Sperm Separation System

Abstract

A device for the collection of motile sperm is disclosed, the device comprising a column having a top, a bottom, and a wall, the wall comprising a mesh layer and a membrane layer, an inlet disposed at the top of the column, and a nosepiece disposed at the bottom of the column.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 425,566, filed November 15, 2022, the entire disclosure of which is incorporated herein by reference. The present application provides an apparatus for separating high-quality motile sperm from dead, dying, and immature sperm, as well as non-sperm cells, commonly found in semen. The apparatus is used to process whole human semen specimens without the need for centrifugation or other equipment, special handling or training, or customized collection containers or culture equipment. The present disclosure provides devices and methods for the efficient preparation of sperm for therapeutic applications. In some embodiments, the devices and methods described herein separate motile sperm from cryoprotectants after warming of the frozen specimen. In some embodiments, the device may be used for processing sperm from livestock semen and for sperm biology research purposes. [Background technology]

[0002] According to the National ART (Assisted Reproductive Technology) Overview published by the CDC in 2019 for in vitro fertilization (IVF) procedures, over 281,000 treatment cycles required the use of processed sperm. Although the number of IUI cycles is unknown in the United States due to the lack of a registry of artificial insemination procedures, many more sperm samples are processed annually for intrauterine insemination (IUI). Assuming trends similar to those in European countries that report IUI results (Ferratti et al., 2013), approximately 28% of all infertile cycles per year, or well over 100,000, are performed in the United States. Regardless of whether the sample is partner- or donor-derived, fresh, or frozen-thawed, the majority of sperm samples (estimated at approximately 400,000 per year) must be processed before use in IVF or IUI.

[0003] Numerous techniques exist for processing sperm for infertility treatment. The general strategy of most processing methods is to separate healthier, more motile sperm from dead and dying sperm, white blood cells, and other potentially harmful components of semen. The higher-quality motile sperm obtained from the separation process are then used for IUI or IVF. Many processing methods utilize one or more centrifugation cycles to concentrate and remove sperm from seminal plasma. A commonly used "density gradient washing" system involves layering semen on top of a column of colloidal solution consisting of either polyvinylpyrrolidone-coated or silane-coated silica particles in a standard 15 cc centrifuge tube (Henkel and Schill, 2003; for review). During centrifugation, motile sperm preferentially migrate through the density gradient solution (DGS) and are concentrated in a diffuse layer at the bottom of the tube. These more motile sperm are collected and "washed" free of the gradient solution with culture medium via another one or two centrifugations. The end result is a relatively high yield of mostly motile sperm with little to no contamination from seminal plasma. Typically, 30-40% of the total number of motile sperm in a semen specimen are recovered after gradient washing, with 75%-90% of the recovered sperm exhibiting active forward movement, or "progressive motility" (World Health Organization, 2021). A drawback of gradient washing, as well as other centrifugation-based cell washing techniques, is that centrifugation induces the generation of reactive oxygen species (ROS; Shekarriz et al., 1995; McKinney et al., 1996), which can damage sperm structures, including DNA (Gonzalez-Marin et al., 2012; Aitken et al., 2022).

[0004] Given the well-documented impact of sperm DNA fragmentation on fertility treatment outcomes (Agarwal et al., 2022; for review; Marinaro (Schneider) and Schlegel, 2023; for review), many experimentalists have opted for less aggressive or more passive sperm selection techniques to minimize ROS generation. In a standard "swim-up" experiment, culture medium is carefully layered over semen or, in some cases, over a layer of sperm concentrated from semen by a single cycle of relatively "gentle" (low g-force) centrifugation. A portion of the motile sperm swims upward into the upper medium. A portion of the Swim Up medium is then sampled, taking care not to disturb the interface between the lower sperm / sperm layer and the medium (Henkel and Schill, 2003; for review). [Prior art documents] [Patent documents]

[0005] [Non-Patent Document 1] CDC, National ART (Assisted Reproductive Technology), 2019 Summary of the Invention [Problem to be solved by the invention]

[0006] The Zymot® "Multi" resin cartridge or "chip" (Demirci et al., 2016; Asghar et al., 2014), which has similar results to standard swim-up and was recently introduced to the market, and the Lenshooke® "CA0" or "Dish" (Hsu et al., 2023) are sperm sorting devices that operate on a similar principle. Sperm from the semen dispensed into the lower chamber swim up through a microporous membrane into the upper chamber of the medium (Demirci et al., 2016; Asghar et al., 2014, 2023). The interface between the semen and the medium is sufficiently secured by the microporous membrane, facilitating easy recovery of sperm from the upper chamber. The advantage of swim-up and sorting devices is that they generally result in the isolation (or sorting) of a highly motile sperm population, with over 90% of these sperm exhibiting progressive motility. The disadvantage of these systems is their inefficiency and low overall number of motile sperm. While there are no reports on the efficiency of a single swim-up column, multiple columns set up in parallel to maximize sperm recovery tend to yield less than 20% of the total number of motile cells found in a semen sample (World Health Organization, 2021). Zymot® "Multi" tips yield an average of 24% of the motile sperm found in the volume of sperm loaded into the device (Demirci et al., 2016; Asghar et al., 2014). However, a single tip can accommodate only about one-third of the volume of an average ejaculate, thus recovering only 8% of the total number of motile sperm found in an average semen sample. The effectiveness of motile sperm recovery using the Lenshooke® "CA0" has not been reported, but, like the Zymot® "Multi," only a portion of the sperm sample can be processed with a single device (Hsu et al., 2023). It is not uncommon for the number of motile sperm recovered from these passive separation methods to be suboptimal for IUI (Samuel et al., 2018). As with swim-up, multiple chips and dishes can be processed in parallel to improve yield, but this is more time consuming and the added expense may be difficult for some programs to justify.

[0007] Currently, the infertility market lacks a passive, low-cost system for separating sperm that is easy to use and provides a high yield of motile cells. Maximizing the yield of motile sperm while minimizing sperm damage is particularly important for men with low sperm counts or unexplained infertility (sperm appear normal, but only a small fraction of sperm in the semen specimen are fully functional). The yield of high-quality sperm after processing can be the difference between success and failure in some infertility treatments. [Means for solving the problem]

[0008] In some embodiments, a sperm separation system (SSS) device is disclosed, the device including a column having a top, a bottom, and walls with a mesh layer and a membrane layer, an inlet disposed at the top of the column, and a nosepiece disposed at the bottom of the column. In some embodiments, the device is configured to accept a fresh or frozen-thawed semen specimen (FIG. 1). After loading with sperm, the SSS device can be inserted into one of a number of culture tube formats routinely used in IVF and andrology laboratories for sperm processing and culture.

[0009] In some embodiments, the mesh layer is configured to provide structural support to the membrane layer.

[0010] In some embodiments, the membrane layer comprises a plurality of micropores.

[0011] In some embodiments, the plurality of micropores comprises micropores having a size of at least 1 micrometer in diameter.

[0012] In some embodiments, the plurality of micropores comprises micropores having a size less than 10 micrometers in diameter.

[0013] In some embodiments, the membrane layer is configured to restrict non-motile sperm, white blood cells, and other round cells.

