is a schematic view of a microscope (enlarged view) depicting the attachment of sperm cells to protein-conjugated microbeads manufactured by the method of FIG. 5A, through protein-protein interactions;

FIG. 6 illustrates method steps for loading selected particles in the form of microbeads into an annular binding chamber of the apparatus of FIGS. 1-4, in accordance with one embodiment of the present invention;

FIG. 7 shows, in an illustrative flow chart, a method according to one embodiment of the invention that first separates motile sperm from a semen sample and then selects motile sperm having a desired fertility index by selectively binding "undesired" motile sperm, allowing the "desired" motile sperm to pass through a binding chamber of a device according to FIGS. 1-4;

FIG. 8 includes three graphs showing performance tests of devices of the present invention, from aspects of sperm recovery, sperm motility, and sperm motility, wherein FIG. 8A is a graph of sperm concentration versus time, and wherein FIG. 8B is a graph of sperm outlet count versus time, and wherein FIG. 8C is a graph of sperm motility and motility versus time, and wherein FIG. 8D is a graph of DNA fragmentation index before and after sperm screening with a device; and

fig. 9A to 9E show schematically and in part detail a second embodiment of a device for sperm separation and screening according to the invention, in comparison to the device of fig. 2A to 2E, having a stacked array of micro-channels extending in a paraboloidal curve between an annular semen collection reservoir located in the upper region or level of the device and an annular binding chamber, the binding chamber being divided into four separate arcuate regions located in the middle lower region or level of the device, having a bottom cover similar to that shown in fig. 2C with an oocyte holding chamber (not shown), the bottom cover being formed in use to hold the bottom of the device therein,

FIG. 9A is a schematic perspective view of the device, including three stacked housing portions, showing microchannels (shown in phantom outline) extending along the height of a substantially cylindrical middle housing portion between upper and base housing portions, which are substantially mirror images of the top housing portion shown in FIG. 2B, but without the microchannels and the bottom housing portion of FIG. 2C;

FIG. 9B is an exploded perspective view of three housing components of the device of FIG. 9A, each manufactured by 3D printing;

FIG. 9C is a side view, partially in section, of the device of FIG. 9A;

FIG. 9D is a bottom view of the top housing portion showing a radially outer annular sperm receiving reservoir or chamber having two adjacent inlets separated by a continuous web connected to a radially inner annular wall surrounding a circular port which in turn communicates with a tubular conduit extending through a mid-height position of the device housing as shown in the translucent portions of FIGS. 9A and 9B and the plan view of FIG. 9E;

FIG. 9E is a top view of the intermediate housing portion with an enlarged schematic detail showing the array of micro-channels in concentric quarter-arc layering regions whereby the micro-channels are separated by the housing web and extend along a substantially parabolic path between the upper ends of the housing portions starting from adjacent semen sample receiving reservoirs of the upper housing portion toward an annular, quarter-arc semen-binding chamber integrally formed in and recessed into the lower end face of the intermediate portion, as schematically shown in FIG. 9F;

Fig. 9F is a bottom view of the intermediate housing part, wherein another enlarged schematic detail is a cross-sectional view taken along the line shown, showing the lower end of the conduit extending between the top and bottom surfaces of the intermediate housing part, the lower end extending to an enlarged, mobile semen outlet chamber in which an oocyte holding chamber is positioned, the oocyte holding chamber being located on the upper surface of the bottom closure plate schematically shown in fig. 9C.

Detailed Description

The following provides a detailed description of embodiments of the invention to assist those skilled in the art in practicing the various aspects of the invention.

Various embodiments of the devices, methods of separating sperm, methods of screening sperm, and methods of fertilization described herein may be practiced in a fertility clinic or research center to study treatment of male infertility In Vitro Fertilization (IVF), intrauterine insemination (IUI), and intracytoplasmic sperm injection (ICSI) applications. The information disclosed herein can be used to isolate motile sperm and subsequently screen sperm for IVF, ICSI, IUI and/or other reproductive treatments. The devices and methods, and variants and methods thereof, may also be used in non-human applications, particularly in agriculture, such as Assisted Reproductive Technology (ART) treatment for breeding of cattle and other animals.

As described in the introductory part of this specification, current sperm screening techniques are very different from in vivo processes within the male and female reproductive systems and may even cause damage to sperm. Of particular concern is DNA integrity, as in both natural and artificial insemination, the chance of conception may be reduced when the DNA Fragmentation Index (DFI) exceeds 30%. Furthermore, unlike somatic cells, the sperm lacks the postspermatogenesis DNA repair mechanism; this weakness makes it urgent to protect the integrity of sperm DNA in ART treatment.

Before describing the preferred embodiment of the present invention in more detail, reference is made to FIG. 1, which schematically illustrates a device 10 for separating motile sperm from a semen sample and subsequently performing a screening procedure to produce motile sperm capable of meeting certain criteria for oocyte in vitro fertilization, and generally illustrates the steps of performing sperm separation and screening during the process using individual structures within the device.

The device 10 comprises the following main functional structures:

(1) A semen sample receiving chamber (also referred to as an inlet reservoir) 12;

(2) A motile sperm directing and separating structure 14 in fluid communication with the inlet reservoir 12, which in the preferred embodiment shown in the other figures is comprised of a plurality of microchannels;

(3) A motile sperm screening chamber 6 (also referred to as a binding chamber) in fluid communication with the separation structure 14, in which chamber a plurality of selection particles 17 bind motile sperm that have passed through the guide structure 14 and have a particular characteristic and are prevented from exiting the chamber 16, whereby the chamber 16 is a grid structure (i.e. a liquid permeable cage) designed to allow unbound motile sperm to swim/pass through the chamber and prevent egress of selection particles 17 and sperm bound to those particles;

(4) A sperm outlet reservoir or chamber 18 in fluid communication with the selection chamber 16, arranged to receive unbound motile sperm that have passed through the binding chamber 16; and

(5) In a preferred embodiment an optional oocyte holding chamber 20, the oocyte holding chamber 20 is in fluid communication with the outlet chamber 18, either arranged within the outlet chamber 18 or as part of the outlet chamber 18, designed for receiving and independently storing oocytes for fertilization by combination with sperm accumulated in the outlet chamber 18.

Fig. 1 also shows a flow chart of a sperm screening process performed within the device 10. The process includes (at step 1) sperm screening based on motility using a radial array of microchannels of a separation structure 14, the separation structure 14 being located between the inlet reservoir 12 and the binding chamber 16. The process further comprises (step 2), using a substrate containing (filled with) bioactive selection particles 17 in a grid-like binding chamber 16, the selection particles 17 being designed to assist in negative screening of sperm by means of sperm trapping (e.g. induced by protein-protein interactions between the sperm plasma membrane and the protein surface of the selection particles). The process also includes (step 3) using a motile sperm accumulation chamber 18 (outlet reservoir) into which unbound motile sperm can enter from the grid-type screening chamber 16. In some embodiments, the outlet reservoir is shaped such that motile sperm cells can be immediately collected. Figure 1 also schematically depicts another process (step 4) wherein motile sperm in the outlet chamber 18 is further moved to an integrated oocyte holding chamber 20 of the device 10, in which chamber an oocyte may be placed, whereby the separated sperm encounters and in combination with the oocyte achieves fertilization, thereby completing at least one in vitro fertilization of the oocyte.

