Method and apparatus for sperm selection

Abstract

A sperm sorting device is disclosed. The sperm sorting device comprises a substrate having a functionalized surface, the functionalized surface comprising a sperm selection agent capable of sorting sperm based on at least one biological or chemical property of the sperm when the sperm is in contact with the functionalized surface.

Description

Priority document

[0001] This application claims priority to Australian Provisional Patent Application No. 2023902280, filed on July 17, 2023, entitled “SPERM Sorting Apparatus and Method”, and Australian Provisional Patent Application No. 2024900010, filed on January 2, 2024, entitled “Method and Apparatus for SPERM Selection”, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This disclosure generally relates to assisted reproductive technologies, and more specifically to the sorting or selection of sperm (sperm cells, sperm) for in vitro fertilization, intracytoplasmic sperm injection and artificial insemination. Background Technology

[0003] In warm-blooded animal species (including humans), fertilization occurs through the fusion of a sperm cell and an oocyte. This fertilized oocyte then divides to form an embryo. Over the past few decades, the use of assisted reproductive technologies (ARTs) has allowed scientists and clinicians to assist in successful reproduction or treat poor fertility, or to store sperm, eggs, or embryos for use in other locations or at other times. Various ARTs exist for human or animal reproduction. For example, in vitro fertilization (IVF) involves collecting eggs, then mixing the eggs and sperm and culturing the embryos before transferring them to the female's uterine cavity to continue development. Intracytal sperm injection (ICSI) involves injecting a single sperm into an oocyte to induce fertilization and embryonic development. Artificial insemination (AI) techniques, including intrauterine insemination (IUI) and intracervical insemination (ICI), are other forms of ART used to promote conception.

[0004] Sperm in ejaculated semen are not all the same. Their physical characteristics are variable, and so is their ability to fertilize an egg and develop into an embryo. It is difficult to distinguish high-quality from low-quality sperm based on their appearance, and the physical characteristics that differentiate them are not well understood. Typically, but not always, a decline in sperm developmental quality is accompanied by changes in morphological characteristics (e.g., the shape of the sperm head or tail) or motility (e.g., the intensity and direction of motility). In standard IVF, the egg is exposed to a pool of motile sperm typically prepared using crude purification or enrichment techniques based on physical characteristics such as morphological features or motility. For example, in standard IVF, sperm are usually selected using "swim-up" or gradient centrifugation techniques. Not all sperm with reduced developmental quality are removed by these techniques, and this is considered a significant cause of fertilization failure. In ICSI, typically only a single sperm is selected based on morphological characteristics and injected into the oocyte. Attention should be paid to the potential adverse health consequences in offspring resulting from the ICSI process. In AI, sperm can be delivered into the female reproductive tract as raw ejaculate (unpurified or enriched), or sperm can be purified or enriched using a range of techniques used in IVF.

[0005] Currently, the inability to clearly distinguish, enrich, or select the most capable sperm for fertilization and embryonic development is a limiting factor in ART. Despite seemingly normal physical characteristics, many sperm possess suboptimal or impaired functional and developmental quality. This can include damage to nuclear or mitochondrial DNA or other critical cellular components, resulting from reactive oxygen species and other causes. Damaged or suboptimal sperm often exhibit apoptosis or a pre-apoptotic state, but they may retain normal morphology and motility. If fertilization occurs with damaged or suboptimal sperm, the sperm may transmit undesirable quality to the fertilized oocyte, leading to embryonic death and / or problems later in development manifesting as birth defects, placental abnormalities, and / or pregnancy complications and / or impaired offspring health. In particular, selecting suboptimal quality sperm for injection involves causing adverse health effects induced by ICSI.

[0006] Other techniques have been used for sperm selection in ART. These include annexin V magnetic activated cell sorting (AV-MACS), which uses paramagnetic annexin V-conjugated microbeads to eliminate sperm with an external phosphatidylserine residue, considered a hallmark of apoptosis. Recently, a biomimetic microfluidic selection platform has been proposed that provides improved sperm quality metrics compared to upstream methods (Warkiani et al., 2022). This platform uses a microfluidic device with superparamagnetic microbeads and neodymium magnets. However, magnetic fields, exposure to microbeads, and other manipulations may pose a risk of damaging or altering sperm. For example, magnetic fields have been shown to increase ROS expression in human cells. Additionally, when used for fertilization, sperm exposed to magnetic microbeads can carry the microbeads into the oocyte. Therefore, the use of microbeads and magnetic fields may affect the fertilization and developmental potential of sperm selected using these techniques and may have adverse consequences for the fetus and offspring.

[0007] Another sperm screening technique already in use is hyaluronic acid (HA)-bound or physiological intracytoplasmic sperm injection (PICSI), which involves sperm binding to a substrate coated with hyaluronic acid, followed by physical removal of the bound sperm for fertilization. Hyaluronic acid-selected sperm exhibit reduced levels of DNA damage and aneuploidy. It has been reported that using hyaluronic acid-based sperm selection for ICSI (so-called physiological ICSI (PICSI)) reduces the proportion of pregnancies ending in miscarriage. However, the effects of PICSI or similar positive selection methods on sperm integrity and subsequent embryonic and fetal development and viability are uncertain. In another technique, sperm with a high negative surface charge, or "zeta potential," are selected for ICSI because they are mature and more likely to have intact chromatin. Furthermore, X-containing sperm carry a greater negative charge. Sperm separation has also been achieved by electrophoresis (see, for example, WO2018 / 071977). The effects of exposure to electric fields and electrophoresis, and subsequent embryonic and fetal development, on sperm integrity are also uncertain.

[0008] Unfortunately, all current techniques for sperm selection in ART are limited by (a) not truly addressing sperm fertilization and developmental capabilities, and / or (b) the risk of sperm damage or harm. Therefore, improvements in sperm preparation techniques are needed to enhance the success rate of assisted reproductive interventions and offspring health outcomes.

[0009] Therefore, there is a need for an apparatus and / or method for sperm selection in ART that improves or reduces one or more of the disadvantages of known apparatus and / or methods. Alternatively or additionally, there is a need for an apparatus and / or method for sperm selection in ART that is a useful alternative to known apparatus and / or methods. Summary of the Invention

[0010] In a first aspect, this document provides a sperm sorting apparatus comprising a substrate having a functionalized surface containing a sperm selector that, when sperm comes into contact with the functionalized surface, the sperm selector is capable of retaining the sperm based on at least one biological or chemical property of the sperm.

[0011] A functional surface can also be considered a functionalized surface because the function has been applied to or assigned to the surface.

[0012] In a second aspect, this document provides a sperm sorting apparatus for removing sperm with reduced retention capacity from a sperm sample intended for ART, the apparatus comprising a substrate having a functionalized surface containing a sperm selector that, when sperm come into contact with the functionalized surface, preferentially removes sperm with reduced retention capacity based on at least one biological or chemical property of the sperm with reduced retention capacity.

[0013] The sperm sorting device of the first or second aspect is particularly suitable for sorting sperm based on fertilization capacity for use in ART.

[0014] In some embodiments of the first and second aspects, a functionalized surface is present in one or more flow channels having an inlet for delivering sperm into the flow channel and an outlet for harvesting sorted sperm from the flow channel, the flow channel providing a flow path for sperm from the inlet to the outlet.

[0015] In certain embodiments of the first and second aspects, the sperm selector is a biological or chemical agent that covalently binds to the substrate, either directly or indirectly. The covalent binding of the sperm selector to the substrate is advantageous because it prevents the transfer of the sperm selector and / or related materials and chemicals to the sperm to be used for fertilization.

[0016] In certain embodiments of the first and second aspects, the sperm selector is a biological or chemical agent that directly or indirectly non-covalently binds to the substrate. The non-covalent binding of the sperm selector to the substrate can occur when it is appropriate for the chemical to be released into a soluble phase or to interact with sperm intended for fertilization, for example when the chemical is a naturally occurring biochemical substance commonly found in semen or the female reproductive tract.

[0017] In certain embodiments of the first and second aspects, the sperm selector comprises a reagent that preferentially captures and / or retains apoptotic sperm or sperm in a pre-apoptotic or damaged state. The reagent may be a biological agent. The biological agent may be selected from, but is not limited to, antiphosphatidylserine antibodies, antifibronectin antibodies, annexin V, and hyaluronic acid. These reagents reproduce the natural biological retention characteristics of abnormal sperm, which exhibit altered membrane characteristics after oxidative stress.

[0018] In certain other embodiments of the first and second aspects, the sperm selector includes agents that promote sperm endurance and / or prime the sperm for fertilization. The agent that promotes sperm endurance and / or primes the sperm for fertilization may be progesterone. Progesterone acts as a sperm chemotaxis and survival factor.

[0019] In some embodiments of the first and second aspects, the sperm sorting device includes a first functionalized surface and a second functionalized surface, the first functionalized surface including a sperm selector that preferentially captures apoptotic sperm, and the second functionalized surface including a sperm selector that promotes sperm tolerance and enables sperm capacitation in preparation for fertilization. In some embodiments of these embodiments, the first functionalized surface and the second functionalized surface are spatially separated from each other on a substrate. In some other embodiments of these embodiments, the first functionalized surface and the second functionalized surface at least partially overlap each other on a substrate.

[0020] In some embodiments of the first and second aspects, the sperm in contact with the functionalized surface is in the form of liquefied semen, which is not mixed with any other material or reagent before contact with the functionalized surface. This advantageously minimizes the risk of any type of contamination and also simplifies the process because it eliminates the need to mix the liquefied semen with reagents and incubate the mixture at room temperature for 15 minutes, as might be required in prior art magnetically activated cell sorting (MACS) and AV-MACS techniques.

[0021] In a third aspect, this article provides a method for preparing semen for fertilization using the sperm sorting apparatus of the first or second aspect, the method comprising: Sperm are brought into contact with the functionalized surface before selection; Separating sperm that are not trapped on the functionalized surface from sperm that are trapped on the functionalized surface; and High-potency sperm are harvested from the sperm sorting device.