[0014] In some embodiments, the inlet includes a loading ring, the loading ring configured to receive the liquid sample.

[0015] In some embodiments, the membrane layer comprises PVP-treated polycarbonate.

[0016] In some embodiments, the mesh layer is configured to restrict debris, clumps of aggregated cells and other matter, and semen clots from penetrating the membrane layer.

[0017] In some embodiments, the mesh layer comprises a plurality of openings, the openings being at least 10 micrometers wide and less than 100 micrometers wide.

[0018] In some embodiments, the mesh layer comprises stainless steel, wherein the stainless steel is configured to limit bacterial penetration through the mesh layer.

[0019] In some embodiments, the wall comprises a plurality of pleats or corrugations.

[0020] In some embodiments, a method for sample separation is disclosed, the method including the steps of receiving a sample into a column by an inlet, the column including a mesh layer and a membrane layer; depositing the column into a receptacle, the receptacle containing a culture medium; capturing a portion of the sample exiting the column through the mesh layer and membrane layer with the culture medium; removing the column from the receptacle; and collecting the captured portion of the sample from the culture medium.

[0021] In some embodiments, the sample used in the method for sample isolation comprises a liquid containing sperm.

[0022] In some embodiments, the captured portion of the sample comprises motile sperm.

[0023] In some embodiments, the method further includes filtering at least one of debris, aggregated cells and other matter, semen clots, non-motile sperm, bacteria, and other non-sperm cells from the sample with a membrane layer, and attaching and filtering them with a mesh layer.

[0024] In some embodiments, the method further includes depositing the column in a second container containing a second medium, and capturing, with the second medium, a second portion of the sample exiting the column through the mesh layer and membrane layer.

[0025] In some embodiments, the container comprises a centrifuge tube, a cell culture tube, a blood tube, a vial, or a cuvette.

[0026] In some embodiments, a method for assembling a device is disclosed, the method including the steps of aligning a mesh layer and a membrane layer, adhering the mesh layer to the membrane layer, forming a two-ply material, and forming a column of a predetermined diameter and length through conforming the two-ply material, the column having a top and a bottom, the membrane layer of the two-ply material forming the outside of the column and the mesh layer of the two-ply material forming the inside of the column, bonding an input piece to the top of the column, and bonding a nosepiece to the bottom of the column.

[0027] In some embodiments, the device is cylindrical and may be available in different diameters and heights for use with different sized tubes. Two different sizes of device were thoroughly tested, a larger one for use with 15 cc centrifuge tubes (Figure 6A) and a smaller one for use with 1.5 ml cryovials and disposable mini-beakers or "dispo-beakers" (Figure 6B).

[0028] In some embodiments, the device may be a square or triangular column for insertion into a cuvette that may be used in conjunction with automated sperm analysis and other cellular assay systems.

[0029] In some embodiments, the device includes an inlet with a loading ring (FIG. 2), a two-ply microporous-wall column (FIG. 5), and a nosepiece (FIGS. 3, 4). The semen sample is loaded into the device through the loading ring and fills the column, saturating the two-ply wall but not overflowing or expelling through the micropores (FIG. 1A). The device is then inserted vertically (nosepiece first) into a culture or centrifuge tube containing culture medium (FIG. 1B).

[0030] In such embodiments, the medium can be configured to receive sperm that migrate out of the device through the two-layer microporous wall (hereafter referred to as "swimout sperm"). The device is left in the upright tube for 15-60 minutes, during which time motile sperm accumulate in the culture tube's culture medium (also referred to as "swimout medium") (Figure 1B). The device can then be removed from the tube and discarded, or placed in another tube to collect additional swimout sperm. In some embodiments, the swimout sperm (sperm remaining in the swimout medium in the culture tube; Figure 1C) are of high quality, with an average forward motility of greater than 90% (Figure 8B; Figure 11A).

[0031] The disclosed methods and devices have several advantages over other passive sperm processing systems. First, a single SSS device (a larger cylindrical version sized for a standard 15cc centrifuge tube; Figures 1 and 6A and 6B) can accommodate ejaculates (semen specimens) up to 4 ml in volume, larger than the average ejaculate volume of 2–3 ml (World Health Organization, 2021). Second, in some disclosed methods and devices, swim-out is performed in a single culture or centrifuge tube, instead of requiring additional equipment, dedicated chambers, or customized tubes, simplifying sperm processing and reducing costs. Third, the disclosed methods and devices can be used with viscous specimens (such as unliquefied semen, frozen-thawed specimens in egg yolk-based extenders, or other diluents) due to the rigid mesh that constitutes the inner layer of the wall (the mesh limits debris and clots from clogging the smaller pores in the polycarbonate membrane that constitutes the outer layer). Fourth, the disclosed methods and apparatus can result in a higher yield of motile sperm compared to other processing methods, at least in part due to the large surface area and vertical configuration of the separation surface (no matter which direction motile sperm move, they eventually come into contact with the column wall). In some embodiments, the disclosed devices and methods can produce an average yield of over 60% of all motile sperm in ejaculate from a single SSS device after 30-60 minutes of incubation (Figures 8A, 10, and 12A). Finally, SSS is easier in both sperm setup and recovery than either of the other passive separation methods, including the DGS wash system or the Zymot® "Multi" (the "Multi" requires the use of two tuberculin syringes: one for sperm loading and one for sperm removal). Care must be taken when loading the semen specimen to ensure that air bubbles are not "injected" into the device, which could become trapped against the separation membrane and interfere with sperm migration to the collection chamber. [Brief explanation of the drawings]

[0032] [Figure 1] 1A-1C illustrate the operation of an SSS device according to an example embodiment. [Figure 2A] 1 is a schematic diagram of a loading ring from the bottom according to an exemplary embodiment; [Figure 2B] 2B is a schematic side view of the loading ring of FIG. 2A. FIG. [Figure 2C] 2B shows a perspective view of the loading ring of FIG. 2A. [Figure 3A] FIG. 3A is a schematic diagram of the top of a nosepiece according to an exemplary embodiment. [Figure 3B] FIG. 3B is a schematic side view of the nosepiece of FIG. 3A. [Figure 3C] 3B shows a perspective view of the nosepiece of FIG. 3A. [Figure 4A] 1 illustrates a nosepiece in accordance with an exemplary embodiment; [Figure 4B] 1 illustrates a nosepiece in accordance with an exemplary embodiment; [Figure 5] 1A through 1E show components and assemblies of an SSS device according to an exemplary embodiment. [Figure 6A] 1 shows different sizes and designs of SSS devices evaluated with human sperm. [Figure 6B] 1 shows different sizes and designs of SSS devices evaluated with human sperm. [Figure 6C] 1 shows different sizes and designs of SSS devices evaluated with human sperm. [Figure 7] Tables A and B show the biocompatibility of the SSS device and the materials that make it up. A: Survival of DGS-washed sperm after 6 hours of co-incubation at 37°C with 1. 2. stainless steel mesh, and 3. polycarbonate membrane was compared to DGS-washed sperm (control) for each experiment. B: Survival of sperm 24 hours after 1. swimming out of the large SSS device, 2. washed through DGS, and 3. remaining in the semen. [Figure 8A] 1 is a graph showing the recovery of motile sperm from a small SSS device in 15 minutes. [Figure 8B] 1 is a graph showing the recovery of motile sperm from a small SSS device in 30 minutes. [Figure 9] 1 is a graph showing the recovery of motile sperm from a large SSS device at 30 and 60 minutes. [Figure 10] FIG. 1 is a graph showing the recovery rate (%R) of motile sperm from a large SSS device as they are transferred through several centrifuge tubes containing fresh culture medium at defined time points; 0, 30, and 60 minutes. [Figure 11A] Graph showing the quality of sperm recovered from the SSS device, i.e., transferred through several centrifuge tubes containing fresh culture medium, at defined time points: 0, 30, and 60 min. Manual count of percent motility (% motility). [Figure 11B] CASA measurement of mean sperm curvilinear velocity (VCL in um / sec). [Figure 11C] Sperm morphology (% normal morphology; %N). [Figure 11D] Percentage of sperm with DNA fragmentation detected by TUNEL assay (% TUNEL positive). [Figure 12A] 1 is a graph showing a direct comparison of DGS and SSS using a large device, showing the efficiency of recovery of motile sperm by the two processing methods. [Figure 12B] The quality of sperm obtained by each method is shown. [Figure 12C] Motile sperm counts (% motility) are shown. [Figure 12D] Shown together with CASA, hyaluronic acid gel permeation assay (HA permeation), and survival after freezing and thawing. DETAILED DESCRIPTION OF THE INVENTION