At least some of the embodiments described herein improve the outcome of conception in an in vitro environment by using a device having what is referred to herein as a sperm guidance (and separation) structure 14 (provided by a radial array of microchannels in the illustrated embodiment) and a sperm screening structure 16 (formed by a grid-type sperm binding chamber), the sperm screening structure 16 containing therein particles 17 that exhibit selective affinity for certain types of sperm, as discussed in more detail elsewhere, the particles 17 being configured to bind those sperm that, while having sufficient motility, exhibit "undesired" factors when such sperm is used in an in vitro oocyte fertilization. In the description of these embodiments, the term "undesired" does not merely mean lack of viability or impaired sperm, but rather represents a negative screening criterion that includes sex selectivity among other factors.

As described above, in the binding chamber 16 filled with selectively binding particles 17 (which may be described in more detail with reference to fig. 5 and 6), a "negative" screening process is achieved by binding undesired sperm, thereby effectively immobilizing the particle-bound sperm, restricting it from exiting the binding chamber 16, while other motile sperm without a particular risk factor do not bind to the particles and swim forward through the binding chamber 16 to the device's outlet reservoir 18 without restriction.

Motile sperm without undesirable characteristics accumulate in the outlet chamber 18 for further processing, such as removal from the device and subsequent use in a freezing or in vivo fertilization procedure. However, as mentioned above, in a particularly preferred embodiment, the oocyte holding chamber 20 is present within the device, either as a separate structure or within the outlet chamber 18, such that motile sperm accumulated in the outlet reservoir 18 may be used to effect in vitro fertilization of an oocyte placed in the oocyte holding chamber 20.

Figures 2 and 3 illustrate one embodiment of the device 10 in more detail, wherein figures 3B through 3E and figures 3J through 3L illustrate various alternative embodiments of the specific configuration of the microchannels 38, the radially extending microchannels 38 forming the sperm separating structure 14 of the device, and figures 3F through 3I illustrate various embodiments of the binding chamber 16. The general description is described above with reference to the schematic diagram of fig. 1.

In describing the device components and structures with reference to the drawings, related terms such as "upper," "lower," "bottom," "top," "radially inner," "radially outer," and the like are used with similar expressions and reference planes to facilitate understanding of the relative positions of the components or structures to one another. It should be understood that these terms are not intended to limit any of the features recited herein unless the context indicates otherwise in the actual use of the apparatus. In a similar manner, the same reference numbers are used in the drawings to denote functionally equivalent structures, although these structures may differ between embodiments.

Hereinafter, the main functional structures 12, 14, 16, 18 and 20 of the device 10 will be described first, and then how the device can be used for performing in vitro fertilization of oocytes will be described in detail.

All of these functional structures 12, 14, 16, 18 and 20 are located within a housing 25, which housing 25 is composed of two generally flat cylindrical plates 22, 24 and is formed, in whole or in part, from a suitable hard-cured, thermoset or thermoplastic polymer using additive manufacturing techniques (i.e., 3D printing) into a concave or convex structure. The preferred material for fabrication is a plastic, such as Cyclic Olefin Copolymer (COC), polycarbonate (PCA), or polymethyl methacrylate (PMMA), but the device may also be fabricated from materials such as Polydimethylsiloxane (PDMS) or photopolymer resins. As indicated by the arrows extending between fig. 2A and 2C, the plates 22, 24 are bonded (or otherwise secured) to one another in face-to-face relation to form a flat cylindrical housing 25. Other manufacturing techniques, such as molding, machining, etc., are not precluded, although the internal construction of the various structures in the device 10 is not so preferred.

Referring first to fig. 2B and 3A, the semen sample chamber 12 is embodied in the form of an annular chamber in which an annular channel is recessed into the bottom surface of the upper housing portion 26 (only for the case where there is a small radially extending web separating the channel into two parts), which bottom surface is covered and closed by the top surface of the lower housing portion 22 after bonding together. Two cylindrical semen sample inlets 28 extend into the sample chamber 12 from the upper surface of the upper housing portion 26 on either side of the web. Thus, semen samples may be deposited into the annular chamber 12 through the inlet 28. Depending on the type of semen from which motile sperm is to be separated and screened, the semen sample chamber must be sized to accommodate buffer and semen introduced into the inlet reservoir 12 at a semen volume of between 50 microliters and 4mL, whereas typically semen is introduced at a volume of between 0.5mL and 1 mL.

Referring then to fig. 3A and the enlarged detail of fig. 3B, 3C and 3E, in the illustrated embodiment, the sperm directing (and separating) structure 14 is formed by a plurality of radially extending radial spokes 38, 38', 38", the micro-passageways 38, 38' and 38" extending between the radially outer annular semen sample chamber 12 and the radially inner sperm binding chamber 16 (described in greater detail below) to provide a plurality of travel paths for motile sperm to travel from the semen holding chamber 12 into the sperm binding chamber 16.

It can be seen that the radially inner "boundary" structure of the annular semen sample chamber 12 is defined along its inner periphery by a plurality of spaced radially outer end faces 54 of radially extending webs 40, 50, the webs 40, 50 standing on a concave inner central face of the upper housing portion 26. The plurality of microchannels 38, 38', 38 "of the guiding and separating structure 14 are defined between circumferentially adjacent pairs of these radially extending webs 40, 50 and by upwardly facing portions of the lower (bottom) housing portion 22 and downwardly facing portions of the upper housing portion 26, which portions extend vertically between the webs 40, 50 when the housing portions 22, 26 are assembled, thereby providing a plurality of closed cross-sectional microchannels.

The web 40 is referred to herein as a microchannel side wall 40 and the web 50 is referred to as a microchannel intermediate wall 50. While the sidewall 40 extends the entire radial length/extension of the sperm guiding (and separating) structure 14 between the annular sample chamber 12 and the radially inwardly positioned sperm binding chamber 16, the wall thickness varies as described below, with the intermediate wall 50 extending only partially and only being present in the radially outer region of the microchannel 38 as shown in fig. 3E and described below, and having a constant wall thickness.

The radially extending side walls 40 and the radially outer end surface 54 of the intermediate wall 50 essentially form a circular grid band which only partially physically separates the annular semen sample holding chamber 12 from the guiding structure 14 formed by the plurality of radially extending micro-channels 38, 38', 38 ".

And then for the construction/composition of the motile sperm guiding structure 14. As described above, the plurality of micro-channels 38, 38', 38 "extend in a radially converging manner, starting from the annular semen sample receiving chamber 12 and extending farther radially inward before the center of the upper housing portion 26, thereby defining a cylindrical void and bordering the radially inner end surface 52 of the sidewall 40. Since it is desirable to (i) accommodate as many microchannels 38, 38', 38 "as possible in the guiding structure 14, (ii) radially converge the network of microchannels 38, and (iii) maintain the width and height of the channels approximately constant to simulate the in vivo oviduct environment, thereby triggering or facilitating the natural wall play of sperm, a configuration/arrangement may be selected whereby adjacent microchannels 38', 38" from the semen holding chamber 12 engage each other along their radial paths and form a microchannel 38 having a similar cross-section as the channels 38', 38 "after the transition or merge region. That is, although the number of inlet points to the guide structure 14 at the annular semen holding chamber 12 is greater, the number of outlet points into the radially inner end of the sperm binding chamber 16 is reduced by an amount equivalent to the number of junction points that the channel has along the radial extension of the guide structure 14.

As shown in the enlarged view of fig. 3E, the number of micro-channels 38', 38 "in the radially outer region of the guiding structure 14 is twice as large as the number of micro-channels in the radially inner region, because the adjacent channels 38', 38" merge downstream into a single channel 38 at the junction 39 starting from the annular semen holding chamber 12. It can be seen that the channels 38' and 38', respectively, are delimited by the side walls 40 and share a common intermediate wall 50 therebetween, which intermediate wall 50 has a limited radial length and ends in a junction zone 39, where the channels 38', 38 "merge into a radially inner channel 38.