[0022] In the fourth aspect, this article provides a method for preparing the sperm sorting device of the first or second aspect, the method comprising: Provide base materials; Depositing a functional film on the surface of the substrate; and A sperm selector is attached to the functional membrane, the sperm selector being able to interact with the sperm based on at least one biological characteristic of the sperm.

[0023] In some embodiments, the sperm sorting apparatus of the first or second aspect further includes a sperm sorting region comprising a surface architecture that interacts with sperm motility. Therefore, in a fifth aspect, this document provides a sperm sorting apparatus comprising a substrate having one or more flow channels having an inlet for delivering sperm into the flow channel and an outlet for harvesting sorted sperm from the flow channel, the flow channel providing a flow path for sperm from the inlet to the outlet, the flow path including at least one biological sperm sorting region capable of sorting sperm based on biological properties and at least one motile sperm sorting region capable of sorting sperm based on physical properties.

[0024] The fifth aspect of sperm sorting devices may be particularly useful for sorting sperm based on fertilization capacity for IVF.

[0025] In some embodiments of the fifth aspect, the at least one biological sperm classification region includes a surface functionalized with one or more biological reagents capable of interacting with sperm. For example, a surface functionalized with one or more biological reagents capable of interacting with sperm may be a surface that preferentially captures apoptotic sperm (including pre-apoptotic sperm and DNA-damaged sperm). A surface preferably capable of capturing apoptotic sperm may be functionalized with an antiphosphatidylserine antibody. Antiphosphatidylserine antibodies replicate the interception of abnormal sperm mediated by immune cells, which exhibit altered membrane characteristics after oxidative stress. In another example, a surface functionalized with one or more biological reagents capable of interacting with sperm may be a surface that promotes sperm endurance and prepares sperm for fertilization by capacitation. A surface that promotes sperm endurance and prepares sperm for fertilization by capacitation may be functionalized with progesterone. Progesterone acts as a sperm chemotaxis and survival factor.

[0026] In some embodiments of the fifth aspect, the sperm sorting device includes a first biological sperm sorting zone, the first biological sperm sorting zone including a surface that preferentially captures apoptotic sperm, wherein the first biological sperm sorting zone is upstream of a second biological sperm sorting zone (i.e., closer to the entrance of the channel), and the second biological sperm sorting zone includes a surface that promotes sperm endurance and enables sperm capacitation in preparation for fertilization.

[0027] In some embodiments of the fifth aspect, the motile sperm sorting region includes a surface architecture that interacts with sperm motility. The motile sperm sorting region replicates aspects of the labyrinthine features of the mammalian oviduct.

[0028] In a sixth aspect, this article provides a method for preparing semen for fertilization using the sperm sorting apparatus of the fifth aspect, the method comprising: The sperm addition step involves adding sperm to the entry point before selection; The sperm sorting step involves, under conditions where the sperm interact with the biological sperm sorting area and the motile sperm sorting area, bringing the sperm in the channel into contact with the biological sperm sorting area and the motile sperm sorting area, and... The harvesting step involves recovering the sperm that has traveled along the channel at the outlet.

[0029] In the seventh aspect, this article provides a method for preparing the sperm sorting device of the fifth aspect, the method comprising: Provide a substrate having one or more flow channels; A functional film is deposited on the surface of one or more flow channels; A biological reagent capable of interacting with sperm is attached to the functional membrane under conditions that at least one biological sperm sorting zone is formed in the channel; and Nanoparticles are covalently attached to the functional membrane under the condition that at least one motile sperm sorting zone is formed in the channel.

[0030] Brief description of the attached figures Schemes of this disclosure will be discussed with reference to the accompanying drawings, in which: Figure 1 The process of surface engineering and characterization is illustrated. (A) Schematic diagram of the deposition of polyoxazoline (PPOx) on a surface using plasma polymerization and its subsequent interaction with biomolecules. (B) The interaction of PPOx with COOH groups on biomolecules leads to the formation of covalent amide bonds. (C) XPS measurement spectrum of a clean glass surface. (D) XPS measurement spectrum of a PPOx-coated glass surface. (E) High-resolution C1s spectrum (50 W, 5 min). (F) Immunofluorescence staining images of progressively increasing concentrations of anti-PS fixed on PPOx-coated glass. Glass coated with PPOx without anti-PS coating is included as a control.

[0031] Figure 2 The results of a study on the biocompatibility of PPOx-coated surfaces with sperm integrity and function are shown. Sperm were incubated with PPOx-coated glass coverslips ('PPOx'), uncoated coverslips ('glass'), or tissue culture plate wells ('TCP') at 37°C and 5% CO2 for 1 hour and 5 hours, respectively, and their motility (A and B) and total motility (C and D) were assessed after incubation for 1 hour (A and C) and 5 hours (B and D). A fourth group contained sperm recovered using standard upstream techniques. The results for each of the four donor semen samples are shown as symbols of different shapes.

[0032] Figure 3The effect of incubation with PPOx-coated, surface-fixed antiphosphatidylserine (anti-PS) coverslips on sperm selection. (A) Representative optical microscopic images showing washed sperm attached to increasing concentrations (50, 100, 200, and 400 μg / mL) of surface-fixed anti-PS antibody. (B) Proportion of sperm adhering to various concentrations of surface-fixed anti-PS after incubation for 30–60 minutes. A density of 1 x 10⁻⁶ μg / mL was used. 6 Washed sperm in G-IVF with preheating and CO2 ventilation TM Plus was incubated at 37°C and 5% CO2 in contact with surface-modified coverslips. (C) Average sperm adhesion on surface-fixed anti-PS at 200 μg / mL (using washed sperm prepared from n=5 different males via upstream). (D) The proportion of sperm undergoing early or late apoptosis (apoptotic sperm) labeled with annexin V was assessed by flow cytometry. (E) The proportion of necrotic (dead) sperm was assessed by flow cytometry using propidium iodide (PI) staining. Results for each of the 5 sperm preparations are shown as symbols of different shapes.

[0033] Figure 4 This section compares the performance of channelized slides with standard upstream techniques used for sperm selection. (A) Schematic images of upstream and channelized slide techniques. (B) Comparison of forward-moving sperm recovery after upstream treatment using channelized slides with different channel heights (0.1, 0.2, 0.4, 0.6, and 0.8 μm). (C) Percentage of sperm DNA fragmentation (%SDF) as determined by Halosperm® analysis. (D, E) Show total sperm motility and forward motility values. Results for each of the five sperm donors are shown as symbols of different shapes. The channel height for CE is 0.4 μm.

[0034] Figure 5The results of mouse sperm selection using functionalized surfaces are shown. (A) Representative flow cytometry plot showing the performance of annexin V and PI in sperm after separation from the epididymis. (B) Representative flow cytometry plot showing the performance of annexin V and PI in sperm that remained unattached after incubation with anti-PS coated coverslips. (C) Representative flow cytometry plot showing the performance of annexin V and PI in sperm after selection using channelized slides. (D) Representative flow cytometry plot showing the performance of annexin V and PI in sperm after selection using anti-PS coated channelized slides (slide height 0.4 mm). (E and F) Performance of annexin V and PI in sperm recovered after incubation with coated or channelized slides. Total sperm motility (G) and progressive motility (H) in sperm recovered after incubation with coated or channelized slides were assessed. (I) Percentage of sperm DNA fragmentation (%SDF) in sperm recovered after incubation with coated coverslips or channelized slides, as determined by flow cytometry and TUNEL staining. Results for each of the six sperm preparations are shown as symbols of different shapes.

[0035] Figure 6 shows a schematic diagram of the fabrication of a gold nanoparticle-coated channelized slide, including regions coated with anti-PS and progesterone ('NGS Device'). (A) Covalent immobilization of COOH-GNP is achieved by surface coating with PPOx, followed by a second functional layer of gold nanoparticles using PPOx; (B) Anti-PS antibody (200 μg mL) is applied. -1 (C) TEM image of 68 nm gold nanoparticles, and (D) Atomic force microscopy (AFM) image of surface-bonded 68 nm gold nanoparticles after 6 h of incubation.

[0036] Figure 7Results of human sperm quality assessment after preparation using an NGS device are shown. (A) Representative flow cytometry plots showing annexin V positivity and PI uptake in sperm from raw semen samples. (B) Representative flow cytometry plots showing annexin V positivity and PI uptake levels in sperm recovered via conventional rapid techniques. (C) Representative flow cytometry plots demonstrating annexin V positivity and PI uptake in sperm recovered via an NGS device. (D) Comparison of annexin V expression and (E and F) sperm percentage with DNA fragmentation (%SDF), as measured by Halosperm® from sperm from raw semen, conventional upstream, uncoated channelized slides, and the NGS device. Total motility (G) and forward motility (H) of sperm prepared using each protocol are also compared. Visual representation of sperm DNA fragmentation in the original sample of liquefied semen as determined by Halosperm® staining (I) Leftmost circle = medium to large halo, indicating unfragmented sperm DNA; second circle from the left = small halo, indicating sperm DNA fragmentation; third circle from the left = no halo, indicating extensive sperm DNA fragmentation; and rightmost circle = degraded sperm). (J) Sperm staining after Halosperm® preparation using standard rapid techniques (top circle = small halo, indicating sperm DNA fragmentation; bottom circle = no halo, indicating extensive sperm DNA fragmentation). (K) Sperm staining after Halosperm® via NGS device. Results from each of the two different semen donors are shown as circles and squares, respectively.