[0033] Figure 1 illustrates the operation of the device in some embodiments. The device can be loaded with semen or other sperm specimen. A standard centrifuge tube is filled with 2-3 ml of medium (Figure 1A). The SSS device can be placed within the tube, and the medium is moved upward and around the walls of the device (Figure 1B). Motile sperm "swim out" from the inside of the device, through a wall made of mesh and microporous membrane, into the medium surrounding the device (Figure 1C). Removal of the device leaves a high yield of motile sperm in the medium. In some embodiments, the SSS device consists of an upper-located loading piece or ring 1 (Figure 2) and a lower-located nose piece 9 (Figure 3), both of which are connected to a centrally located pillar 17 with a two-layered wall (Figure 5).

[0034] 2A-2C are schematic diagrams of a loading element or ring according to an exemplary embodiment. In FIG. 2A, a view of the loading ring 1 is shown from a bottom view, with the inner wall 4 of the loading ring 1, the outer wall 3 of the loading ring 1, and the outermost edge 2 of the rim of the loading ring 1. The rim is discontinuous and has a span or arc 5 of approximately 50 degrees where the rim is absent. In some embodiments, the arc 5 may include an angle greater or less than 50 degrees. In FIG. 2B, a side view of the loading ring shows the relative thickness 6 of the rim and the relative height of the stem 7. The stem 7 is the portion of the ring that can be inserted into the top of a filtration column, such as filtration column 17 (FIGS. 5C, D), and the rounded edge 8 of the stem 7 facilitates insertion into the filtration column. A 3D perspective view (FIG. 2C) clearly shows the absence of the rim span or arc 5, a feature that allows the device to be easily grasped with force to remove it from the centrifuge tube. The span or arc 5 also allows for the attachment of a thin "fingerstrip" to aid in the insertion and removal of the device.

[0035] Figures 3A-3C are schematic diagrams of a nosepiece 9 according to an exemplary embodiment. In Figure 3A, a top view shows the outer wall of the stem 10 of the nosepiece 9, the outer edge of the "fitting ridge" 11, and the widest point of the variable segment 12 of the nosepiece 9. In Figure 3B, a side view of the nosepiece 9 shows the relative heights of the stem 10 and the "mating ridge" 11. The stem 10 is inserted into the bottom of a filtration column, such as filtration column 17 (Figures 5C and 5D), and the rounded edge 13 of the stem 10 facilitates insertion. The "mating ridge" 11 is similar in thickness to the double-layered wall of the column, facilitating adhesion with the UV-catalyzed acrylate. The shape of the variable segment 12 (including the tip 14) can be changed without altering the fit between the nosepiece and the column. A 3D perspective view (Figure 3C) clearly shows the circumferential "mating ridge" 11 and indicates the nosepiece is closed.

[0036] Figures 4A-B show two prototype variable segments 12 according to an exemplary embodiment. The SSS device with a narrower, rounded nosepiece (Figures 4A, 6C) was tested with four different types of 15 ml conical centrifuge tubes. In each case, insertion of the SSS device with the narrower nosepiece resulted in adequate displacement of culture medium around the device column wall. The wider, more tapered nosepiece (Figures 4B, 6C) was designed for use with a specific brand of centrifuge tube, which rarely has a narrow conical tip. The dimensions of the stem 10 and "mating ridge" 11 of both nosepieces are identical, allowing for attachment to device columns of the same diameter.

[0037] In some embodiments, a method for assembling the device is disclosed, including aligning a mesh layer and a membrane layer, adhering the mesh layer to the membrane layer, forming a two-ply material, and forming a column of a predetermined diameter and length through the conformation of the two-ply material, the column having a top and a bottom, the membrane layer forming the exterior of the column, and the mesh layer forming the interior of the column; bonding an input component to the top of the column; and bonding a nosepiece to the bottom of the column (FIG. 5). In some embodiments, the mesh layer is configured to provide structural stability to the membrane layer and the column. Furthermore, conforming the two-ply material of the device to various three-dimensional shapes allows for a greater surface area for sperm separation for a given fill volume. In some embodiments, the two-ply material may include pleats or corrugations to further increase surface area and improve sperm separation.

[0038] Figures 5A-5E illustrate the assembly of an SSS device in some embodiments. First, a mesh layer 15 and a membrane layer 16 are placed side-by-side in rectangular sheets of the same size to form a double wall. In some embodiments, the mesh layer 15 may be stainless steel. In some embodiments, the membrane layer 16 may be polycarbonate. The mesh layer 15 and the membrane layer 16 may be adhered to each other using a thin bead of UV-cured acrylate along the edges of the cut material to form a single two-ply sheet from the two materials. In some embodiments, conforming the two-ply material involves curling the two-ply material around a cylinder of a specific diameter and length, with the membrane layer 16 facing outward (Figure 5B) and the mesh layer 15 facing inward, and then clamping the two-ply material to form a cylindrical column 17 (Figure 5C). The cylindrical shape of the two-ply sheet may be stabilized with a thin bead of UV-cured acrylate applied longitudinally along the overlap area. The method further includes removing the post 17 from the cylindrical form and fitting the stem 7 of the loading ring 1 and the stem 10 of the nosepiece 9 onto either end of the two-ply post 17 (FIG. 5D). The column may be bonded to the load ring 1 and nosepiece 9 using a thin circumferential bead of UV-cured acrylate or epoxy (FIG. 5E), where the acrylate bead may overlap the "mating ridge" 11 on the nosepiece 9. Other permutations of assembly may include the use of fasteners, ties, bands, and / or washers to secure various elements of the SSS device, as well as heat fusing, ultrasonic welding, and / or infrared welding of the membrane and / or fasteners. In some embodiments, the two-ply material may be formed into posts of various shapes, such as triangular prisms, rectangular prisms, or any other suitable shape.