It should also be noted that while the intermediate wall (or web) 50 has a smooth side facing the microchannels 38', 38", the side wall 40 has two rectangular ribs 44 on opposite sides facing the respective channels 38', 38", the two rectangular ribs 44 extending/protruding perpendicularly from the central web 43 of the side wall 40 and extending parallel to each other radially inward from the annular semen holding chamber 12, as the central web 43 becomes thicker, they become narrower (at 44 '), as shown by the merging junction 39 of fig. 3E. In a top plan view, the side walls 40 taper from the junction 39, at the junction 39 the channels 38', 38 "merge into a radially inner channel 38 towards their radially inner ends. This is best illustrated in the specific embodiment shown in fig. 3J through 3L.

The function of the ribs 44 will be described with reference to fig. 3D, the ribs 44 also being generally referred to herein as "surface features" and extending along a portion of the length of the microchannel 38 in the illustrated embodiment, but in other embodiments the ribs 44 may extend along the entire radial length of the microchannel 38, with fig. 3D showing three of the various other structural forms of the opposing surfaces of the opposing side walls 44 (and intermediate walls 50, if desired) of the microchannels 38, 38', 38 "shown in fig. 3B, 3C and 3E. In comparison with fig. 3B, 3C and 3E, fig. 3D shows a microchannel cross-section embodiment omitting intermediate wall 50, and furthermore, as indicated by the dashed vertical lines, only half of the cross-section of wall 40 is shown.

First, it is apparent that motile sperm swimming in a closed channel having, for example, a rectangular cross-section will be limited by four boundary surfaces. If the cross-section is small enough to simulate the in vivo environment of motile sperm swimming in a substantially stagnant fluid, such as a microchannel forming a rectangular cross-section of, for example, 100 μm by 75 μm, the natural swimming characteristics of sperm will result in wall-swimming behavior along four boundary surfaces as described above. However, if the cross-section is larger, only the more mobile sperm will migrate toward and then swim along the boundary surface. That is, sperm selectivity based upon motility is affected not only by the size of the passage of sperm from the entrance point upstream to the exit point, but also by the number of boundary surfaces present within the passage cross-section and along its length. Thus, according to one aspect of the invention, at least one wall providing a boundary surface has at least one structure selected from the group consisting of recesses 42 and/or protrusions 44 extending along at least a portion of the length of the channel 38 and providing at least one additional boundary surface along which motile sperm may swim toward the binding chamber 16 at the radially inner end of the guide structure 14/microchannel 38 based on wall-walk behavior (as compared to a channel without such a surface structure).

The boundary surface as used or defined herein is defined by the intersection of any two planes that meet in such a way that they form a corner/angle therebetween (the angle need not be stepwise discontinuous but may include rounded edges), the included angle between the individual boundary surfaces preferably being between 30 degrees and 150 degrees.

Then for the right one of the three microchannel cross-sectional embodiments shown in fig. 3D, a portion of the upper housing portion is identified as 26, and as previously described, the side wall 40 is upstanding and integral with the base plate of the upper housing portion 26. The lower housing portion 22 closes the channel 38 at the bottom (otherwise the channel 38 would be open). Thus, the upper housing 26 provides a top boundary of the channel 38, the lower housing portion 22 provides a bottom boundary thereof, and the opposing wall 40 provides a side boundary thereof. Three rectangular cross-section ribs 44 extend/protrude from and are integrally formed on the side 48 of each wall 40 (in the embodiment of fig. 3B and 3C, only two such ribs). Assuming that the sides 48 generally provide a boundary surface along the entire height of the channel 38 for motile sperm to travel based on wall-play, it should be noted that the ribs 44 effectively subdivide or divide the two boundary surfaces along the channel height, however, the upper and lower surfaces of the ribs 44 define additional boundary surfaces 49 along which boundary surfaces 49 motile sperm may travel once entering the channel 38. Thus, the presence of six ribs 44 will add a total of twelve additional boundary surfaces along which motile sperm may swim. It should be noted that the ribs 44 are integrally formed with the side walls 44 and project into the channel 38. It will be apparent, however, that a C-shaped slot 42 is defined between the two ribs 44 and the side 48 of the side wall 40. In other words, the number of surface features that identify the increased boundary surface is dependent upon whether the spacing between the opposing end faces 45 of the ribs 44 or the side faces 48 of the side walls 44 is considered to define the interior width of the channel 38.

The central microchannel cross-sectional view of fig. 3D shows a different embodiment of such surface features present in a microchannel that increases the number of boundary surfaces along which motile sperm travel, wherein the sidewall 44 is stepped 46 along the height of the sidewall 40, wider at the lower shell end and narrower at the web portion at the upper end of the sidewall.

Finally, the rightmost microchannel 30 cross-sectional configuration embodiment illustrates that ten additional boundary surfaces provided by the sidewall surface structural elements may also be provided by the corresponding curved cross-sectional ribs 44 (or complementarily shaped grooves 42 defined between adjacent ribs 44).

It should be appreciated that other embodiments of such additional boundary surfaces may exist. For example, if the cross-section of the channel is rectangular, the top or bottom of the micro-channel 38 will also be the surfaces forming the normal boundary surfaces, and it is conceivable to provide structural elements on these surfaces to increase the number of boundary surfaces. It is also worth noting that the "basic" channel cross-sectional configuration is not limited to rectangular cross-sections. For example, the geometry may be circular, triangular or hexagonal.

In terms of size, the microchannels 38 may be between 2mm and 15mm in length and 50 μm to 5000 μm in height, but typically between 100 μm and 1000 μm. As mentioned above, assuming a rectangular channel geometry (only one possibility), the width of the micro-channel may be between 50 and 5000 μm, typically between 50 and 250 μm. The free space (or distance) between the oppositely disposed (or facing) surface structure elements 42, 44 should also be selected to be of a size that facilitates motile sperm travel to the additional boundary surface 49 disposed at these structures, and may be set to a minimum of 50 μm (which may be higher). Eventually, the choice of the maximum and minimum dimensions of the micro-channel will also be partially influenced by the sperm phenotype to be isolated and screened from the semen sample, as well as the semen source (e.g. bovine, human, canine, etc.). As noted above, the width of the microchannels may also be non-uniform throughout the length of the device, creating non-uniform boundaries or boundaries of convergence and divergence for sperm to travel along.

Those skilled in the art will appreciate that the specific geometric arrangement, size, or configuration of the microchannel paths may vary. Although separation of sperm cells from human and animal semen samples using devices having 5 to 5000 microchannels has been observed to exhibit acceptable performance, the specific performance may vary from sample to sample being processed. In this case, one or more of these factors may be varied depending on the volume, concentration and viscosity of the sample to improve the sperm separation process.

The sperm binding chamber 16 will next be described with reference to figures 2B, 2D, and 3F to 3J. The binding chamber 16 is substantially annular in configuration, through which liquid can pass from the inlet to the outlet side, and the binding chamber 16 is disposed downstream of the guide structure 14 (when viewed in terms of the motile sperm traveling through the various functional structures of the device 10) and upstream of the outlet reservoir 18. It is primarily designed to receive and contain a large number of selection particles 17 therein, which selection particles 17 have a combined selectivity for motile sperm having passed through the guiding structure 14, having a predetermined, undesired marking. The binding chamber 16 and/or the selection particles 17 are also designed to substantially confine sperm bound to the particles in the chamber 16 and prevent such sperm from traveling further toward the outlet reservoir 18 without impeding the passage of motile sperm that are not bound to the selection particles. The binding chamber 16 filled with binding particles 17 can be regarded as a filter, which serves as a dual function, firstly as an additional physical barrier similar to the cumulus cell mass through which sperm must pass during fertilization of an egg in a natural body, and secondly for capturing DNA (or other) damaged sperm.