[0037] Figure 8Results of the study on the role of different selective components of the NGS device in contributing to optimal sperm selection and quality are shown. Channelized slides were coated with all the components described in Example 6 ('NGS device', e.g., antiphosphatidylserine (anti-PS), gold nanoparticles (GNP), and progesterone (P)), with GNP and P but without anti-PS ('Chan_GNP_P'); with GNP and anti-PS but without P ('Chan_GNP_anti-PS'); with antiPS and P but without GNP ('Chan_anti-PS_P'); with P but without anti-PS or GNP ('Chan_P'); with anti-PS but without P or GNP ('Chan_anti-PS'); with GNP but without anti-PS or P ('Chan_GNP'); or uncoated ('Chan'). Unselected sperm ('raw') and sperm collected via standard upstream techniques ('upstream') were used for comparison. The quality of the recovered sperm was measured by the proportion of sperm stained with annexin V (A) and the proportion of PI-positive sperm after recovery following exposure to different sperm selection strategies (B). Results from each of the two different sperm donors are shown as circles and squares, respectively.

[0038] Figure 9 Images of anti-Fn IF staining at a concentration of 200 μg / mL are shown on a plasma-polymerized PPOx surface after passivation (backfilling) with 5% HSA (12 h reaction time). The presence of functional anti-Fn molecules is revealed through interaction with FITC-labeled secondary antibodies. A) IF staining of anti-Fn on a plasma-polymerized PPOx surface, B) IF staining of anti-Fn on a clean glass surface.

[0039] Figure 10 The images show fluorescence images of sperm and monocytes stained with DAPI using a rabbit anti-human monoclonal label against Fn. A) Monocytes incubated with an isotype-matched rabbit IgG antibody control prior to treatment with FITC-labeled secondary antibody. B) Monocytes stained with anti-Fn and associated secondary antibody IF. C) IF staining of primitive sperm (including sperm and monocytes).

[0040] Figure 11 Flow cytometry results showing Fn expression on sperm surface. A) Isotype-matched rabbit IgG antibody control. B) Fn expression. C) Percentage of sperm from the 5 participants expressing Fn on the plasma membrane.

[0041] Figure 12 The image shows sperm attaching to an anti-Fn coated surface. A) Bright-field image of sperm attached to an anti-Fn coated surface. B) Percentage of attached sperm in the three participants.

[0042] Figure 13 The image shows the detection of sperm apoptosis using annexin V-FITC / PI staining. A) Fluorescence imaging of annexin V and PI-positive sperm populations. B) Quantification of annexin V / PI-positive sperm populations by flow cytometry. C, D) Flow cytometry data from primitive sperm and swim-up sperm populations: "No dye" is considered a control where cells were not stained. "PI only" means the sample was stained with PI only. "Annexin V only" means the sample was stained with Alexa Fluor only. TM 488-conjugated Annexin V staining. "Annexin V+PI", sample used with Alexa Fluor. TM Annexin V staining conjugated with 488 and PI. The x-axis represents annexin V fluorescence; the y-axis represents PI fluorescence. In each figure, the lower left quadrant shows cells that are negative for both annexin V and PI; the upper left shows necrotic PI-positive annexin-negative sperm; the lower right shows early apoptosis annexin V-positive PI-negative sperm; and the upper right shows late apoptosis or necrosis annexin V and PI-positive sperm.

[0043] Figure 14 Fluorescence imaging of annexin V-FITC bioconjugated on a plasma-polymerized PPOx surface (200 μg / ml concentration and 12 h reaction time). The surface was passivated with 5% HSA overnight and washed three times for 1 h before image capture.

[0044] Figure 15 Displaying sperm apoptosis detected using annexin V-FITC / PI staining. A, B) Bright-field imaging of attached sperm from two different participants on an annexin V-coated surface. C) Percentage of attached sperm from two different participants on an annexin V-coated surface. D, E) Quantification of annexin V / PI-positive sperm populations by flow cytometry. C, D) Flow cytometry data from unattached sperm populations on an annexin V-coated surface: "No dye" is considered a negative control, where cells are not stained as positive. "PI only," samples were stained with PI only. "Annexin V only," samples were stained with Alexa Fluor only. TM 488-conjugated Annexin V staining. "Annexin V+PI", sample used with Alexa Fluor. TM 488-conjugated annexin V and PI staining. The x-axis represents annexin V fluorescence; the y-axis represents PI fluorescence. In each figure, the lower left quadrant shows cells that are negative for both annexin V and PI; the upper left shows necrotic PI-positive annexin-negative sperm; the lower right shows early apoptosis annexin V-positive and PI-negative sperm; and the upper right shows late apoptosis or necrosis annexin V and PI-positive sperm.

[0045] Figure 16 Flow cytometry analysis showing changes in ROS production during primordial ejaculation, upstream sperm, and after incubation with an annexin V-coated surface. A) Control with unstained sperm. B) Primordial ejaculation sample. C) Upstream sperm. D) Unattached sperm on the annexin V-coated surface. E) Percentage of ROS-positive sperm population (n=1).

[0046] Figure 17 The effect of incubating sperm with annexin V-coated glass coverslips on sperm selection is shown. Representative (AD) images of attached sperm on coverslip surfaces coated with 10, 50, 100, and 200 μg / ml annexin V are presented. (E) The proportion of sperm with annexin V fixed to the surface was measured after an incubation period of 45–60 minutes. G-IVF was performed using preheated and CO2-ventilated G-IVF systems in contact with surface-modified glass coverslips. TM The incubation density in Plus medium is 1-2 × 10⁻⁶. 6 Washed human sperm were used in the experiments. Incubation was performed at 37°C and 5% CO2. (F) Average sperm adhesion to surface-fixed annexin V at a concentration of 200 μg / mL using sperm samples obtained from a total of 6 different men. (GI) The proportion of apoptotic or necrotic sperm in each treatment group was assessed by flow cytometry using FITC-labeled annexin V and PI staining. Representative flow cytometry histograms depict annexin V and PI staining in the original sample (G), upstream sample (H), and sample (I) incubated with annexin V-coated coverslips (concentration of 200 μg / mL). The proportion of apoptotic or necrotic sperm staining was quantified by FITC-labeled annexin V (J) and PI staining (K). Results obtained from each of the six different sperm preparations are shown using different symbols to depict each individual. *P≤0.01 and **P≤0.001.

[0047] Figure 18 The effects of incubating human sperm with HA and anti-FN coated glass coverslips were shown. This was achieved using G-IVF in preheated and CO2-ventilated conditions. TM The incubation density in Plus medium is 1-2 × 10⁻⁶. 6Washed sperm (n=3 individual samples per experiment) were used in the experiments. Incubation with coated coverslips was performed at 37°C and 5% CO2. (A) Representative image of sperm attached to a coverslip coated with 300 μg / ml HA. (B) The proportion of sperm attached to the surface-fixed HA was measured after an incubation period of 45–60 minutes. (C) Representative image of sperm attached to a coverslip coated with 40 μg / ml anti-Fn. (B) The proportion of sperm attached to the surface-fixed anti-Fn was measured after an incubation period of 45–60 minutes. Representative flow cytometry histograms depict annexin V staining in the original sample (E), the upstream sample (F), and the sample (G) incubated with HA-coated coverslips (300 μg / ml). Representative flow cytometry histograms depict annexin V staining in the original sample (H), upstream sample (I), and sample (J) incubated with anti-Fn coated coverslips (100 μg / ml). (K) The proportion of sperm not attached to annexin V-labeled HA-coated coverslips was calculated. (L) The proportion of sperm not attached to annexin V-labeled anti-Fn-coated coverslips was calculated.

[0048] Figure 19 The effects of sperm interactions with a range of coatings on glass coverslips were assessed. Glass coverslips were treated to produce plasma-polymerized amine or PPOx membranes, then coated with GNP and anti-PS antibodies, or with anti-PS antibodies without GNP, resulting in four different surface coating variants (NH2, GNP, PPOx, anti-PS; PPOx, GNP, PPOx, anti-PS; NH2, anti-PS and PPOx, anti-PS). Perm prepared upstream was incubated with the coated coverslips, and apoptosis was assessed by annexin V staining and motility parameters compared to an upstream control. (AG) Representative flow cytometry scatter plots for each sperm preparation. (H, I) Proportions of sperm labeled with annexin V and PI indicate apoptotic and necrotic populations, respectively. (J, K) Total sperm motility and progressive sperm motility assessed after collecting recovered sperm from different groups.

[0049] Figure 20The effects of previous G protein coating on anti-PS fixation and sperm-surface interaction are shown. (A) Schematic diagram of G protein fixation on the surface and its interaction with anti-PS and subsequently with sperm. (BE) Confirmation of G protein availability on PPOx-coated surfaces using anti-Fn labeled with Alexa Fluor 488. Fluorescent images depict the interactions between different anti-Fn concentrations (2, 5, 20, and 50 μg / ml) and the G protein-coated surfaces. The corresponding control reaction is shown in the upper left corner of each image. Scale bars are measured at 100 μm. (FH) Evaluation of G protein functional groups fixed to PPOx-coated glass coverslip surfaces by mass spectrophotometry. Mass spectrophotometric images show the relative densities of available anti-PS molecules in solution before and after incubation with different surfaces, as shown in the figure. Lower residual anti-PS concentrations are evident in solutions incubated with PPOx plus G protein-coated surfaces (H) compared to PPOx alone (G) or the control without incubation with the coated surface (F). (I) Mass spectrophotometric analysis of the relative density of anti-PS in solutions at a concentration of 50 μg / ml before and after incubation with different surfaces, as shown in the figure. (JK) Flow cytometry analysis of sperm recovered from channelized slides prepared with and without G proteins in the coating method, including (J) the proportion of sperm labeled with annexin V (apoptotic population) and (K) the proportion of sperm labeled with PI (necrotic population). Results from each of the three different sperm formulations are presented using different symbols corresponding to each formulation. Data were analyzed by ANOVA and post-hoc t-tests. *P < 0.01.