[0039] In some embodiments, the loading ring 1 and nosepiece 9 comprise polypropylene (FIG. 6A). Polypropylene is relatively inexpensive, easily mass-produced by injection molding, and can be heat sterilized. In some embodiments, small ball bearings of galvanized steel or other material may be embedded within the nosepiece; thus, the device is configured to displace medium within a centrifuge tube or other vial or tube in a manner that ensures that the medium extends the length of the wall of the device's column (FIG. 1B).

[0040] In some embodiments, medical-grade commercial and engineering resins with appropriate density (specific gravity greater than that of water) can be used for the loading ring and / or nosepiece. In some embodiments, the loading ring and / or nosepiece can be formed using injection molding or 3-D printing, so that the size, shape, and fine contours of the loading ring and nosepiece can be customized to optimize the fit of the SSS device into many different types and brands of culture tubes, vials, cuvettes, or other containers used to contain, process, and evaluate cells. Variations in the plastic parts of the SSS device are shown in Figure 6C, which are based on the different variable segments 12 of the nosepiece (Figure 5). These illustrative examples allow the SSS device to easily sink to the bottom of a centrifuge tube without the need for additional steel bearings or other weights.

[0041] The two-layered wall of the SSS device can include an inner stainless steel filter mesh layer 15 and an outer membrane layer 16, with the mesh layer 15 and membrane layer 16 each containing a plurality of microscopic openings and pores (FIG. 5A). In some embodiments, the mesh layer comprises surgical-grade stainless steel. In some embodiments, the composition of the stainless steel promotes bacterial adhesion. In further embodiments, the mesh layer comprises nylon or other flexible plastic. In some embodiments, the membrane layer comprises sterile polycarbonate or a polymer such as polyamide, cellulose acetate, polypiperazine amide, or the like.

[0042] 6A-C show a device for collecting motile sperm according to an exemplary embodiment, the device including a column having a top, a bottom, and a wall including a mesh layer and a membrane layer, an inlet disposed at the top of the column, and a nosepiece disposed at the bottom of the column. In some embodiments, the membrane layer includes a plurality of micropores. In some embodiments, the mesh layer includes a plurality of openings configured to restrict penetration of debris, clumps of aggregated cells and other materials, and semen clots into the mesh layer. In some embodiments, the micropores in the membrane layer have a diameter of about 5 μm, and the openings in the mesh layer have a size of about 25 μm. In some embodiments, the mesh layer includes polycarbonate, and the membrane layer includes stainless steel.

[0043] Individual elements of the device, as well as the fully assembled device, were tested for sperm toxicity (Figure 7A-B). Three components of the SSS device, the stainless steel mesh, polycarbonate membrane, and UV-cured adhesive, were separately incubated with sperm washed by DGS in a medium optimized for sperm handling (VitroLife®, Inc.; human oviductal fluid containing 10% serum supplement or HTF10%S). One ml of washed sperm suspension, adjusted to 10 x 10 motile sperm / ml in HTF10%S, was continuously incubated with the device components for 6 h at 37 °C. There was no difference in sperm survival compared to washed sperm in controls, as determined by a manual motility count (% motility) of at least 400 sperm or the quality of progressive motility assessed using a 0-4 scoring system (Figure 7A). Sperm toxicity was also determined in the fully assembled, larger (4 ml volume) device (Figure 6A). Semen was loaded into the device, and the sperm were allowed to swim out in HTF 10% S in a standard 15 ml centrifuge tube for 60 min. The device was removed from the centrifuge tube, and the swim-out sperm were incubated in swim-out medium for an additional 24 h at room temperature. The viability and vitality of the swim-out sperm exceeded that of sperm in semen and sperm washed into HTF 10% S via DGS (Figure 7B). Viability was determined by manual motility counting (% motility) of at least 400 sperm, and sperm motility vitality was determined using computer-assisted sperm analysis (CASA; for a review, see Amann and Waberski, 2014) of sperm curvilinear movement (VCL or VelocityCL) (Figure 7B). VCL values ​​are reported in microns / second and represent the average of at least 500 sperm motility trajectories digitally captured and processed by CASA. All experiments were performed using 3-4 semen specimens, one from each of 3-4 different donors. All data in the tables are reported as the mean + / - standard error of the mean (sem). All subsequent studies use the same determination method for both % motility and VCL.

[0044] Initial swim-out experiments were performed using a small SSS device (1.8 ml capacity; Figure 6B). Preliminary testing included evaluation of membranes with different pore sizes (5 μm and 8 μm) and stainless steel meshes with different mesh sizes (25 μm and 50 μm). All combinations produced similar results with respect to the number and quality of swim-out sperm, but the combination of a 5 μm pore-size polycarbonate membrane and a 25 μm mesh-size stainless steel mesh produced swim-out media with minimal detectable microscopic debris from seminal plasma (determined at 200x magnification). Similarly, in a series of experiments, temperature was evaluated for swim-out efficiency. RT (23°C) was compared with 30°C and 37°C. Higher recovery rates of swim-out sperm were observed at both elevated temperatures compared with RT, but no difference in swim-out efficiency was observed between 30°C and 37°C. All subsequent swim-out tests were performed at 30°C using a device consisting of a 25 μm stainless steel mesh and a polycarbonate membrane with 5 μm pore size.

[0045] The mini-SSS device was evaluated for swim-out efficiency over 15 and 30 minutes. Semen specimens from each donor were allowed to liquefy at room temperature for 30 minutes. Two devices were loaded with 1 ml of semen from the same ejaculate. The devices were placed in cryovials containing 1 ml of HTF 10% S, and one was incubated for 15 minutes and the other for 30 minutes. Swim-out time observations were paired and based on 12 total semen specimens, four specimens from each of four donors. On average, over 40% of the total number of motile sperm introduced into the mini-SSS device were recovered in the swim-out medium after 15 minutes, and on average, nearly 65% ​​of the total number of motile sperm introduced into the mini-SSS device were recovered in the swim-out medium after 30 minutes (Figure 8A). Columns and error bars represent the mean + / - s.e.m.

[0046] The large SSS device was evaluated for swim-out efficiency over 30 and 60 minutes. Semen specimens were processed almost immediately after receiving them from the donor, an average of approximately 9 minutes after specimen collection. In most cases, the samples were not completely liquefied. The samples were gently mixed with 0.5–1.0 ml of HTF 10% S before loading into the SSS device. In some experiments, the semen specimen was split into two portions; in other cases, the entire semen specimen was loaded into a single device. The semen sample used to load the SSS device was never less than 2.5 ml. Swim-out time observations were unpaired and based on a minimum of three to a maximum of six specimens from seven different donors. On average, the percentage of motile sperm loaded into the large SSS device recovered in swim-out medium after 30 and 60 minutes was 48% and 63%, respectively (Figure 9). Columns and error bars represent the mean + / - s.e.m.