The radially inner ends 52 of the microchannels 38 of the guide structure 14 provide a rounded grid boundary structure 34 radially outward of the chamber 16, as shown in fig. 2D, which is an enlarged, protruding detail of the top housing portion 26 shown in fig. 2B, as also seen in fig. 3F-3I. In other words, the inlet side of the binding chamber 16 is bounded or defined by the downstream ends of the walls 44 of the plurality of microchannels 38 (thereby providing a grille-inlet structure). The outlet side at the radially inner end of the annular bonding chamber 16 is defined by a circular array of closely spaced cylinders or posts 56, the cylinders or posts 56 being arranged in a pattern defining a grid outlet structure 36. Thus, the binding chamber 16 may be suitably described as a "cage structure" into which motile sperm may enter from the inlet-side plurality of microchannels 38 and from which particle-bound sperm are prevented from exiting at the outlet-side by a plurality of blocking posts 56, the blocking posts 56 having a spacing 58 between circumferentially adjacent posts 56, the spacing 58 being sufficiently small to accommodate (i.e., prevent passage of) the sperm-binding selection particles 17 accommodated in the chamber 16.

As can be seen more clearly in fig. 2D, the radially inner end portions 52 of the walls 40 separate the microchannels 38 adjacent the binding chamber 16, which are configured such that (i) the microchannels 38 narrow toward their respective discharge ends, and (ii) together define a grid boundary structure capable of inhibiting the re-entry of motile sperm that have exited the microchannels 38 into the microchannels 38. In other words, the end cross-section of the microchannel 38 narrows and provides an inner surface along which motile sperm can travel out of the microchannel 38 into the binding chamber 16 in a preferred converging path, such that it is substantially (but not absolutely) impossible for motile sperm to reenter the microchannel 38 due to the narrowing of the cross-section. For example, assuming that the microchannel side walls are substantially rectangular in vertical cross section, the thickness of the ends widens, whereby the microchannels are substantially triangular in plan view of the wall ends.

As regards the posts 56, they may be integrally formed with the upper housing portion 26 by additive manufacturing techniques. Although a post 56 having a cylindrical configuration as shown in fig. 2D and 3F/3G may be used that functions similarly to the shape of the end 52 of the microchannel defining sidewall 40, in a preferred embodiment, a plurality of blocking posts 56 are shaped and/or arranged relative to one another in a manner that may limit the retrieval of motile sperm that have exited the binding chamber 16 back into the binding chamber. The gap (spacing) 58 between the blocking posts 56 needs to be wide enough to allow unbound motile sperm to pass through the binding chamber 16 toward the radially inward outlet reservoir 18. In other words, the grating structure 36 provided by the circular array of closely spaced pillars 56 provides a boundary structure 36 between the junction chamber 16 and the outlet chamber 18 that does not obstruct the passage of liquid. In a preferred embodiment, the gap 58 between adjacent blocking posts 56 is between 10 and 100 μm. Also, the cross-section of the end of the microchannel 38 needs to be approximately small to accommodate the selection particles 17 in the binding chamber 16.

The shape of the blocking post 56 is not limited to any particular geometry, but is preferably a shape that is capable of directing sperm toward the outlet reservoir 18 and preventing sperm from re-entering the binding chamber 16. Suitable cross-sectional shapes include trapezoids, semi-circles, triangles, chevrons, semi-circles, and crescent shapes. An example of a crescent cross-sectional shape is shown in fig. 3I.

As shown in fig. 2A and 2D, the selection (or binding) particles 17 may be filled into and removed from the binding chamber 16 through an inlet 32 in the upper housing plate 26 that is connected to the chamber 16. The upper aperture of the inlet 32 of the binding chamber outside the device may be between 0.5 and 5mm and the lower aperture of the inlet may be between 0.5 and 5 mm. The inlet may be funnel-shaped or cylindrical, but is preferably funnel-shaped, and more preferably also gradual, to enable better flow of the selected particles 17 into the binding chamber 16 and to reduce the likelihood of clogging during filling.

Binding chamber 16 in device 10 enables sperm expressing predetermined, undesired biomarkers on the surface of the sperm's outer membrane to be bound in binding chamber 16. This may be achieved, for example, by providing selective particles 17 coated with ligands that are capable of binding to and capturing sperm expressing these predetermined, undesired biomarkers. These markers can be expressed at any point on the sperm anatomy, such as the head, middle segment (mitochondria housing sperm), and tail. The term "surface marker" refers to a protein that, for example, migrates to the outer layer of the plasma membrane of a sperm and interacts with the local environment of the sperm.

One problem with the sperm cell separation method used in prior art document US'655 is the lack of selectivity for sperm cells having characteristics other than motility based. This problem is solved by the device 10, which device 10 comprises a binding chamber 16 in which selective particles 17 are accommodated. The selection particles 17 may be in the form of pellets, preferably microspheres or microbeads. Microbeads are formulated/manufactured to enable molecular screening of sperm cells based on the presence of conjugated ligands on the surface of the microbeads. Conjugated ligands comprising proteins capable of attaching to labels selected from lectins (e.g. peanut lectin and pea lectin), antibodies and annexin A5 (preferably annexin A5 the ligand may also be a drug, including imidazoquinoline amines (e.g. resiquimod, imiquimod and gardelmod).

These conjugated ligands bind to motile sperm expressing the label through chemical bonds, for example, through interaction with a spermatogenic protein-protein. In a preferred embodiment, the selection particles 17 may be coated with annexin A5, annexin A5 being a cellular protein of the annexin group. Annexin A5 is capable of binding to the outer membrane phosphatidylserine expressed on the surface of motile sperm passing through the device. In other embodiments, the selection particles may be coated with antibodies in order to target specific antigens expressed on the surface of motile sperm.

In some embodiments, the diameter of the selected particles 17 used may be between 50 μm and 300 μm, which will also determine the spacing 59 between the blocking posts 56 in the radially inner grid circular boundary structure 36 of the binding chamber 16, as well as the maximum of the height/width of the micro-channels 38 providing the radially outer grid circular boundary structure 34 of the binding chamber 16. Preferably, the particles are chosen to be spherical with a diameter between 150 μm and 200 μm. The gap 59 between the pillars 56 is between 6 μm and 100 μm. The size and diameter of these columns are factors that determine the desired distance between them and the size of the microbeads used. The shape, size and bulk density of the particles 17 in the binding chamber 16 are selected so as not to substantially impede the passage of motile sperm exhibiting undesired indicia upon exiting the binding chamber 16, but need to be large enough to adequately contain motile sperm exhibiting undesired characteristics in the binding chamber 16.

The ligand may be "attached" to the surface of the selection particles 17 (prior to injection into the device 10) using various methods known in the art, and in the present case, the preferred form is achieved by a polymer coating method. The ligand polymer typically consists of a metal complex having affinity for the protein and the surface of the carboxylated microbead. In some embodiments, the strong polyvalence bond formed with the carboxyl group of the electron enables the polymer adhesion layer to remain stably on the surface of the selection particle 17. In one embodiment, to immobilize the antibody protein, the polymer layer utilizes a ligand to bind the Ab Fc domain.