[0050] Figure 21 Comparative analyses of glass and polystyrene substrates used for surface modification and assembly into channelized slide devices for sperm selection are shown. (AD) Representative flow cytometry scatter plots, (A) without dye; (B) raw sperm or (C) sperm preparation recovered from a tissue culture plastic channelized slide (TCP-NGS) device or (D) sperm preparation recovered from a glass channelized slide (Glass-NGS) device. (EF) Flow cytometry analysis showing the proportion of sperm in raw semen or recovered sperm preparations (from TCP or glass channelized slide devices) labeled with annexin V (E) and PI (F), indicating apoptotic and necrotic populations, respectively. (G, H) Total sperm motility and progressive sperm motility assessed after collecting and recovering sperm from different groups.

[0051] Figure 22Evaluation of channelized slides with functionalized surfaces used for porcine sperm selection is shown. (A) Photomicrograph showing attached porcine sperm immobilized in a channelized slide with functionalized surfaces and resisting PS capture. (BD) Representative flow cytometry scatter plots of raw sperm (B), sperm prepared upstream (C), and sperm recovered from the channelized slide with functionalized surfaces (D). (EF) Flow cytometry analysis showing the proportion of sperm in raw semen or recovered sperm preparations labeled with annexin V (E) and PI (F), indicating apoptotic and necrotic populations, respectively. (G) Overall sperm motility assessed after collecting recovered sperm from different groups.

[0052] Figure 23 The effect of adding microchannels to the NGS device on sperm selection efficiency is shown. (A) Schematic diagram showing the addition of microchannels to the slide before NGS assembly. (B) Microscopic image of the microchannels on the slide. (C) Profile measurement of the surface morphology and depth distribution of the microchannels on the slide. (DG) Representative flow cytometry scatter plots of raw sperm (D), sperm prepared by sliding upwards (E), and sperm recovered from an uncoated NGS device with microchannels (F) and a coated NGS device with microchannels (G). (HI) Flow cytometry analysis showing the proportion of sperm in raw semen or recovered sperm preparations labeled with annexin V (H) and PI (I), indicating apoptotic and necrotic populations, respectively. (J, K) Total sperm motility and progressive sperm motility assessed after collecting recovered sperm from different groups. (l) Proportion of sperm with DNA fragmentation determined by Halosperm assay. Representative images of sperm (MO) in raw semen (M), upstream (N), and after recovery from an NGS device with added microchannels.

[0053] Implementation Plan Description The following definitions are provided for the specific terms used in the following written description.

[0054] As used in the specification and claims, the singular forms “a (a, an)” and “the” include plural references unless the context clearly specifies otherwise.

[0055] In the specification and claims, an item, object, device, apparatus, system, method, or process may be "comprising" (open-ended) or "substantially composed of" the listed elements and other components or elements described herein. As used herein, "comprising" means the recited element or its structural or functional equivalent, plus one or more other elements not recited. Unless the context otherwise requires, the terms "having" and "comprising" should also be interpreted as open-ended. As used herein, "substantially composed of" means that the invention may include components other than those listed in the claims, but only if the addition does not substantially alter the essential and novel features of the claimed invention.

[0056] The term "about" or "approximately" means within an acceptable range of a particular value as determined by someone skilled in the art, which will depend in part on how the value is measured or determined, for example, limitations of the measurement system. For example, "about" may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly concerning biological systems or processes, the term may mean within orders of magnitude of the value, preferably within 5 times, and more preferably within 2 times. Unless otherwise stated, the term "about" indicates within an acceptable range of error for a particular value, for example, ±1-20%, preferably ±1-10%, more preferably ±1-5%. In a further embodiment, "about" should be understood to mean + / - 5%.

[0057] When a range of values ​​is provided, it should be understood that every intermediate value between the upper and lower limits of the range, as well as any other stated value or intermediate value within the stated range, is covered within this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and are also covered within this invention, subject to any specific excluded limits within the range. Where the range includes one or two limits, the range excluding any or both of those included limits is also included in this invention.

[0058] Unless otherwise stated herein, all percentages and ratios used herein are by weight of the total composition. Unless otherwise stated, all temperatures are in degrees Celsius. Unless otherwise stated, all measurements were performed at 25°C and normal pressure.

[0059] All ranges listed herein include endpoints, including those listing "ranges between two values." Terms such as "about," "approximately," "basically," and "around" should be interpreted as modifying terms or values ​​so that they are not absolute, but not in the context of existing technology. These terms will be defined by the context and the terms they modify, as understood by those skilled in the art. This includes at least the extent of expected experimental, technical, and instrumental errors of the given technique used to measure the values.

[0060] When used herein, the term "and / or" when used in a list of two or more items means that any one of the listed characteristics may be present, or any combination of two or more of the listed characteristics may be present. For example, if a composition of the present invention is described as comprising characteristics A, B, and / or C, then the composition may comprise characteristic A alone; characteristic B alone; characteristic C alone; a combination of A and B; a combination of A and C; a combination of B and C; or a combination of A, B, and C.

[0061] This article discloses a sperm sorting device 10, the implementation of which is shown in [illustration]. Figure 6A In section B, the sperm sorting device comprises a substrate with a functionalized surface containing a sperm selector that, when sperm come into contact with the functionalized surface, can sort sperm based on at least one biological or chemical property of the sperm.

[0062] This article also discloses a sperm sorting device 10 for retaining sperm with reduced ability from a sperm sample to be used in ART. The device 10 includes a substrate 12 having a functionalized surface 14, the functionalized surface 14 including a sperm selector that is capable of preferentially retaining sperm with reduced ability based on at least one biological or chemical property of the sperm with reduced ability when the sperm comes into contact with the functionalized surface 14.

[0063] In the female reproductive tract, several biological mechanisms work together to select sperm with optimal characteristics and ensure that substandard sperm cannot fertilize oocytes. In fact, sperm must navigate a biological barrier course as they pass through the cervix, uterine cavity, and fallopian tubes. Along the way, they interact with immune cells and epithelial cells, and many cells are isolated (physically restricted) or eliminated by the action of these cells or their secretions, resulting in only a small percentage reaching the ampulla of the upper fallopian tube, where fertilization occurs. Along this route, the most capable and best-suited sperm enhance their ability to pass the final test of penetrating the clear oocyte band. The sperm sorting device 10 disclosed herein integrates molecular characteristics to provide a simple, reliable, and efficient selection of the best-quality sperm in ejaculate in a format suitable for application in human and animal clinical ART laboratories.

[0064] Device 10 provides a relatively simple sperm sorting technique. Many existing sperm sorting techniques involve multiple steps, such as incubation and the application of magnetic fields. These steps can potentially lead to sample loss, and the overall efficiency of the method may be affected. The liquefaction of semen with reagents and the incubation of the mixture may also have clinical considerations. In contrast, the device and method disclosed herein use immobilized sperm selectants (e.g., immobilized antibodies) to isolate sperm without mixing any reagents with the semen. This reduces the risk of sample contamination. Furthermore, the device and method disclosed herein do not require the use of any magnetic fields that could affect the fertilization potential of sperm.

[0065] Furthermore, some existing technological devices, such as the one disclosed by Warkiani et al., require the use of strong magnets and are therefore unsuitable for home use. The device and method disclosed in this paper are simpler to use and can be used for home preparation of sperm for AI via self-application.

[0066] Sperm selectors that can interact with sperm can be any molecule that has molecular interactions with sperm. Reagents that can interact with sperm can be biomolecules. Biomolecules can be, for example, selected from amino acids, peptides, proteins, steroid hormones, aptamers, nucleic acids, DNA molecules, RNA molecules, antibodies, growth factors, antimicrobial agents, antithrombotic agents, and cell adhesion proteins. Biomolecules can be in the form of particles or nanoparticles containing biomolecules.

[0067] Sperm selectors can be agents that preferentially capture and / or retain apoptotic sperm or sperm in a pre-apoptotic or damaged state. These agents can be biological agents, such as antiphosphatidylserine antibodies, antifibronectin antibodies, annexin V, and hyaluronic acid. These agents replicate the immune cell-mediated aspect of abnormal sperm retention, which exhibits altered membrane characteristics after oxidative stress. Alternatively or additionally, sperm selectors can be any agent that promotes sperm endurance and / or prepares sperm for fertilization. A suitable agent for promoting sperm endurance and / or preparing sperm for fertilization is progesterone. Progesterone acts as a sperm chemotaxis and survival factor.

[0068] Sperm selectors may include secreted and surface-bound proteins and biological agents, including cytokines and / or other immunomodulatory molecules, which participate in the interception of naturally occurring sperm and / or the killing of sperm by female immune cells or epithelial cells in the reproductive tract after secretion into the semen.

[0069] The sperm selector can be covalently attached to the substrate surface, or it can be non-covalently attached. Covalent attachment to the substrate surface can be facilitated by a functional membrane and by a reaction between one or more functional groups on the functional membrane and the sperm selector. For example, amino groups in the functional membrane can react with carboxyl, carboxylic, or aldehyde groups of the sperm selector. If desired, the reaction between one or more functional groups on the functional membrane and the sperm selector can be promoted or catalyzed. For example, the reaction between one or more functional groups on the functional membrane and the sperm selector can be promoted by a coupling agent such as a carbodiimide coupling agent (e.g., EDC, DCC, DIC), a triaminophosphonium coupling agent (e.g., BOP, PyBOP, PyBrOP), or a tetramethylammonium / tetramethylureon coupling agent (e.g., HATU, HBTU, HCTU). If desired, a crosslinking agent portion can be used between the functional membrane and the sperm selector.

[0070] There are considerable benefits associated with the covalent bonding of the sperm selector to the substrate surface, and this ensures that no chemicals are transferred to the sperm that will be used for fertilization.