[0047] Four of the seven donors had consistently good-quality semen specimens; specimens had normal concentrations of progressively motile sperm combined with normal levels of morphologically normal sperm, as determined by the 5th edition of the WHO Laboratory Manual for Semen Analysis. In contrast, three of the seven donors had poor specimen quality, consistently with fewer than normal numbers of progressively motile sperm and / or morphologically normal sperm. When the same data were stratified based on WHO "normal" (all observations from four "good" specimen donors) and WHO "abnormal" (all observations from three "poor" specimen donors), motile sperm recovery appeared to be highly dependent on overall semen quality. Recovery of WHO "normal" specimens at 30 and 60 minutes exceeded that of WHO "abnormal" specimens by 14% and 21%, respectively. The mean recovery of motile sperm in swim-out medium for WHO "normal" specimens at 30 and 60 minutes was 57% and 73%, respectively (Figure 9). Columns and error bars represent the mean + / - sem.

[0048] In another series of experiments using a larger SSS device, pre-liquefied semen (as described for the experiment in Figure 9) was loaded into a single device and then transferred to a new centrifuge tube containing fresh HTF with 10% serum supplement at 30 and 60 minutes, then incubated for an additional 60 minutes (120 minutes after loading the SSS device; Figure 10). Each swim-out fraction, as well as the semen remaining in the device (the "spent" fraction), were assessed for total motile sperm counts. The mean cumulative recovery (%) after 30, 60, and 120 minutes, and the % of total motile sperm in the spent fraction, are shown in Figure 10. Observations of the swim-out fraction were paired and based on 10 total semen samples (two samples from each of five donors). Columns and error bars represent the mean + / - s.e.m.

[0049] The quality of sperm in the initial semen specimen, each swim-out fraction, and the spent fraction is shown in Figure 11A-D. The percentage of motile sperm recovered in the first 30 min of swim-out (94.3 ± 0.8%) was higher than that recovered in the next 30 min (92.1 ± 1.1%) and the final 60 min (87.9 ± 1.9%), with very few motile sperm remaining in the spent fraction (13.8 ± 1.5%; Figure 11A). Similarly, sperm vitality, as determined by mean curvilinear velocity (VCL), was highest in the first 30 min of swim-out fraction compared to subsequent swim-out fractions. All swim-out fractions contained sperm with superior VCL, on average, compared to both the initial semen specimen and the spent fraction (Figure 11B). Morphology was determined by microscopic evaluation of stained sperm fixed on glass slides. For all experiments, a minimum of 200 sperm were evaluated from every swim-out fraction. Sperm classified as morphologically normal have only minor variations in the shape and dimensions of both the flagellum and head (Kruger et al., 1986, 1991; WHO laboratory manual, 2021). The swim-out fraction contained a higher percentage of sperm with normal morphology compared to either the semen or spent fraction (Figure 11C). TdT-mediated dUTP nick end labeling (TUNEL; Mochizuki, H. et al., 1994; Portela-Carilau et al., 1994) has been used to identify DNA damage, particularly DNA fragmentation, in sperm using fluorescence microscopy (Agarwal et al., 2016; for review; Ribeiro et al., 2017). TUNEL assays were performed in a subset of treatments for each of the five donors. At least 200 sperm per treatment per donor were evaluated for the presence of nuclear labeling ("TUNEL positive") using a fluorescent microscope. The percentage of sperm selected by swim-out with evidence of DNA fragmentation was significantly lower than the percentage of sperm in the semen and spent fractions (1.7 ± 0.6%, 13.2 ± 1.9%, and 18.8 ± 3.7%, respectively; Figure 11D). Columns and error bars represent the mean ± s.e.m.

[0050] The SSS and DGS sperm washing systems were directly compared on the same semen specimen for the efficiency of motile sperm recovery and the quality of the recovered sperm (Figure 12A-D). Semen specimens were received an average of 11 minutes after collection and divided into equal volumes of 1 ml of HTF 10% S. Half of the sample was loaded into a large SSS device for a 60-minute swim-out, while the other half was allowed to fully liquefy over the next 15-20 minutes and then loaded onto the top of a column of DGS (double layer, 45 / 90%) in a standard 15cc centrifuge tube. The DGS washing system involved an initial cycle of centrifugation at 300 x g for 15 minutes, followed by a second cycle of centrifugation at 300 x g for 10 minutes with fresh HTF medium (to remove residual DGS; Henkel and Schill, 2003). The swim-out medium was evaluated after 60 minutes. One fraction of semen was loaded onto a large SSS device using swim-out (SwimOut) for 60 minutes, while the other fraction was washed using DGS. The recovery efficiency of motile sperm by the two processing methods is shown in Figure 12A. Similar to what was observed in previous experiments, 63.1 ± 9.8% (mean ± SEM) of the total number of motile sperm loaded onto the SSS device was recovered in the swim-out medium after 60 minutes, which was nearly double the number of motile sperm recovered with DGS treatment (34.4 ± 4.4%; mean ± SEM). The experiment was performed using four semen specimens, one from each of four donors.

[0051] The quality of sperm isolated by the SSS and DGS methods is shown in Figures 12A-D. Both the percentage of motile sperm (Figure 12A) and sperm curvilinear velocity (Figure 12B) were higher for Swim Out Sperm (sperm isolated via SSS) than for sperm isolated using DGS. Sperm were also evaluated for their ability to penetrate gels made from long polymers of hyaluronic acid (HA). HA gels are similar in viscosity and charge to cervical mucus (Aitken et al., 1992), and sperm HA penetration assays have been used to measure sperm functional and structural competence (Aitken et al., 1992, 2006; Tollner et al., 2008, 2011). The motile concentration of processed sperm was adjusted to 10 x 106 / ml in HTF 10% S, and then 8 µl of the sperm suspension was added to the slide chamber containing HA. Using a microscope equipped with a grid eyepiece, sperm reaching a region of HA exactly 3 mm from the sperm suspension / HA interface were counted over a 15-minute period. On average, HA penetration by swim-out sperm (SSS) was 35% greater than that of DGS-treated sperm (Figure 12C). Treated sperm were frozen in vials over liquid nitrogen vapor in an egg yolk-based Tris / Tes-buffered diluent, with added glycerol at a final concentration of 5%. Frozen specimens were stored in liquid nitrogen for at least 2 weeks and then warmed at 37°C for 15 minutes. The ability of swim-out sperm to survive cryopreservation (as determined by % motility and VCL) was superior to sperm isolated by DGS (Figure 12D). Columns and error bars represent the mean + / - s.e.m. Exemplary SSS Devices

[0052] The SSS device described herein offers flexibility and adaptability for use with a wide range of culture tubes and vessels used in mammalian sperm processing. The large-scale device prototype in Figures 6A and 6C (left) is contoured to work with various types of conical centrifuge tubes. Similarly, the small-scale device prototype in Figure 6B works with a variety of small vials, including "cryo-vials" and "mini-dipo-beakers." Users do not need to purchase customized vessels or chambers for collecting swim-out sperm; rather, they can choose from a variety of different products commonly available and commonly used in IVF and male reproductive laboratories. Such flexibility can accommodate laboratory preferences and have cost advantages. Furthermore, the device can be modified in terms of shape and size without changing the underlying principles and effectiveness of the design. Thus, the SSS device can be adapted for use with new sperm processing systems and culture vessels, including non-cylindrical vessels. The device's adaptability is useful for other applications, including assisted reproduction in animals, novel semen collection systems, sperm biology research, and sperm cell diagnostics (see Examples).