The selection particles 17 are typically introduced into the binding chamber 16 and suspended in a solution (carrier liquid) suitable for anchoring to one or more molecular entities on the surface of the selection particles 17. Typically, the binding chamber 16 is designed to hold 10 to 1000. Mu.l of solution, including microbeads 17. The distance between the radially outer and radially inner grill circular boundary structures 34, 36 of the bonding chamber 16 may be between 0.1 and 2 mm.

The sperm outlet reservoir 20 is described below as being located radially inward of the radially inner grill circular boundary structure 36 of the binding chamber 16. Essentially, it is a cylindrical void in the center of the housing 25, surrounded by a cylindrical array of posts 56, with the inlet 30 formed through the material thereof, and the plate-like upper housing portion 26 is formed along the central axis of the flat cylindrical housing 25. The upper surface of the lower housing portion 22 provides a bottom surface of the reservoir 20. Preferably, a chemoattractant is introduced into the outlet reservoir 20 through inlet 30, such as progesterone, RANTES, neotame, p-tert-butylphenylpropionaldehyde, atrial natriuretic peptide, hyaluronic acid, or a combination thereof, which aids in drawing motile sperm that are not bound to the selection particles 17 out of the binding chamber 16.

In the embodiment shown in figures 2 and 3, a plurality (nine in this example) of individual oocyte holding structures 24 are co-located in a 3 x 3 square array within the central region of the sperm outlet chamber 18, each oocyte holding structure 24 being adapted to receive and hold an oocyte for fertilization in combination with the oocyte using motile sperm accumulated in the outlet chamber 18. The preservation structure 18 is advantageously manufactured using 3D printing during the manufacturing of the lower housing portion 22. The holding structure 24 may be said to collectively define the oocyte holding chamber 20.

Each holding structure 24 is formed of eight arcuate wall sections or posts arranged in a circular, cylindrical fashion with a small spacing therebetween, but large enough to allow motile sperm to enter the interior of the oocyte holding compartment. Although eight arcuate wall sections are shown in this embodiment, in another preferred embodiment the oocyte holding chamber includes six columns. Also, the appropriate spacing between the wall portions is selected to limit the passage of oocytes contained therein through the gap. In general, a pitch of 10 μm to 200 μm is sufficient, but preferably 30 μm to 50 μm. It should be noted that the oocyte holding structure is located below the inlet 30 through which the oocyte is to be inserted into the structure, and the height of the structure may be the same as or greater than the height of the micro-channel. However, in a preferred embodiment, the height of the holding structure 24 and the height of the extended sperm outlet chamber itself is between 0.5 and 5 mm. The number of oocyte holding chambers may vary depending on the quality of the clinical specimen and the time required to store the oocyte throughout the In Vitro Fertilization (IVF) cycle. The distance between the oocyte storage structures 24 is typically between 0.1mm and 1 mm.

It should also be noted, therefore, that the sperm outlet chamber 18 and the oocyte holding structure 24/oocyte holding chamber 20 are in fluid communication whereby the outlet chamber 18 is filled with a suitable buffer liquid of a suitable viscosity (as is the case with the micro-channel 38 of the directing structure 14 and the semen holding chamber 12) in addition to the chemoattractant, to create a virtually non-flowing stagnant liquid, allowing sperm to travel from the semen holding chamber 12, through the directing structure 14 and the combining chamber 16, through various separation and screening regions within the device 10, into the sperm holding chamber 18 and the oocyte holding structure 24. Preferably, the sperm buffer is a hydroxyethylpiperazine ethylene sulfate (HEPES) based buffer or sperm cryoprotectant medium.

Before starting with a description of the preferred method of separating and screening sperm from a semen sample using the apparatus 10 described above, and the method of in vitro fertilization using the apparatus, another embodiment of such an apparatus 100 will be described very briefly with reference to the series of drawings included in fig. 9.

Fig. 9A to 9E schematically and in part in detail show a second embodiment of an apparatus 100 for sperm separation and screening in accordance with the present invention. In contrast to the device of fig. 2A to 3J, the device 100 is made up of three stacked housing components 126, 123 and 122, which are also manufactured using 3D printing as previously described.

The device 100 has a plurality of concentrically stacked microchannel layers 114 (similar to the layers in onions, see fig. 9E and 9F, rather than a planar layer with a single radially extending microchannel) to provide the sperm guiding structure 14, the microchannel layers 114 extending in a parabolic curved pattern in a cylindrical intermediate housing portion 123. The layer 114 extends between an annular semen collection reservoir 112 and an annular bonding chamber 116, the annular semen collection reservoir 112 being located/defined at an upper region or level of the device 100, the annular semen collection reservoir 112 being provided at an upper circular plate-like housing portion 126, the annular bonding chamber 116 being divided into four separate arcuate regions formed at a lower region or level of a cylindrical intermediate housing portion 123 (see fig. 9F). A cylindrical sperm outlet holding chamber 118 is also formed in the intermediate housing portion 123 below the binding chamber. A bottom circular baseplate (bottom housing part) 122, similar to that shown in figure 2C, carries an oocyte holding structure (shown schematically at 124 only in figure 9C) which is located in the sperm outlet chamber 118 (and thus has the dual function previously mentioned) and provides the bottom of the device 100 on which the oocyte holding structure is located in use.

Fig. 9D shows the top housing portion 126 in a bottom view to show the radially outer annular sperm receiving reservoir or chamber 112 having two adjacent inlets 128, the inlets 128 being separated by a continuous web 129, the web 129 being connected to a radially inner annular web 131 surrounding a circular port 130, the circular port 130 in turn being in communication with a tubular conduit 132, the tubular conduit 132 extending through the height of the middle 123 of the housing 125 of the device 100, as shown in the translucent portions of fig. 9A and 9B and the plan view of fig. 9E. The conduit 132 communicates with the sperm outlet chamber and allows motile sperm to be withdrawn from the chamber when desired.

In fig. 9A and 9C, the six-layer structure 114 is shown in dashed outline extending along the height of the intermediate housing portion 123, each layer including a plurality of circumferentially adjacent microchannels 138, while fig. 9E and its enlarged isometric detail and enlarged detailed side view of fig. 9F more clearly illustrate the geometry and layout of the curved microchannel layer 114. It will be appreciated that the upper ends of the six microchannel layers 114 all fall within the outline (or boundary) of the annular semen sample receiving chamber 112, such that sperm dispensed within the chamber 112 may enter each microchannel 138, advancing it toward the binding chamber 116 at the lower elevation of the device 100.

The configuration of the microchannels 138 themselves is similar to the microchannels 38, 38', 38 "described above with reference to fig. 2 and 3.

The device 100 is characterized by different functional units/structures 112, 114, 116 and 118/120 located at different heights (also referred to as areas) within the device housing 125. As shown, the device 100 has within its cylindrical housing 125 a first distinct region comprising the inlet reservoir 112, a second region comprising the micro-channel of the guiding structure 114, a third region comprising the binding chamber 116, a fourth region comprising the outlet reservoir 118 and a fifth region 120 comprising the oocyte holding chamber, note that the third, fourth and fifth regions may preferably be arranged in a common region, meaning that the oocyte holding chamber will be within the range of the outlet reservoir, and the outlet reservoir 116 (as in the case of the embodiment shown in fig. 2 and 3) may be at the same height, but radially outwards surrounding the outlet chamber 118 in an annular manner and have a similar boundary structure as described before. For example, when the outlet reservoir is designed as a substantially cylindrical disc chamber, one or more separate wall structures may be present within the chamber, each wall structure being adapted to receive an oocyte, and motile sperm being able to pass through to contact the oocyte.