[0071] The functionalized surface 14 can be present on the substrate 12 in any suitable form. Advantageously, the functionalized surface 14 is configured on the substrate 12 such that a sperm sample can pass through the functionalized surface 14 under conditions where lower-capacity sperm are isolated on the functionalized surface 14 and higher-capacity sperm pass through the functionalized surface 14 without isolation. This can be done in a variety of ways, including positioning the functionalized surface 14 in a channel that can introduce sperm. In some embodiments, the functionalized surface 14 is present in one or more flow channels 16 having an inlet 18 for conveying sperm into the flow channel 16 and an outlet 20 for harvesting sorted sperm from the flow channel 16. Thus, the flow channel 16 provides a flow path for sperm from the inlet 18 to the outlet 20. In the illustrated embodiment, the sperm sorting device 10 includes a glass microscope slide 30, characterized by a 500 μm × 5 cm flow channel 16 and a 100 μL reservoir at the inlet 18 and outlet 20 of the flow channel 16. The lower glass surface of flow channel 16 is functionalized with (1) a covalently bound antiphosphatidylserine (anti-PS) antibody and (2) ionically (non-covalently) bound progesterone on the substrate surface. The antiphosphatidylserine forms a first functionalized surface 14a, which replicates immune cell-mediated interception of abnormal sperm (which exhibits altered membrane characteristics after oxidative stress), and the progesterone forms a second functionalized surface 14b, wherein progesterone is a survival factor that promotes sperm endurance and prepares sperm for fertilization by capacitation.

[0072] Biological reagents, such as antiphosphatidylserine antibodies, antifibronectin antibodies, annexin V, and hyaluronic acid, are covalently bound to the surface of flow channel 14 by surface functionalization with a functional membrane capable of covalently binding to antiPS antibodies.

[0073] Any suitable substrate can be used, provided that the functional membrane can be formed on its surface and can be maintained on its surface under typical operating conditions. Suitable substrate materials include glass, silicon, ceramics, metals, plastics, polymer materials, paper, paper laminates, cellulose, carbon fibers, biomaterials, surfaces containing biomolecules, surfaces containing small organic molecules, surfaces containing inorganic molecules, etc. Plastics can be selected from: polycarbonate, polyethylene, polypropylene, polystyrene, polytetrafluoroethylene, polyethylene terephthalate; polyethylene naphthalate, tetrafluoroethylene-hexafluoropropylene copolymer, polyvinyl fluoride, nylon, polyvinyl chloride, copolymers of the above and mixtures thereof. In one embodiment, the substrate is glass. In other embodiments, the substrate is polystyrene.

[0074] The substrate has one or more flow channels 16. The one or more flow channels 16 can be of any suitable size, geometry, or configuration. The one or more flow channels 16 can be open or closed, such as channels typically used in "flow-through" type diagnostic devices. In some embodiments, the substrate includes one or more microfluidic flow channels or microchannels 16. As used herein, the terms "microfluidic," "microchannel," and variations thereof refer to a chip, device, apparatus, substrate, channel, or related device containing a fluid control feature having at least one submillimeter dimension.

[0075] Optionally, the inner surface of one or more flow channels 16 may be coated with a material having different properties from the substrate material, such as poly(dimethylsiloxane) (PDMS) or epoxy resin.

[0076] Optionally, one or more flow channels 16 may be defined by three-dimensional (3D) patterns or elements to alter surface morphology and enhance the interaction between sperm and functionalized surface 14. As a non-limiting example, functionalized surface 14 (such as flow channel 16) may include nanoparticles, such as gold nanoparticles, covalently bonded to the surface. Alternatively, or additionally, functionalized surface 14 (such as flow channel 16) may include grooves or etched surfaces to which a functional layer is applied. 3D printing can be used to fabricate a series of surfaces having some form of 3D patterning or morphology, on which functional layers can be added.

[0077] The sperm sorting device 10 is particularly suitable for sorting sperm to perform ART in humans and other mammals, including rodents (rats, mice, hamsters, guinea pigs, etc.), primates, apes (including monkeys, great apes, etc.), monkeys (including marmosets, baboons, etc.), great apes (including gorillas, chimpanzees, orangutans, gibbons, etc.), farm and livestock animals (including sheep, goats, pigs, cattle, horses, etc.), track animals, endangered species of animals (including pandas, etc.), and pets such as dogs, cats, etc.

[0078] Functional membranes may contain any inorganic, organic and / or biological materials, molecules or mixtures of molecules that can be bonded to the substrate surface via nonionic bonding.

[0079] Alternatively, the functional membrane may comprise any inorganic, organic, and / or biological material, molecule, or mixture of molecules, which may be covalently or ionicly bonded to the substrate surface and contain one or more functional groups that can be covalently or ionicly bonded to at least one sperm selector used for functionalizing the surface 14. One or more functional groups of the functional membrane are preferably capable of bonding to carboxylic acid groups, carboxylic ester groups, amino or amide groups on a biomolecule.

[0080] Examples of chemicals suitable for covalently linking biological sperm selectors include oxazolines, amines, epoxy resins, aldehydes, acid anhydrides, thiols, EDC / NHS-related chemicals, click chemicals, isocyanates, nitriles, and imines.

[0081] In some embodiments, the functional membrane is a polymer. Polymers can be formed using classical polymerization techniques. Thus, as is known in the polymer field, polymers can be formed on the surface of a substrate by polymerizing suitable starting monomers or prepolymers using a suitable polymerizing agent.

[0082] In certain other embodiments, the functional membrane can be formed by plasma polymerization. Thus, in some embodiments, the functional membrane is a plasma polymer formed by plasma polymerization of one or more functional starting materials. Starting materials containing oxazoline, amine, epoxy, aldehyde, acid anhydride, thiol, isocyanate, nitrile, and imine groups can be plasma polymerized using the conditions described herein to form a plasma polymer containing oxazoline, amine, epoxy, aldehyde, acid anhydride, thiol, isocyanate, nitrile, and imine groups, which can then be reacted with a biological reagent capable of interacting with sperm to form covalent bonds therewith.

[0083] Non-limiting examples of starting materials that may be used include 2-substituted oxazolines, 4-substituted oxazolines, 5-substituted oxazolines, 2,4-disubstituted oxazolines, 2,5-disubstituted oxazolines, 4,5-disubstituted oxazolines, 2,4,5-trisubstituted oxazolines, allylamine, propionaldehyde (i.e., propionaldehyde), glycidyl methacrylate, and allyl glycidyl ether.

[0084] In some embodiments, the functional membrane is a plasma-polymerized polyoxazoline (“PPOx”). As used herein, the term “polyoxazoline” refers to a homopolymer or copolymer formed from at least one oxazoline starting material or monomer. The polyoxazoline polymer may or may not contain a complete oxazoline moiety. The polyoxazoline polymer may be a copolymer formed by plasma polymerization of at least one oxazoline starting material or monomer and at least one comonomer. The comonomer may be selected based on the desired properties it can provide to the polyoxazoline polymer and / or its suitability for plasma polymerization (e.g., its vapor pressure or volatility). The comonomer may be selected from, but is not limited to: silanes, siloxanes, fluorocarbons, hydrocarbons, reactive functional monomers, organic monomers, and unsaturated monomers such as N-vinylpyrrolidone, hydroxyethyl methacrylate, acrylamide, dimethacrylamide, dimethylaminoethyl methacrylate, acrylic acid, methacrylic acid, vinyl-substituted polyethylene glycol or polypropylene glycol, vinylpyridine, and vinyl sulfonic acid.

[0085] Plasma-polymerized polyoxazoline polymers and functional films on substrate surfaces can be prepared by exposing the substrate surface to plasma containing oxazoline monomer vapor while polymerizing oxazoline monomers to form plasma-polymerized polyoxazoline polymers on the substrate surface.

[0086] Conditions required for the polymerization of oxazoline monomers to form plasma-polymerized polyoxazoline polymers may include a power of about 10 W to about 50 W, a deposition time of about 1 minute to about 7 minutes, and / or about 0.5 to about 3 x 10⁻⁶. -1 A monomer pressure of mbar. A power of approximately 50 W for about 2 minutes is particularly suitable for polymerizing oxazoline monomers to form plasma-polymerized polyoxazoline polymers, as these conditions provide stable plasma-polymerized polyoxazoline polymer films with thicknesses greater than approximately 20 nm, such as approximately 20 nm to 40 nm or 20 nm to 30 nm. Depending on the plasma deposition equipment design and power coupling efficiency, a range of other deposition conditions will be applicable.

[0087] A plasma containing oxazoline monomer vapor is formed under reduced pressure in a vacuum chamber. Therefore, the step of exposing the surface of a substrate to a plasma containing oxazoline monomer vapor may include placing the substrate in the chamber, sealing the chamber, forming the plasma in the chamber, introducing the oxazoline monomer vapor into the chamber, and maintaining the substrate at a temperature suitable for oxazoline monomer polymerization to form a polymer film on the surface. Those skilled in the art will understand that plasma is one or more electrically excited ionized gases that, upon excitation (e.g., ignition), create a highly reactive environment that can alter the properties of materials directly exposed to plasma discharge. The plasma deposition step can be operated at a wide range of pressures (e.g., from 10 mTorr to above atmospheric pressure (e.g., 10 x atmospheres or higher)). The plasma may consist of a combination of inert gases (e.g., helium, neon, argon, krypton, xenon, radon) and oxazoline monomers (or other suitable monomers required depending on the chemical properties of the functional membrane). The plasma can be formed at a range of frequencies (low-frequency direct current (DC) and alternating current (AC), pulsed DC, radio frequency (RF), and microwave).

[0088] The plasma polymerization conditions described above can also be used to form plasma polymers from other functional starting materials as needed.