[0053] The SSS device results in excellent sperm separation and collection from semen samples, leaving behind dead sperm as well as viable sperm that are likely damaged and nonfunctional. First, sperm exiting the device are highly motile. The motility rates of swim-out sperm recovered at 15 and 30 minutes were 96.2 ± 1.9 and 94.3 ± 0.8 (mean ± standard error), respectively (Figure 8B). These sperm that swim out within 30 minutes exhibited vigorous motility as determined by VCL measurements, which was on average more than twice as frequent as the motile sperm that failed to exit the semen in the SSS device after 2 hours (Figure 11B). Similarly, separation through the device favors sperm with normal morphology and higher structural integrity of their DNA (Figures 11C, D). On average, more than 16% of swim-out sperm had normal shape, which is more than twice the percentage of morphologically normal sperm remaining in the semen (Figure 11C). Similarly, less than 2% of swim-out sperm had detectable levels of DNA fragmentation, a level 10-fold lower than that observed for sperm remaining in semen (Figure 11D). The characteristics of sperm exiting the SSS device appear comparable to those reported for sperm sorted with Zymot® Multi protopes, with average % motile, % morphologically normal, and % DNA fragmentation of 90.4, 17.6, and 2.1, respectively (Asghar et al., 2014). Furthermore, extending the swim-out period beyond 30 minutes can increase the recovery rate of motile sperm from semen (Figure 10), with only a slight decrease in overall sperm quality (Figure 11A-C).

[0054] In some embodiments, one advantage of SSS devices is the bonding of a rigid yet malleable mesh to an ultrathin microporous membrane. The underlying (inner ply) mesh material provides structural stability to the device and can act as a scaffold for forming the overlying membrane into several possible three-dimensional shapes. Various types of microporous membranes, including PCTE, have been used in sperm separation and sperm migration studies, whereby motile sperm migrate across a flat or planar membrane separating two chambers, a configuration similar in principle to that of Zymot® "Multi" and Lenshooke® "CA0" (Hong et al., 1991; Lee et al., 1989; Chijioke (Berkeley) et al., 1988; Raoof (Ham) et al., 1987). In contrast, the microporous membrane of SSS devices is not constrained to a single plane or a specific shape; therefore, the SSS configuration may allow for a larger surface area for sperm separation for a given fill volume. A two-layer design may create a pleated or corrugated membrane surface to further increase surface area and improve sperm separation.

[0055] One advantage of the SSS device is its efficiency in separating motile sperm compared to other passive separation systems. While Zymot® "Multi" is likely the more efficient of the widely used passive systems, it does have some limitations regarding the recovery of motile sperm. First, after 30 minutes, the return rate of sorted sperm in the upper chamber (collection chamber) back into the lower chamber (loading chamber with semen) exceeds the migration rate of sperm from the semen into the collection chamber (Asghar et al., 2014). Consequently, the instructions for use with Zymot® "Multi" suggest removing the collection medium before 30 minutes for best results. The authors of (Hsu et al., 2023) made a similar recommendation for Lenshooke® "CA0." While not intending to be bound by theory, the inventors believe that a simple explanation for this limitation of the "Multi" and "CA0" devices is that all sperm, both motile and non-motile, gradually settle to the bottom of the container, tube, or chamber by gravity, which is precisely how a mobile sedimentation system for sperm sorting works (Shany and Tavori (Horton), 2012; Kang et al., 2019; Meitei (Ham) et al., 2021). Motile sperm in the sperm in the lower chamber of the Zymot® "Multi" and Lenshoek® "CA0" devices eventually detach from the filter membrane and become unavailable for sorting. Second, Zymot® "Multi" appears to have limited effectiveness with viscous samples, showing improved effectiveness with a four-fold dilution of the sperm specimen (Asghar et al., 2014). Third, Zymot® accommodates a fill volume of only 850 μl, approximately one-third the volume of an average ejaculate.

[0056] SSS devices may not suffer from a degradation in performance over time. Due primarily to the vertical arrangement of the filtration components of the SSS device, gravity-induced sperm sedimentation does not present a drawback and may promote higher sperm recovery rates. In some embodiments, as swim-out sperm (sperm outside the device) settle, they will migrate toward the bottom of the tube, below the microporous membrane area, where there is no possibility of sperm exchange between the swim-out medium and semen within the device. In such embodiments, as motile sperm within the device settle, they continue to migrate outward through the microporous wall. Support for these possibilities is provided by the observations in Figures 8 and 9, where sperm recovery rates continuously increase over time with no apparent difference in sperm accumulation rate when compared to an experiment in which the SSS device was transferred to a tube containing fresh medium at various time intervals (Figure 10).

[0057] Viscous samples do not appear to limit the performance of the SSS device. Pre-liquefied semen samples of various viscosities are routinely loaded into the SSS device with good results (Figures 9-12). This is due to the ability of the inner stainless steel mesh to prevent gelled semen debris and clumps from clogging the pores of the outer polycarbonate membrane. Of note for the Zymot® "Multi" and Lenshooke® "CA0" devices is the requirement to allow sperm to liquefy for 30 minutes before loading a portion of the sperm sample into the device. In contrast, semen specimens can be loaded into the SSS device immediately, if not immediately after collection. As semen liquefaction occurs gradually over 5-20 minutes, sperm are "released" and become more motile. The SSS device mobilizes sperm into the medium as soon as they become "available," allowing them to be easily rescued from potentially toxic components of seminal plasma.

[0058] Another feature that distinguishes the SSS device from most sperm sorting devices on the market is its ability to accommodate the entire ejaculate. Preliminary testing of materials for column construction determined that the mesh and microporous membrane work together to counteract the effects of hydrostatic pressure on fluid filtration. In some embodiments, there is no fluid flow from the inside to the outside of the device, regardless of the volume of medium contained in the collection tube on the outside of the device or the volume of semen loaded inside the device. Taller devices can be devised to accommodate semen volumes greater than 4 ml. The ability to process the entire ejaculate with a single device has utility for IUI and for handling specimens from species with larger average ejaculate volumes.

[0059] Isolation of motile sperm using SSS may be less harmful to sperm and more physiological than DGS. In addition to demonstrating higher recovery rates using the SSS device, split-ejaculation studies directly comparing DGS and SSS methods indicate that SSS is superior for preserving sperm quality, function, and survival (Figures 12A-D and 7B). Compared to DGS, SSS may result in the collection of a population of sperm that is more motile in terms of both percent motility and VCL (Figure 12A). SSS-treated sperm survived better than DGS-treated sperm after 24 hours of incubation at RT (Figure 7B) and after freezing and thawing using standard cryopreservation methods (Figure 12D). After adjusting for motile sperm concentration, swim-out and SSS-treated sperm were compared side-by-side in adjacent "microslide" chambers for their ability to penetrate an HA gel simulating cervical mucus. DGS resulted in a lower rate of sperm HA penetration compared to both sperm in semen and sperm treated with SSS (Figure 12C). It has previously been reported that centrifugation of sperm with DGS alters the sperm interface by removing sperm coating proteins and carbohydrates (Zoca (Horton) et al., 2022), which significantly reduces the net negative sperm interface charge (Simon (Horton) et al., 2016). Similarly, alteration of the sperm surface by enzymatic removal of negatively charged sugars or removal of glycocalyx components via chemically induced capacitation results in a significant decrease in sperm's ability to penetrate HA gels and cervical mucus in vitro (Tollner et al., 2008, 2011). The difference in sperm HA penetration observed here between treatment methods may in part reflect the superiority of the SSS method for preserving sperm surface properties important for migration through mucus. [Example]

[0060] The following examples are offered to illustrate, but not to limit, the present disclosure.