It should also be appreciated that while fig. 9A-9F illustrate layers 114 extending within a truncated parabolic (or similar) envelope, the multi-layer 3D channel array may be defined by a truncated conical envelope having straight micro-channels (rather than vertically curved micro-channels in a plane perpendicular to the horizontal plane). More closely, as in the first embodiment of the device 10, the regions (whether at the same or different elevations) are in fluid communication with each other, the buffer solution fills the various structures to enable (a) motile sperm to be separated from the semen sample within the microchannel or guide structure 114, (b) motile sperm that have entered the binding chamber 116 and have a predetermined characteristic to be separated from motile sperm bound to the selection particles 117 and thus retained in the binding chamber 116, and (c) unbound motile sperm to reach the outlet reservoir 118. The (unbound) sperm so screened may then be removed from the outlet reservoir 118 and/or allowed to "swim" into the oocyte holding chamber 120 where fertilization is effected in combination with one or more oocytes received therein.

As already indicated with reference to fig. 1, the present invention also provides a method for separating and/or screening motile sperm from a semen sample, comprising the steps of: providing a device 10, 100 according to the first or second embodiment described above; filling the microchannels 38, 138 of the guiding structure 14, 114 and the semen sample inlet reservoir 12, 112 and motile sperm outlet reservoir 18, 118 of the device with sperm buffer solution to create a substantially non-flowing liquid environment within the various functional regions/structures of the device for motile sperm to travel therein; introducing a semen sample into the inlet reservoir 12, 112; waiting for a predetermined period of time; and withdrawing sperm accumulated in the outlet reservoirs 118, 118 for further processing or use.

The solution containing the selection particles 17 is injected into the binding chamber 16, 116 through a dedicated inlet before the sperm buffer or semen is introduced into the device. The solution containing the selection particles 17 is introduced into the device by a continuous slow injection and the reservoir is filled to the desired percentage by this injection.

The device itself may be evacuated and then pre-filled with a buffer solution (to maintain sperm motility and viability) that may be pre-heated to a desired temperature, such as room temperature or near core body temperature, to simulate an in vivo environment.

During the sperm separation and screening process, the device may be placed under controlled atmospheric conditions at room temperature. For example, after injection of a semen sample, the levels of oxygen and carbon dioxide in the atmosphere and humidity may be preset to optimal levels to allow sperm to travel through the micro-channel to the collection chamber outlet.

The method may further comprise covering the sperm outlet and the selective particle (microbead) inlet with a closure prior to injecting the semen sample into the annular holding chamber, and exposing only the outlet reservoir prior to placing the oocyte. For example, the closure may be a lid, tape, a special plug or snap-in lid.

In another broad aspect, in at least one embodiment described herein, there is provided a method of oocyte in vitro fertilization using separated and screened motile sperm obtained from a semen sample, comprising the steps of: providing an apparatus according to the present invention; filling the microchannel or guide structure (as the case may be) with sperm buffer solution, as well as the inlet reservoir and outlet reservoir of the device; filling the binding chamber with a selection particle having a selectivity for binding motile sperm having a predetermined undesired characteristic associated with fertilization of the oocyte, such as expression of an apoptosis-related marker, expression of a sex-selective related marker, or having a permeable membrane, thereby confining the motile sperm bound to the particle in the binding chamber without impeding passage of motile sperm not bound to the selection particle; introducing the oocyte into an oocyte holding chamber; introducing a semen sample into an inlet reservoir; waiting a period of time to allow the screened motile sperm to accumulate in the outlet reservoir; introducing motile sperm from the outlet reservoir into the oocyte chamber; and allowing the selected motile sperm sufficient time to fertilize an oocyte contained in the oocyte holding compartment.

In some embodiments, the bare oocyte is directly aspirated into the oocyte holding chamber/holding structure 24, 124 of the device 10, 100 with a pipette.

The placement of the oocyte is preferably performed after the device is filled with a solution comprising the selection particles (if used), the sperm buffer, and the semen sample. After oocyte placement, the outlet reservoir may optionally be sealed with mineral oil and the oocyte holding chamber covered.

As previously described, the chemoattractant may assist in directing unbound sperm out of the selection particle tolerance/binding chamber 16, 116 and into the oocyte storage region of the device. The chemoattractant may be added as a concentrated drop to the middle of the outlet, either before or after oocyte placement. This can cause the chemoattractant to diffuse out of the gradient, providing the sperm with a chemical gradient that follows its travel.

There are a number of methods that can be used to manufacture the device according to the invention, and figure 4 schematically illustrates one potential manufacturing method using 3D printing techniques. This process includes 3D printing, cleaning of the resin material, curing of the resin material, bonding of the relevant housing parts 22, 26, 122, 123, 126 of the device 10, 100 by double sided tape (if it is desired that the device be detachable) or with permanent adhesive when pressure is applied.

Fig. 5 shows an example of a production process of protein-coated microbeads used as selection particles 17. As shown in fig. 5, in a first microfluidic mixing module, microbeads (in this case polystyrene cores) are mixed with a polymer solution. After a subsequent washing step, the conjugated microbeads are added to the second module assembly along with the antibody or protein solution in a second mixing module to produce fully coated microbeads. The antibody-conjugated microbeads are now capable of binding externally expressed surface markers, such as those found on sperm. As shown in fig. 5B, sperm cells are firmly attached to protein-conjugated microbeads by protein-protein interactions.

Fig. 6 schematically illustrates an exemplary method for loading microbeads (selected particles) into the binding chamber (reservoir) 16 of the device 10. Briefly, the method includes steps such as resuspending the bead solution, pipetting the solution with a pipette, and injecting the solution into the binding chamber inlet (in a smooth manner). The volume of the bead reservoir can be tailored to accommodate different solution volumes. For example, 200 to 300. Mu.l of the solution may be injected. In addition, the volume of the reservoir can be adjusted to ensure that the microbeads adequately fill the area of the chamber. The concentration of beads used in the bead solution depends on the size of the beads. The concentration and volume of the solution may be adjusted according to the desired sample and size. One example of this process includes injecting a high concentration of microbeads into the chamber to completely fill the chamber or reservoir. The end of the flow chart also shows a clear image of the microbead reservoir 16 loaded with protein-coated microbeads 17 and the boundary structures 34 and 36 described with reference to fig. 2 and 3.

Fig. 7 is a schematic flow chart illustrating a process for performing a method of separating sperm and fertilising an oocyte using the apparatus 10 described above. This involves filling the device 10 with sperm buffer in a vacuum chamber, loading a semen sample into the semen reservoir 12, dripping a chemoattractant into the central outlet 18 (the function of which is seen in the accompanying inset), and finally placing the oocyte into a holding structure 24 located within the oocyte holding chamber 20 surrounded by the annular sperm outlet chamber 18. As previously described, the addition of a chemoattractant may enhance the directing of sperm travel to the oocyte holding compartment 20 by chemical gradients, causing sperm to be redirected in response to changes in concentration.

Figures 8A, 8B and 8C show performance measurements (performance in terms of sperm recovery, motility and vitality) using the device 10 manufactured according to the illustrations of figures 2 and 3, according to an exemplary embodiment of the present invention. The concentration of sperm recovered from the sperm collection chamber of the device is typically a function of time, the length of the sperm travel path from the semen holding chamber through the directing structure, the binding chamber, and finally into the sperm collection (or outlet) chamber, and the starting concentration of the semen sample. In the lead configuration, sperm are separated primarily based on motility, and in the binding chamber, negative screening is performed by binding and retaining "defective" or "undesired" sperm in the binding chamber.