[0089] The oxazoline monomer can be a substituted oxazoline having a substituent at any one of the 2-, 4-, or 5-positions of the oxazoline ring, or any combination of these substituents. Any of these oxazolines can be used to form a plasma-polymerized polyoxazoline polymer, provided that they are vapors under the plasma deposition conditions used. In embodiments, the oxazoline monomer is selected from 2-substituted oxazolines, 4-substituted oxazolines, 5-substituted oxazolines, 2,4-disubstituted oxazolines, 2,5-disubstituted oxazolines, 4,5-disubstituted oxazolines, and 2,4,5-trisubstituted oxazolines. The substituents on the oxazoline ring can be selected from: halogens, OH, NO2, CN, NH2, optionally substituted C1-C12 alkyl, optionally substituted C2-C12 alkenyl, optionally substituted C2-C12 alkynyl, optionally substituted C3-C12 cycloalkyl, optionally substituted C2-C12 heterocycloalkyl, optionally substituted C2-C12 heterocycloalkenyl, optionally substituted C6-C18 aryl, optionally substituted C1-C18 heteroaryl, optionally substituted C1-C12 alkoxy, optionally substituted C2-C12 alkenyloxy, optionally substituted C2-C12 heteroalkoxy, optionally substituted C3-C12 cycloalkoxy, optionally substituted C3-C12 cycloalkenyloxy, optionally substituted C1-C12 Heterocyclic alkoxy groups, optionally substituted C2-C12 heterocyclic alkenoxy groups, optionally substituted C6-C18 aryloxy groups, optionally substituted C1-C18 heteroaryloxy groups, optionally substituted C1-C12 alkylamino groups, SO3H, SO2NH2, SO2R, SONH2, SOR, COR, COOH, COOR, CONR ' NRCOR ' NRCOOR ' NRSO2R ' 、N RCONR ' R " And NRR'. In some embodiments, the oxazoline monomer comprises a 2-substituted oxazoline. In a specific embodiment, the oxazoline monomer is a 2-alkyl-2-oxazoline. The alkyl substituent can be C1-C1. 12 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, etc.

[0090] In a specific embodiment, the oxazoline monomer is selected from 2-alkyl-2-oxazoline and 2-aryl-2-oxazoline. The alkyl substituent of the 2-alkyl-2-oxazoline can be C1-C2. 10Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, etc. The alkyl substituents can be optionally substituted. The aryl substituents in 2-aryl-2-oxazoline can be C5-C. 10 Aryl groups, such as optionally substituted phenyl, optionally substituted naphthyl, optionally substituted thiophene, optionally substituted indolyl, etc.

[0091] In some other embodiments, the functional membrane is a plasma-polymerized amine. The amine polymer can be formed by plasma polymerization of at least one unsaturated amine monomer, such as allylamine.

[0092] The surface of the substrate can be treated before the deposition of the plasma-polymerized polyoxazoline polymer. For example, the surface can be treated by cleaning with detergent, water, or a suitable solvent. Alternatively, the surface can be treated by exposing it to air in the plasma chamber to activate it.

[0093] The reactivity of the oxazoline ring present at the ω-terminus of polyoxazolines has been used for conjugation with proteins and drugs in solution. It is believed that the reactivity of the oxazoline ring leads to the formation of covalent amide bonds through reaction with carboxylic acid functional groups. Plasma-deposited packages are allowed to retain the complete oxazoline ring on the surface, which is not typically the case when using other surface preparation techniques. This retention of the reactive chemical functional groups then allows for convenient and rapid covalent coupling of proteins, antibodies, etc.

[0094] Optionally, the functional membrane may also include additional binding portions capable of covalently binding to the functional membrane, such as plasma-polymerized polyoxazoline or amine polymers, and also capable of binding to sperm selectors of interest. These additional binding portions may comprise any inorganic, organic, and / or biological material, molecule, or mixture of molecules. For example, G proteins can be used for antibody attachment to surfaces due to their strong affinity for the Fc region of immunoglobulin (antibody) proteins.

[0095] Sperm can be delivered to inlet 18 (and therefore to channel 16) in any suitable form, including as a semen sample, sperm, and fluid. The semen sample used can be a fresh semen sample or a stored semen sample. Any storage method can be used to store the semen sample, such as freezing the semen sample.

[0096] To sort sperm in the sperm sorting device 10, a sperm preparation medium is introduced into the channel. Any suitable sperm preparation medium can be used, containing a fluid medium supporting cell culture, such as IVF medium, sperm preparation medium, and / or isotonic saline solution. The sperm preparation medium may contain substances for further protecting the sperm, such as substances protecting the sperm from free oxygen radicals. Sperm-protecting substances may be, for example, EDTA, albumin, and / or proteins. Examples of liquid culture media that can be used are described in PCT / EP97 / 06721, which is incorporated herein by reference. A range of suitable sperm preparation media are also commercially available. For example, commercially available G-IVF from Vitrolife can be used. TM Plus medium. In use, sperm delivered to inlet 18 can migrate from inlet 18 to outlet 20 in the sperm preparation medium, and in doing so, must interact with functionalized surfaces 14 (which may include a single functionalized surface 14 or may include a first functionalized surface 14a and a second functionalized surface 14b) before reaching outlet 20, which can be recovered at outlet 20.

[0097] Optionally, the sperm to be sorted in the sorting device 10 may be pre-screened. Any suitable known sperm sorting method may be used for pre-screening, such as (for example) standard rapid methods for separating motile sperm.

[0098] For use with the sperm sorting device 10, semen can be introduced into the reservoir at inlet 18, and commercial sperm preparation media can be introduced into the reservoir at outlet 20 and flow channel 16. The sperm sorting device 10 can then be incubated under suitable conditions, after which sperm can be recovered from the reservoir at outlet 20. Any suitable incubation conditions can be used and can be determined by those skilled in the art. For example, incubation conditions can be 37°C and 6% CO2 for 45-90 minutes. The sperm recovered from outlet 20 is ready for use in ART and has already had its seminal plasma components removed (no further acceleration by centrifugation or washing, etc., is required).

[0099] This paper also discloses a method for preparing semen for fertilization using the sperm sorting device disclosed herein. The method includes contacting sperm with a functionalized surface prior to selection. Sperm not isolated on the functionalized surface are then separated from sperm already isolated on the functionalized surface, and high-potential sperm are harvested from the sperm sorting device.

[0100] This document also discloses a method for preparing a sperm sorting device as disclosed herein. The method includes: providing a substrate; depositing a functional membrane on the surface of the substrate; and attaching a sperm selector to the functional membrane, the sperm selector being capable of interacting with the sperm based on at least one biological characteristic of the sperm.

[0101] This article also discloses the sperm sorting device 10, the implementation of which is shown in [illustration]. Figure 6B The device 10 includes a substrate 12 having one or more flow channels 14, each flow channel 14 having an inlet 16 for delivering sperm into the flow channel 14 and an outlet 20 for harvesting sorted sperm 22 from the flow channel 14. The flow channel 14 provides a flow path for sperm from the inlet 16 to the outlet 20. The flow path includes at least one biological sperm sorting region 22 capable of sorting sperm based on biological characteristics and at least one motile sperm sorting region 24 capable of sorting sperm based on physical characteristics.

[0102] Advantageously, Figure 6B The sperm sorting device 10 disclosed herein reproduces the naturally occurring sperm selection process in the human female reproductive tract after sexual intercourse. In the female reproductive tract, several biological mechanisms work together to select sperm with optimal characteristics and ensure that substandard sperm cannot fertilize oocytes. In fact, as sperm navigate the tortuous labyrinth of the cervix, uterine cavity, and fallopian tubes, they must pass through a kind of biological barrier track. Along the way, they interact with immune cells and epithelial cells, and many cells are isolated (physically restricted) or eliminated by the action of these cells or their secretions, or they cannot pass through the physical barriers of the cervix, uterus, and lower fallopian tubes, resulting in only a small proportion reaching the ampulla of the upper fallopian tube where fertilization occurs. Along the way, the most competent and suitable sperm approach survival factors and become ready or permitted to increase their competitive advantage over other sperm, thereby enhancing their physical ability to pass the final test of penetrating the clear oocyte band. The sperm sorting device 10 disclosed in this article integrates a combination of physical and molecular characteristics to provide a simple, reliable, and effective selection of the best quality sperm in semen in a format suitable for use in human and animal clinical IVF laboratories.

[0103] exist Figure 6BIn the illustrated embodiment, the sperm sorting device 10 includes a glass microscope slide 30, characterized by a 500 μm × 5 cm flow channel 14 and a 100 μL reservoir at the inlet 16 and outlet 20 of the flow channel 14. The lower glass surface of the flow channel 14 is functionalized with (1) gold nanospheres and (2) antiphosphatidylserine (anti-PS) antibodies covalently bound to the glass and gold surfaces and ionically (non-covalently) bound progesterone. The flow channel 14 and the gold nanospheres provide a motile sperm sorting zone 24 with a complex structure reproducing the fallopian tube maze. Antiphosphatidylserine forms a first biological sperm sorting zone 22a, which reproduces the interception of abnormal sperm mediated by immune cells (which exhibit altered membrane characteristics after oxidative stress), and progesterone forms a second biological sperm sorting zone 22b, wherein progesterone is a survival factor that promotes sperm endurance and prepares sperm for fertilization by capacitation.

[0104] Gold nanospheres and antiphosphatidylserine (anti-PS) antibodies are covalently bound to the surface of the channel by surface functionalization with a functional membrane that can covalently bind to the gold nanospheres and anti-PS antibodies.

[0105] This article also reveals a method of using, such as Figure 6B The sperm sorting apparatus 10 disclosed herein discloses a method for preparing semen for fertilization. The method includes a sperm addition step of adding sperm to an inlet prior to selection, followed by a sperm sorting step of contacting the sperm in the channel with the biological sperm sorting zone and the motile sperm sorting zone under conditions that allow the sperm to interact with them. In a harvesting step, the sperm that have traveled along the channel are recovered at an outlet.

[0106] This article also reveals a method for preparing such as Figure 6B The method of the sperm sorting device 10 disclosed herein includes providing a substrate having one or more flow channels, and depositing a functional membrane on the surface of the one or more flow channels. A biological reagent capable of interacting with sperm is attached to the functional membrane under conditions forming at least one biological sperm sorting region in the channel. Nanoparticles are then covalently attached to the functional membrane under conditions forming at least one motile sperm sorting region in the channel.