[0061] Example 1. IUI: For intrauterine insemination, it is standard practice to remove sperm from seminal plasma by "washing" them into culture medium by two or more centrifugations, with or without a density gradient solution. The SSS device replaces these repeated centrifugation activities. Sperm that swim out of the SSS device can be concentrated into a smaller volume of medium suitable for fertilization in a single cycle of centrifugation. Sperm isolated by the SSS device are superior in both number and quality (% motility, VCL, and viability) to sperm isolated by DGS (Figure 12), which is important because the number of actively motile sperm per minute influences IUI outcomes (Ombelet et al., 2007).

[0062] Example 2. Preparation of sperm for cryopreservation: The SSS device can be a valuable component of a sperm processing routine for cryopreservation. Dead and dying sperm, immature sperm, and leukocytes found in semen can be sources of ROS and hydrolytic enzymes that can further damage viable sperm during the freezing and thawing process. SSS-treated sperm have superior post-thaw survival and progressive motility compared to DGS-treated sperm (Figure 12D).

[0063] Example 3. IVF / ICSI: A time-course study demonstrates that sperm exiting the SSS device early are of the highest overall quality (Figure 8B, Figures 11A-D). After 15 minutes, the number of swim-out sperm exceeds sufficient numbers for conventional IVF and IVF with intracytoplasmic sperm injection (ICSI). SSS can replace swim-up and other passive separation systems that are more difficult to set up. Thus, incubation of semen in the SSS device can be shortened to 15 minutes or less to obtain the highest quality sperm for IVF procedures where the number of recovered motile sperm is less important.

[0064] Example 4. Research Applications It is well established in mammalian reproduction research that ejaculation, particularly the delivery of sperm and seminal plasma, occurs in multiple pulsatile waves, resulting in various successive fluid fractions. Up to 80% of the sperm in the ejaculate are delivered in the first 20% of the released fluid fraction, referred to as the "sperm-rich" fraction. The remaining fluid in the ejaculate, primarily derived from the seminal vesicles, is delivered in subsequent fractions rich in proteins necessary for inducing semen gelation. During intercourse, the sperm-rich fraction is propelled to the cervix and mixes with cervical mucus. The majority of the semen continues to the vaginal vault, forming a clot (a loose gelatinized plug) that is retained there, potentially blocking the cervix and helping to prevent sperm from flowing back into the vagina. Sperm from the cervix gradually migrate to the upper reproductive tract. Small "waves" of highly competent sperm eventually reach the vicinity of the egg in the distal or lateral fallopian tube, one of which is the fertilizing sperm (Suarez and Pacey, 2006; for review; Yanagimachi, 1994; for review).

[0065] The conditions experienced by sperm when processed for either assisted reproduction or research are very different. Sperm donors collect specimens by masturbation into a single specimen cup, resulting in the mixing and prolonged incubation of sperm in all semen fractions. Sperm are exposed to and trapped in the clot or gelling fraction, which greatly reduces their motility. For this reason, it is common practice to delay evaluation of sperm in semen for 30 minutes, allowing sufficient time for most specimens to liquefy. Sperm typically remain in the semen for 60 minutes, during which time technicians evaluate the semen for sperm motility, number, morphology, semen volume, etc. (WHO Laboratory Manual, 2010). Short-term exposure to seminal plasma (30–60 min) has been shown to alter sperm function, and exposure for more than 2 h can result in permanent sperm damage and death ( Yavas and Selub, 2004 ; Punjabi et al., 2021 ; Iemmolo (Horton) et al., 2005 ).

[0066] The SSS device can recover sperm from semen even before liquefaction is complete. The SSS device can be loaded before liquefaction is complete (in the majority of data presented in this application, the SSS device was loaded with semen within 11 minutes of collection). Sperm can be recovered from the device within minutes, thus requiring very little time for co-incubation with seminal plasma. Preliminary studies indicate the feasibility of directly collecting semen into a device with a dedicated funnel and collection tube. The sperm-rich fraction enters the SSS device first, and the sperm begin to migrate almost immediately through the device into the medium, avoiding direct interaction with the coagulated fraction. Physiologically, these sperm more closely resemble sperm that enter the cervix during mating and are therefore a better source of cells for studies of IUI and sperm biochemistry and function.

[0067] Example 5. Assisted Reproduction in Animals: Human sperm share a common morphology or shape with sperm from many other mammals. Specifically, sperm from dogs, horses, cattle, and pigs are very similar to those of human sperm in terms of head and flagellum dimensions. Based on the basic principles of sperm handling in humans and these species, the inventors anticipate the application of the SSS device in artificial insemination and IVF techniques, such as those present in animal breeding programs. Although variations in mesh and membrane pore size may be necessary for each species to optimize device performance, the inventors predict that the SSS device design described herein should be useful for sperm preparation in the animal breeding industry.

[0068] Example 6. Patients with poor semen quality: Our studies using smaller SSS devices suggest that sperm separation efficiency may increase as the device becomes smaller. This is not surprising, since the ratio of polycarbonate membrane surface area to semen loading volume increases with decreasing device diameter. Many subfertile men have smaller-than-average ejaculate volumes and fewer motile sperm. Smaller SSS devices, with significantly higher recovery efficiency, may be more suitable for use in these patients than larger devices.

[0069] Example 7: Removal of frozen-thawed sperm from cryoprotectant. Most sperm specimens frozen for long-term storage are contained in vials or straws with a total volume ranging from 0.5 ml to 1.0 ml, including the volume of added cryoprotectant. It is common practice in fertility clinics to wash frozen-thawed sperm from the cryoprotectant by centrifugation, or swim-up, before use in IUI or IVF. Small SSS devices are used for this purpose and can replace these other, less efficient methods. Furthermore, due to the inner layer of stainless steel mesh, SSS devices can be used with more viscous cryoprotectant diluents, such as those made from egg yolk.

[0070] Example 8. Relevance to Sperm Diagnostics: Automated male fertility diagnostics is a growing industry with numerous computer-based systems employing optical sensors / digital capture interfaces that are seeing increasing market acceptance. AI greatly expands the capabilities of sperm cell analysis systems, extending far beyond motility analysis and morphology. We envision the use of smaller SSS devices in cuvettes, flat-sided containers with excellent optical properties used for a variety of fluorometric and spectroscopic / spectrophotometric applications. Sperm separated by SSS devices exhibit more physiological behavior and better preserve potential surface markers of fertility compared to sperm prepared by DGS and other centrifugation methods. The efficiency of smaller SSS devices translates into a convenient means for providing a sufficiently large number of motile sperm (relevant sperm for fertility analysis) within minutes of loading the sperm specimen. Meanwhile, sample "noise," nonviable sperm, nonsperm cells, and debris in the seminal plasma are limited by the device and removed along with it prior to analysis.