The average concentration (mL) of sperm recovery is directly related to the initial concentration of the original semen. Even with different samples from the same person, the concentration of the person's original semen may vary greatly. Sperm recovery increases with increasing sample incubation time within the device and results in sperm concentrations higher than those required for conventional in vitro fertilization.

In all variants, the motility of sperm at the outlet was higher than 95%, while the viability was near 100%, as shown in figure 8B, but prolonged sperm placement lost energy. This shows a significant increase in motility and vitality of the recovered sperm population compared to the original semen; the activity exceeded 95% and was always close to 100% in multiple tests. Due to the mixed screening mechanism, only highly mobile and healthy sperm cells can be recovered from the device. The percentage of viable sperm cells in the overall device was high over time, which also indicates that the 3D printed material used in this example was biocompatible.

Fig. 8D shows the results of DNA fragmentation of untreated primary sperm compared to sperm screened using a device according to the invention. The results showed that sperm screened by the device exhibited lower DFI than untreated sperm, indicating that the device was able to screen for higher quality sperm with greater chance of conception.

The above embodiments are intended to be described by way of example only and not by way of limitation. For example, while the illustrated device incorporates one or two inlet reservoirs, it is possible to incorporate more inlet reservoirs, multiple different oocyte holding chambers, and different numbers of layers within the stacked configuration. Furthermore, while various embodiments of devices and methods for separating sperm are presented, these embodiments may also be used with other eukaryotic or prokaryotic cells having autonomous motility or motility.

It must also be noted that, as used in the specification and the claims, the singular forms "a," "an," and "the" include plural referents unless otherwise specified. Thus, for example, a "channel inlet" may include more than one channel inlet.

Throughout this specification the use of the terms "comprises" or "comprising" or grammatical variants thereof shall be taken to specify the presence of stated features, integers, steps or components but do not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof that are not specifically mentioned.

The term "about" as used herein is to be understood to be within normal tolerances in the art, e.g., within two standard deviations of the mean, unless specified or apparent from the context. "about" is generally understood to be within a typical% of a measured value or specified value. Unless the context indicates otherwise, all numerical values set forth in the specification and claims may be modified by the term "about.

The ranges provided herein are to be understood as shorthand for all values that fall within the range. For example, a range of 1 to 50 should be understood to include any number, combination of numbers, or subranges from the group of 1, 2, 3, 4, etc. to 48, 49, or 50.

Reference is made to:

aitken, r.j., bronson, r., smith, t.b., and De iulies, g.n. "source and meaning of DNA damage in human sperm: diagnostic strategies and scarecrow spurious evaluations ". Mol. Hum. Reprod.19,475-485 (2013).

Aitken,R.J.,Bronson,R.,Smith,T.B.&De Iuliis,G.N.The source and significance of dna damage in human spermatozoa；a commentary on diagnostic strategies and straw man fallacies.Mol.Hum.Reprod.19,475–485(2013).

Davies, m.j. Et al, "reproduction technique and risk of birth defects". Obstet. Gynecol. Surv.67,527-528 (2012).

Davies,M.J.et al.Reproductive technologies and the risk of birth defects.Obstet.Gynecol. Surv.67,527–528(2012).

Esteves, S.C.2015, "clinical management of non-obstructive azoospermia sterile males", J.Asian male science, vol.17, 3 rd, pages 459-70.

Esteves,S.C.2015,‘Clinical management of infertile men with nonobstructive azoospermia’, Asian Journal of Andrology,vol.17,no.3,pp.459–70.

Kashaninejad, n., shiddiky, m.j.a. & Nguyen, n. -t. "progress of assisted reproduction technology based on microfluidics: from sperm sorter to on-chip reproductive system ". Adv.biosystem.2, 1700197 (2018).

Kashaninejad,N.,Shiddiky,M.J.A.&Nguyen,N.-T.Advances in Microfluidics-Based Assisted Reproductive Technology:From Sperm Sorter to Reproductive System-on-a-Chip.Adv.Biosyst.2,1700197(2018).

Mcdowell, S.et al, "advanced sperm screening technique for assisted reproduction". Cochrane library.1-40 (2014) doi:10.1002/14651858.CD010461.pub2.Www.Cochrane ibrary.com

Mcdowell,S.et al.Advanced sperm selection techniques for assisted reproduction.Cochrane Libr.1–40(2014).doi:10.1002/14651858.CD010461.pub2.www.cochranelibrary.com

Muratori, M, tarozzi, N, cambi, M, boni, L, iorio, A.L., passaro, C, luppino, B, nadalini, M, marchiani, S, tamburino, L, forti, G, maggi, M, baldi, E, borini, A.2016, "change in DNA fragmentation level during density gradient sperm screening of assisted reproductive technologies", "journal of medicine (U.S.), volume 95, stage 20, pages 1-9.

Muratori,M.,Tarozzi,N.,Cambi,M.,Boni,L.,Iorio,A.L.,Passaro,C.,Luppino,B.,Nadalini, M.,Marchiani,S.,Tamburrino,L.,Forti,G.,Maggi,M.,Baldi,E.&Borini,A.2016,‘Variation of DNA Fragmentation Levels during Density Gradient Sperm Selection for Assisted Reproduction Techniques’,Medicine(United States),vol.95,no.20,pp.1–9.

Muratori, M., tarozzi, N., carpentiero, F., danti, S., perrone, F.M., cambi, M., casini, A, azzari, C, boni, L., maggi, M., borini, A. & Baldi, E.2019, "sperm screening by Density gradient centrifugation and swimming: effect on fragmentation of live sperm DNA "," science report ", volume 9, phase 1, pages 1-12.

Muratori,M.,Tarozzi,N.,Carpentiero,F.,Danti,S.,Perrone,F.M.,Cambi,M.,Casini,A., Azzari,C.,Boni,L.,Maggi,M.,Borini,A.&Baldi,E.2019,‘Sperm selection with density gradient centrifugation and swim up:effect on DNA fragmentation in viable spermatozoa’,Scientific Reports,vol.9,no.1,pp.1–12.

Oleszczuk, K., augustinsson, L., bayat, N., giwercman, A. And Bungam, M. "prevalence of high DNA fragmentation index in male partners of infertile couples of unknown origin". In Men science 1,357-360 (2013).

Oleszczuk,K.,Augustinsson,L.,Bayat,N.,Giwercman,A.&Bungum,M.Prevalence of high DNA fragmentation index in male partners of unexplained infertile couples.Andrology 1,357–360 (2013).

Oseguera-Lwave, I., ruiz-Di az, S., ramos-Ibeas, P.and Perez-Cerezales, S. "New sperm screening techniques for improving the results of in vitro fertilization and intracytoplasmic sperm injection". Cell and developmental biology front (2019).

Oseguera-López,I.,Ruiz-Díaz,S.,Ramos-Ibeas,P.&Pérez-Cerezales,S.Novel Techniques of Sperm Selection for Improving IVF and ICSI Outcomes.Front.Cell Dev.Biol.7,(2019).

Robinson, L. et al, "effect of sperm DNA fragmentation on convection yield: systematic review and meta analysis ". Human reproduction 27,2908-2917 (2012).

Robinson,L.et al.The effect of sperm DNA fragmentation on miscarriage rates:A systematic review and meta-analysis.Hum.Reprod.27,2908–2917(2012).