[0107] Example Example 1 - Preparation of antibody-coated, plasma-polymerized polyoxazoline (PPOx) films on glass channel slides This embodiment demonstrates (1) the feasibility of applying a PPOx film to functionalize the surface of a glass channel slide, and (2) the suitability of PPOx-coated channel slides for coating antibodies in a bioactive form that allows for the development of sperm selection devices.

[0108] Antiphosphatidylserine (anti-PS) antibodies were selected for coating studies to mimic the surface properties of neutrophils, which function in vivo in the female reproductive tract to isolate suboptimal sperm from ejaculation. Neutrophils bind to sperm undergoing early or late apoptosis, leading to loss of sperm plasma membrane integrity and externalization of the phosphatidylserine (PS) portion. Neutrophils recognize surface PS via the expression of their annexin V, which binds to sperm-associated PS and causes neutrophils to isolate sperm within neutrophil extracellular traps (NETs).

[0109] Materials and methods Plasma polymerization of channelized glass slides Using established standard methods (Macgregor–Ramiasa et al., 2017, Ramiasa et al., 2015), 2-methyl-2-oxazoline (Sigma-Aldrich, Australia) was deposited onto oxygen plasma-etched Sticky-Slide I Luer (Ibidi) substrates. ® Plasma polymerization was performed on a standard glass slide in a bell-shaped chamber reactor coupled with capacitive coupling. A plasma power of 50 W and a plasma density of 0.8 × 10⁻⁶ were used. -1 Plasma deposition was performed at a pressure of mbar to deposit the oxazoline precursor for 2 minutes. The resulting plasma-deposited polyoxazoline (PPOx) surface coating generates irreversible covalent amide bonds upon reaction with carboxylic acid groups on the protein.

[0110] Surface characterization X-ray photoelectron spectroscopy (XPS) (SPECS SAGE, Germany, monochromatic Mg Kα radiation source, 15 kV, 10 mA) was used to define the atomic composition of the PPOx film. Measurement spectra were obtained at a passage energy of 40 eV, a resolution of 1 eV, and a measurement angle of 54° between the X-ray irradiation and the analyzer. Atomic concentrations were determined by analyzing the spectral peak areas using Casa XPS software version 2.3.10 (Casa Software).

[0111] Water contact angles were measured after PPOx plasma polymerization on a glass slide. Oxygen plasma-etched glass was used as a control. Following surface polymerization, 5 μL MilliQ (Eppendorf) water droplets were formed on the surface using a motor-driven micro-injector. Triple evaluations were then performed at three points along the surface, and the process was monitored using a camera. Contact angles were measured from images of the droplets using ImageJ software.

[0112] The thickness of the PPOx film was measured using the Variable Angle Spectroscopic Ellipsometer (JA Woolam Co. Inc., USA), and the data were analyzed using WVASE32 software (NIH) with the Drop Analysis plugin.

[0113] Antibody immobilized onto PPOx-coated surface Next, the suitability of PPOx-coated channelized slides for coating with antiphosphatidylserine (anti-PS) antibodies was evaluated. The PPOx-coated channelized slide surfaces were washed three times for 15 minutes each with sterile Dulbecco phosphate-buffered saline (PBS, pH 7, Sigma Aldrich). The anti-PS antibody (clone 1H6, catalog number: 05-719, Sigma Aldrich) was diluted in PBS to concentrations ranging from 10 to 400 μg / ml. Depending on the surface area to be coated, 20–70 μl of anti-PS was gently aspirated onto the PPOX-coated glass substrate and allowed to bind overnight at 4°C. The antibody solution was then aspirated, and unreacted PPOx groups on the surface were passivated (backfilled) with 2% human serum albumin (HSA, VitroLife) for 5 hours at 4°C.

[0114] Immunofluorescence analysis was performed to detect anti-PS antibodies immobilized at different concentrations on PPOx-coated surfaces. After passivation with 2% HSA for 5 hours, the coated surfaces were washed with sterile PBS before use. The slides were then incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse antibody (Abcam, diluted 1 / 500 in PBS for 30 minutes at room temperature). Secondary antibodies were incubated on PPOx-coated channelized slides without prior anti-PS antibody coating as a negative control.

[0115] result Compared to alternative techniques for binding antibodies to surfaces, plasma-based systems offer advantages such as substrate independence, solvent-free operation, reproducibility, and scalability (Sainz-García et al., 2022, Macgregor-Ramiasa et al., 2017). Oxazoline-based plasma deposition coatings are biocompatible and facilitate irreversible covalent binding of biomolecules via their COOH groups (Macgregor-Ramiasa et al., 2017). Figure 1 (A and B).

[0116] In this embodiment, 2-methyl-2-oxazoline was used as a precursor to generate a PPOx film on a glass substrate, and the resulting PPOx film formed a hydrophilic surface with a water contact angle of 56.3 ± 4.8 (Table 1).

[0117] Table 1 - Effect of PPOx coating on surface wettability measured by water contact angle

[0118] The thickness of the plasma-deposited PPOx film was measured using ellipsometry and found to be approximately 26 nm. This thickness was further determined by comparing the film with a culture medium (G-IVF) used for sperm capture. TM The membrane thickness was remeasured after 24 hours of incubation with the Plus (Vitrolife) solution to confirm membrane stability. No significant changes in membrane thickness were observed (Table 2), supporting the stability of the coating during incubation with the sperm biocompatible solution.

[0119] Table 2 - In relation to G-IVF TM Elliptic measurements of PPOx film thickness before and after incubation in Plus medium.

[0120] Spectrum measurements were performed using a typical XPS ( Figure 1 D) The atomic composition of the PPOx film was determined to be oxygen (8.5%), carbon (73%), and nitrogen (18%), with the reference XPS measurement spectrum of the uncoated glass surface shown in [image / data]. Figure 1 In C, no silicon was detected in the XPS spectrum, indicating that the glass substrate is covered by a conformal, non-porous PPOx envelope. Based on high-resolution C1s XPS spectroscopy, approximately 30% of the carbon was determined to be in the CO environment, while 10% was in the C=O environment. Figure 1 E). These reactive chemical functional groups are formed due to the structural breakage and recombination of the oxazoline precursor.

[0121] Immunofluorescence analysis confirmed that, with increasing absorption in a concentration-dependent manner, the anti-PS antibody was successfully deposited on the functionalized glass surface. Figure 1 F). No fluorescence signal was detected on the negative control glass. Immunofluorescence staining showed an anti-PS concentration-dependent increase in staining intensity and uniform fluorescence distribution.

[0122] Example 2 - Biocompatibility assessment of PPOx-coated glass with human sperm In this embodiment, the biocompatibility of PPOx-coated glass with human sperm was determined, and it was confirmed that PPOx coating did not have an adverse effect on sperm motility parameters.

[0123] Materials and methods Semen preparation In short, after a 2-4 day ejaculation withdrawal period, semen was collected from four healthy donors via masturbation. Within 2 hours of collection, the semen was applied to a glass coverslip. Preheated and aerated G-IVF was then separated using standard upstream methods. TM Motile sperm were collected in Plus medium and then added to a PPOx-coated glass coverslip in a 1 ml culture well.

[0124] Sperm cultured using PPOx-coated incubation To evaluate the biocompatibility of the PPOx-coated surface, 100 μL LG-IVF was used. TM Plus medium with 1×10 6 Sperm washed at a concentration of [specific concentration] was added to PPOx-coated glass coverslips in polystyrene tissue culture grade Pepper plates. PPOx-coated and uncoated standard glass coverslips were then placed in 4-well plates (Nunc [specific format]). TM The surface was washed three times with a large amount of sterile PBS for 15 minutes each time to remove any excess unreacted PPOx groups. Potential microbial contaminants on the surface were then cleaned by incubation in sterile PBS with 200 μg / mL streptomycin (Thermofisher), 200 U / mL penicillin (Thermofisher), and 500 ng / mL amphotericin B (Thermofisher) for 2–3 hours. G-IVF was then used at 37°C and 5% CO2. TM Plus (Vitrolife) is applied to the surface of covered or uncovered coverslips at a depth of 2×10. 6 Sperm were incubated at different well densities for 1 and 5 hours. Additional sperm were incubated in uncovered culture dishes (TCP). Sperm motility and viability were assessed in all groups at 1 and 5 hours after incubation (at 37°C and 5% CO2).

[0125] Sperm motility assessment Necrotic sperm were quantified using propidium iodide (PI) staining absorbed by cells with damaged plasma membrane integrity. Sperm were collected into 5 mL flow cytometry (FACS) tubes after 1 and 5 hours of incubation. Next, 4 μL of propidium iodide (PI)-RNase A staining solution (Thermofisher) was added to the sperm preparation, gently mixed, and incubated in the dark for 5 minutes. Control tubes containing unstained sperm cells were used to calibrate the flow cytometry settings. Sperm motility was analyzed using an excitation wavelength between 400 and 600 nm and an emission wavelength between 600 and 700 nm on a FACS Canto II flow cytometer (BDBiosciences).

[0126] Assessment of sperm motility According to WHO Guideline V (World Health Organization, Geneva, 2010), sperm motility was assessed after 1 hour and 5 hours of incubation. Sperm motility was evaluated using a CASA® semi-automated semen analyzer (Microptic, Spain, Barcelona). Briefly, 2 μL of sample was added to a Leja 10 slide and scanned using an optical microscope (10x objective). The proportions of progressive (STR > 80%, where STR = linear velocity / average path velocity * 100), non-progressive (STR < 80%), and immotile sperm were calculated using a preset human counting / motility program. Total motility was calculated by the proportion of forward-progressing and non-progressive sperm. At least 200 sperm were evaluated from at least five fields of view.

[0127] result No significant harmful or cytotoxic effects of PPOx coating were observed in functional measurements of sperm quality, including sperm motility (PI uptake) and total motility after short or long incubation. Figure 2 The results indicate that the plasma-polymerized PPOx membrane is biocompatible with sperm, and there is no loss of sperm motility or viability after contact with the PPOX-coated glass. This suggests that the coating is suitable for sperm selection and related biomedical applications.