[0071] Accordingly, implementations of the present disclosure may relate to one of the enumerated exemplary embodiments (EEE) listed below.

[0072] EEE1 is an apparatus for the collection of motile sperm, the apparatus comprising a column having a top, a bottom, and a wall, the wall comprising a mesh layer and a membrane layer, an inlet located at the top of the column, and a nosepiece located at the bottom of the column.

[0073] EEE 2 is the device of EEE 1, wherein the mesh layer is configured to provide structural support to the membrane layer.

[0074] EEE 3 is any of the devices of EEE 1-2, wherein the membrane layer comprises a plurality of micropores.

[0075] EEE4 is the device of EEE 3, wherein the plurality of micropores comprises micropores having a size of at least 1 micrometer in diameter.

[0076] EEE5 is the device of EEE 3, wherein the plurality of micropores comprises micropores having a size less than 10 micrometers in diameter.

[0077] EEE6 is any of the devices EEE 1-5, wherein the membrane layer is configured to restrict non-motile sperm, white blood cells, and other round cells.

[0078] EEE7 is the apparatus of any one of EEE1-6, wherein the inlet comprises a loading ring, the loading ring configured to receive the liquid sample.

[0079] EEE 8 is any of the devices of EEE 1-7 wherein the membrane layer comprises PVP-treated polycarbonate.

[0080] EEE 9 is any of devices EEE 1 to 8, in which the pillar is a circular cylinder, a triangular prism, or a rectangular prism.

[0081] EEE10 is the device of any of EEE1-9, wherein the mesh layer is configured to restrict debris, clumps of aggregated cells and other material, and semen clots from penetrating the mesh layer.

[0082] EEE11 is the device of any of EEE1-10, wherein the mesh layer comprises a plurality of openings, the openings being at least 10 micrometers wide and less than 100 micrometers wide.

[0083] EEE12 is the device of any of EEE1-11, wherein the mesh layer comprises stainless steel, the stainless steel being configured to restrict bacterial penetration through the mesh layer.

[0084] EEE 13 is the device of any of EEE 1-12, wherein the wall further comprises a plurality of pleats or corrugations.

[0085] EEE 14 is a method for sample separation, comprising the steps of receiving a sample by an inlet into a column comprising a mesh layer and a membrane layer as described in any of EEE 1 to 13, depositing the column in a container containing a culture medium, capturing with the culture medium a portion of the sample that passes through the mesh and membrane layers and exits the column, removing the column from the container, and collecting the captured portion of the sample from the culture medium.

[0086] EEE 15 is the method of EEE 14, wherein the sample comprises a fluid containing sperm.

[0087] EEE 16 is the method of EEE 15, wherein the captured portion of the sample contains motile sperm.

[0088] EEE17 is the method of any of EEE 14-16, further comprising filtering at least one of debris, agglutinated cells and other matter, semen clots, non-motile sperm, bacteria and other non-sperm cells from the sample with the membrane layer; and attaching and filtering at least one of debris, agglutinated cells and other matter, semen clots, non-motile sperm, bacteria and other non-sperm cells from the sample with the mesh layer.

[0089] EEE18 is the method of any of EEE 14-17, further comprising placing the column in a second container containing a second medium, and capturing with the second medium a second portion of the sample that exits the column through the mesh layer and membrane layer.

[0090] EEE19 is the method of any of EEE 14 to 18, wherein the container comprises a centrifuge tube, a cell culture tube, a blood tube, a vial, or a cuvette.

[0091] EEE 20 is a method of assembling a device described in any of EEE 1 to 13, the method including the steps of aligning a mesh layer and a membrane layer; adhering the mesh layer to the membrane layer; forming a two-ply material; and conforming the two-ply material to form a column of predetermined diameter and length, the column having a top and a bottom, the membrane layer of the two-ply material forming the outside of the column and the mesh layer of the two-ply material forming the inside of the column; bonding an input piece to the top of the column; and bonding a nose piece to the bottom of the column.

[0092] While the SSS device has been described in terms of one or more embodiments, it should be understood that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the invention.

Claims

1. 1. A device for the collection of motile sperm, comprising: a column having a top, a bottom, and a wall, the wall including a mesh layer and a membrane layer; an inlet located at the top of the column; and and a nosepiece located at the bottom of the column.

2. 10. The device of claim 1, wherein the mesh layer is configured to provide structural support to the membrane layer.

3. 10. The device of claim 1, wherein the membrane layer comprises a plurality of micropores.

4. 4. The device of claim 3, wherein the plurality of micropores comprises micropores having a size of at least 1 micrometer in diameter.

5. 4. The device of claim 3, wherein the plurality of micropores comprises micropores having a size of less than 10 micrometers in diameter.

6. 10. The device of claim 1, wherein the membrane layer is configured to restrict non-motile sperm, white blood cells, and other round cells.

7. 10. The device of claim 1, wherein the inlet comprises a loading ring, the loading ring configured to receive the liquid sample.

8. 10. The device of claim 1, wherein the membrane layer comprises PVP-treated polycarbonate.

9. The device of claim 1, wherein the pillar is a circular pillar, a triangular pillar, or a rectangular pillar.

10. 10. The device of claim 1, wherein the mesh layer is configured to restrict debris, clumps of aggregated cells and other matter, and semen clots from penetrating the mesh layer.

11. 10. The device of claim 1, wherein the mesh layer comprises a plurality of openings, the openings being at least 10 micrometers wide and less than 100 micrometers wide.

12. 10. The device of claim 1, wherein the mesh layer comprises stainless steel, the stainless steel configured to restrict bacterial penetration through the mesh layer.

13. The device of claim 1 , wherein the wall further comprises a plurality of pleats or corrugations.

14. 1. A method for sample separation, comprising: receiving a sample through an inlet into a column including a mesh layer and a membrane layer; placing the column in a vessel containing a medium; capturing, with the medium, a portion of the sample that passes through the mesh layer and membrane layer and exits the column; removing the column from the vessel; and and collecting the captured portion of the sample from the medium.

15. 15. The method of claim 14, wherein the sample comprises a liquid containing sperm.

16. 16. The method of claim 15, wherein the captured portion of the sample comprises motile sperm.

17. 15. The method of claim 14, further comprising filtering at least one of debris, aggregated cells and other material, semen clots, non-motile sperm, bacteria and other non-sperm cells from the sample with a membrane layer, and attaching and filtering with said mesh layer.

18. placing the column in a second container containing a second medium; and 15. The method of claim 14, further comprising capturing a second portion of the sample that exits the column through the mesh layer and membrane layer with a second medium.

19. 15. The method of claim 14, wherein the container comprises a centrifuge tube, a cell culture tube, a blood tube, a vial, or a cuvette.

20. 1. A method for assembling a device, comprising: aligning a mesh layer and a membrane layer; bonding the mesh layer to the membrane layer to form a two-ply material; forming a column of a predetermined diameter and length by conforming two plies of material, the column having a top and a bottom, a membrane layer of the two plies of material forming an exterior of the column and a mesh layer of the two plies of material forming an interior of the column; Binding the input component to the top of the column; and connecting a nosepiece to the bottom of the column.

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