Rappa, k.l. et al, "sperm processing for advanced reproductive technology: current state of the art "Biotechnol. Adv.34,578-587 (2016).

Rappa,K.L.et al.Sperm processing for advanced reproductive technologies:Where are we todayBiotechnol.Adv.34,578–587(2016).

Sakkas, d., ramalingam, m., garrido, n., and barrett, c.l.r. "sperm selection in natural conception: what we can learn from the natural mother to improve the outcome of assisted reproduction? "., human reproduction, update 21, 711-26 (2015).

Sakkas,D.,Ramalingam,M.,Garrido,N.&Barratt,C.L.R.Sperm selection in natural conception:what can we learn from Mother Nature to improve assisted reproduction outcomesHum.Reprod.Update 21,711–26(2015).

Wade, J.J., macLachlan, V. And Kovacs, G. "success rate of in vitro fertilization has improved significantly over the past decade". Aust. New Zeal. J. Obstet. Gynaecol.55,473-476 (2015).

Wade,J.J.,MacLachlan,V.&Kovacs,G.The success rate of IVF has significantly improved over the last decade.Aust.New Zeal.J.Obstet.Gynaecol.55,473–476(2015).

The world health organization "examination and treatment of human semen" 286 (2010) doi 10.1038/aja 2008.57

World Health Organisation.Examination and processing of human semen.World Health 286 (2010).doi:10.1038/aja.2008.57.

Claims

1. A device for separating motile sperm from a semen sample, the device comprising:

an inlet reservoir for receiving a semen sample;

a guide structure formed from a plurality of micro-channels disposed in fluid communication with and between the inlet reservoir and the binding chamber, the micro-channels including at least one wall defining a boundary surface; wherein the at least one wall has at least one surface structure having one or more recesses and protrusions extending over at least a portion of the length of the microchannel and providing at least one additional boundary surface along which motile sperm travel toward the binding chamber;

wherein the recess comprises at least one groove extending along part or all of the length of the microchannel along one or more axially straight, saw tooth, wavy, or a combination thereof, wherein the depth of the groove is constant or variable, and the cross-section of the groove defines at least one sharp or rounded edge, the surface structure being disposed in and extending along the wall;

Wherein the protrusion comprises at least one rib extending along a portion or the entire length of the microchannel along one or more paths that are axially straight, zigzag, wavy, or a combination thereof, wherein the height of the rib is constant or variable, and the cross-section of the rib defines at least one sharp or rounded edge, the surface structure being disposed in and extending along the wall;

a binding chamber disposed in downstream fluid communication with the inlet reservoir via the guide structure, the binding chamber in downstream fluid communication with the outlet reservoir; wherein the binding chamber is arranged for receiving a selection particle having a selectivity for binding motile sperm that (i) has passed through the guiding structure, and (ii) has a predetermined, undesired marker, the binding chamber and/or the selection particle being designed to confine sperm bound to the selection particle in the binding chamber; wherein the binding chamber is bounded on an inlet side by downstream ends of the plurality of microchannels and on an outlet side by a plurality of blocking posts to define a cage-like structure;

A grid boundary structure, the grid boundary structure being formed of a plurality of closely spaced blocking posts, into which active sperm are disposed from a plurality of microchannels on an inlet side and from which sperm bound to selected particles are prevented from flowing out on an outlet side by a plurality of blocking posts;

an outlet reservoir for collecting motile sperm separated from the sample; wherein the end of the microchannel at the outlet reservoir or the binding chamber is shaped to inhibit motile sperm from moving back into the microchannel, and/or wherein the plurality of blocking posts are shaped and/or arranged relative to one another to inhibit motile sperm that has exited the binding chamber that has not bound the particles from returning into the binding chamber.

2. The device of claim 1, wherein the at least one wall comprises one or both of the groove and the rib arranged in a manner including a rectangular, semi-circular, or stepped sidewall configuration along a length or width of the microchannel.

3. The device of claim 1, wherein the selection particles are contained in the binding chamber, the selection particles comprising a bead conjugated with a ligand comprising a protein attached to a label selected from the group consisting of: lectin, anti-CRISP, annexin A5, anti-chemokine receptor antibody and anti-CD 63, anti-CD 9, ALIX or TSG101; or comprises a drug selected from the group consisting of resiquimod, imiquimod, and gardimmod.

4. The device of claim 1, wherein the gap between adjacent ones of the occlusion posts is wide enough to allow motile sperm to pass therethrough and small enough to prevent outflow of sperm and/or selected particles bound to the particles.

5. The device of claim 1, wherein the plurality of microchannels have a width between 10 μιη and 5000 μιη and a height between 10 μιη and 5000 μιη.

6. The device of claim 1, wherein the length of the microchannel is between 0.1mm and 15 mm.

7. The device of claim 1, wherein two or more of the microchannels merge into a single microchannel from the inlet reservoir to an outlet reservoir or junction chamber.

8. The device of claim 1, comprising 5 to 5000 of said microchannels, and being arranged in a single layer or in a multi-layer stack.

9. The device of claim 1, wherein the micro-channel extends between the inlet reservoir and the outlet reservoir in a radial, parallel, spiral, serpentine, or combination thereof.

10. The device of claim 1, wherein one or more chemoattractants are applied to the outlet reservoir, or a sufficient amount of buffer is applied at least to the microchannel.

11. The device of claim 1, further comprising an oocyte holding chamber in fluid communication with the outlet reservoir and configured to receive screened and/or separated motile sperm from the outlet reservoir.

12. A device according to claim 11, having a first region comprising the inlet reservoir, a second region comprising the microchannel or the guide structure, a third region comprising the binding chamber, a fourth region comprising the outlet reservoir and a fifth region comprising the oocyte holding chamber, and wherein one or more of the first to fifth regions are located at a different height than another one or more of the first to fifth regions, which regions are in fluid communication such that (a) motile sperm can be separated from the semen sample by the microchannel or the guide structure when received in the inlet reservoir, (b) motile sperm having predetermined characteristics can be separated from motile sperm bound to the selection particles and contained in the binding chamber, (c) motile sperm not bound to the selection particles can reach the outlet reservoir, and (d) screened oocyte sperm can be retrieved from the outlet reservoir and/or contacted with an oocyte held in the holding chamber and/or can be removed from the outlet and holding chamber.

13. A method of screening and/or separating motile sperm from a semen sample comprising the steps of:

providing an apparatus according to any one of claims 1 to 10;

filling the microchannel or the guide structure, as appropriate, with sperm buffer solution, and the inlet reservoir and the outlet reservoir of the device;

introducing a semen sample into the inlet reservoir;

waiting for a predetermined period of time; and

recovering motile sperm accumulated in the outlet reservoir.

14. A method for in vitro fertilization of an oocyte with motile sperm isolated and screened from a semen sample, comprising the steps of:

providing an apparatus according to claim 11 or 12;

filling the microchannel or the guide structure, and the inlet reservoir and the outlet reservoir of the device, as appropriate, with sperm buffer solution;

filling the binding chamber with the selection particles having a selectivity for binding to motile sperm having a predetermined undesirable characteristic associated with fertilization of an oocyte;

introducing an oocyte into the oocyte holding chamber;

introducing the semen sample into the inlet reservoir;

Waiting a period of time to allow the screened motile sperm to accumulate in the outlet reservoir and the oocyte holding chamber; and

allowing the selected motile sperm sufficient time to fertilize an oocyte contained in the oocyte holding compartment.