[0128] Example 3 - Preparation and use of immobilized anti-PS device and the effect of immobilized anti-PS on sperm selection The ability of PS-resistant coverslips to isolate surface-membrane PS in suboptimally expressed sperm was investigated to remove sperm in early or late apoptosis and generate sperm-rich populations with maximum motility and fertilization capacity. In methods commonly used to identify cells in early or late apoptosis, the proportion of sperm expressing surface-membrane PS was determined by incubating sperm with annexin V, the natural ligand of surface-membrane PS. Furthermore, unselected and selected sperm were evaluated using PI, which is absorbed by cells in late apoptotic and necrotic cells.

[0129] Materials and methods Anti-PS fixation on PPOx plasma-polymerized surfaces PPOx-coated glass coverslips (prepared as described above) were washed with sterile PBS to remove any contamination and uncrosslinked, soluble low molecular weight fractions from the surface of the plasma polymer. Anti-PS was then immobilized on the PPOx-coated surface. Anti-PS was diluted to concentrations of 10, 50, 100, 200, and 400 μg / mL (in PBS) and incubated overnight at 4°C with PPOx-coated channelized slides. After incubation, the antibody solution was aspirated, and the remaining active polymer surface was blocked by adding 2% HSA solution at 4°C for 5 hours. The coverslips were then thoroughly rinsed three times with PBS for 15 minutes each time to remove any unbound HSA and anti-PS. Immobilization of the anti-PS antibody on the PPOx substrate was confirmed using immunofluorescence (IF) staining (as described above).

[0130] Sperm attachment The effect of incubating sperm with coverslips immobilized with anti-PS at different densities (10, 50, 100, 200, and 400 ug / mL) on sperm attachment was then evaluated. In short, human semen was collected from six healthy donors via masturbation after 2–4 days of ejaculation withdrawal. Edible sperm were separated using standard upstream methods, and then washed sperm (1 × 10⁻⁶) were collected. 6 / mL) briefly held in preheated and ventilated G-IVF TM In Plus medium, then add 1×10 to 100 μL. 6 Sperm was added to a coverslip. The sample was applied to a slide within 2 hours of collection. Once sperm was added, the channelized slide was incubated at 37°C and 5% CO2 for 30–60 minutes. Then, the sample was processed using G-IVF. TM Plus washing to remove sperm. The number of sperm attached to the surface was quantified using an optical microscope, and the percentage of sperm attached was calculated by dividing the number of attached sperm by the total number of sperm added to the coverslip.

[0131] Annexin V / PI assay Then, using annexin V / PI immunofluorescence staining and flow cytometry, the proportion of sperm labeled with annexin V (an early apoptosis marker) and PI (a cell viability marker) was assessed in the original ejaculate, sperm obtained using conventional rapid techniques, and residual unbound sperm populations collected after incubation with a fixed anti-PS surface. Annexin V-Alexa Fluor was used for this purpose. TM 488 (Thermofisher) and PI / RNase A solution were used to detect sperm cells undergoing translocation from the inner lobe to the outer lobe of the plasma membrane (annexin V). + Sperm) and apoptotic / necrotic sperm (PI) +Sperm). In short, the harvested sperm were first washed with 1 mL PBS (350 x g, 5 min, 25 °C), then washed with 500 μL annexin binding buffer (ABB) containing 50 mM HEPES, 700 mM NaCl, 12.5 mM CaCl2, pH 7.4 (Thermofisher). The sperm pellet was resuspended in ABB and distilled at 2 x 10⁻⁶ ppm in FACS tubes. 6 The final sperm density was aliquoted into 200 μL ABB solutions. The sperm samples were then gently mixed, and 5 μL of annexin V-FITC (final concentration 2.5 μg / mL) and 4 μL of PI (final concentration 2 μg / mL) were added. The mixture was then incubated at room temperature for 5 minutes. After incubation, the sperm samples were washed in 500 μL ABB solutions, resuspended in ABB, and analyzed by flow cytometry within 10 minutes.

[0132] result To investigate the effect of incubating sperm with a specific concentration of surface-fixed anti-PS antibody on the proportion of sperm attaching to the coated surface, sperm were incubated on coverslips coated with 50, 100, 200, or 400 μg / mL anti-PS. Then, optical microscopy was used. Figure 3 A) The proportion of sperm bound to the surface was visualized and quantified manually. It was found that the concentration of attached anti-PS antibodies had a significant impact on sperm attachment. Figure 3 B) exhibits a concentration-dependent effect on sperm chelation. Maximum sperm attachment was observed using an anti-PS concentration of 200 μg / mL, and no significant increase in sperm attachment density was detected using anti-PS concentrations higher than this.

[0133] The proportion of sperm attached to coverslips coated with 200 μg / mL anti-PS was then assessed in six individuals to determine whether the anti-PS-coated slides effectively selected sperm from a range of male samples and to evaluate inter-individual variability. The proportion of sperm attached to the anti-PS-coated surface was found to range from approximately 12% (minimum) to 22% (maximum). Figure 3 C). Furthermore, the adhesion of sperm to surfaces coated with anti-PS antibodies was shown to be specific to the anti-PS reagent, as negligible sperm binding was observed after incubation with coverslips coated with individual PPOx, unrelated isotype-matched control antibodies, or bovine serum albumin (BSA). Figure 3 C).

[0134] To investigate the effect of surface-fixed anti-PS incubation on the proportion of PS-expressing sperm (used to specify sperm populations in early or late apoptosis), unconnected sperm were stained with annexin V and PI and assessed by flow cytometry. The annexin V-positive sperm population was found to be significantly reduced compared to virgin sperm and sperm isolated using conventional rapid techniques. Figure 3 D). This also applies to the proportion of PI-positive sperm (used to specify a sperm population with damaged surface membrane integrity, indicating late-stage apoptotic or necrotic sperm). Figure 3 E).

[0135] Example 4 - Characteristics of sperm separated from channelized slides compared with conventional upstream techniques The quality of selected sperm cells using channelized slides coated with PPOx and anti-PS was compared with that of the standard upstream cyclotron technique. Quality was measured as % total motility and % forward motility and DNA fragmentation (as determined by Halosperm® analysis). A second objective was to investigate whether channelized slides with different channel heights exhibited different efficacy in selecting optimal quality sperm.

[0136] Materials and methods Evaluation of sperm recovery rates on slides with different channel heights To compare the performance of channelized slides and upward sliding selection techniques and to evaluate the effect of channeled slide height on sperm selection and recovery, slides with channel heights of 0.1, 0.2, 0.4, 0.6, and 0.8 mm were evaluated. Briefly, human semen was collected from five healthy donors via masturbation after 2–4 days of ejaculation abstinence, with samples applied to slides within 2 hours of collection. Semen samples were held at 37°C for 30–60 minutes to liquefy. Then, the semen was processed using G-IVF with an appropriate volume of... TM Plus loading channels to prepare channelized glass slides for use. As shown in Table 3, the added G-IVF TM The volume of the plus depends on the channel height. Channel loading is performed slowly and gently, especially on slides with low channel heights (0.1 and 0.2 mm) to avoid leaving air inside the channel. Any residual air will impede or completely prevent the gradual movement of sperm through the channel. Next, 60-100 μL of raw liquefied semen is loaded into the inlet orifice and 60-100 μL of LG-IVF is used. TMThe outlet wells were filled with Plus medium. The slides were then incubated at 37°C and 5% CO2 for 45–60 minutes to allow progressively motile sperm to swim through the channels from the inlet well to the outlet well. After incubation, the supernatant from the outlet well was carefully collected, and sperm count and motility were assessed. The second portion of liquefied semen was prepared using a standard upstream method. The proportion of recovered sperm classified as progressively motile was then compared with that of sperm separated using conventional rapid techniques.

[0137] Table 3 - Channel Loading Volume Based on Channel Height

[0138] Evaluation of sperm DNA fragments (Halosperm) DNA fragmentation levels were assessed using the Halosperm G2 kit (Halotech®) according to the manufacturer's instructions. In brief, 100 μL of agarose was placed in a 90°C water bath for 30 minutes, then incubated at 37°C for at least 5 minutes prior to use. Then, 50 μL of sperm sample was gently mixed with the gel, avoiding air bubbles. Next, 8 μL of the mixed sample was pipetted onto an agarose-coated slide and covered with a coverslip. To solidify the gel, the slide was placed at 4°C for 5 minutes. The coverslip was then gently removed, and the slide was vertically immersed in Solution 1 at room temperature for 7 minutes. The slide was then immersed in Solution 2 for 20 minutes and washed with distilled water for 5 minutes. 70% and 100% ethanol solutions were applied to the slide for two minutes each to dehydrate it. After air drying, the slide was incubated in Solutions 3 and 4 for 7 minutes each step. To assess the percentage of sperm DNA fragmentation, the slide was imaged using an optical microscope at x20 magnification. At least 100 sperm cells per slide were scanned using Sperm Analyzer (SCA) software (Microptic LS) to measure halo size. Halo size is known to correlate with the degree of sperm DNA fragmentation, with large halos representing intact DNA and small or absent halos representing fragmented DNA. The percentage of sperm DNA fragmentation (%SDF) was calculated as the number of sperm cells with fragmented DNA relative to the total number of sperm cells analyzed.

[0139] result Regardless of whether 0.1μM, 0.2μM, 0.4μM, or 0.8μM channels were used, the recovery of progressively motile sperm after selection using channelized slides was comparable to the recovery of sperm obtained from conventional rapid acquisition, although there was a trend towards lower recovery in the case of shallower channels. Figure 4 B). When based on DNA fragmentation ( Figure 4 C) and athleticism Figure 4When assessing markers of sperm quality (D and E), sperm recovered from channelized slides were comparable to those from conventional upstream development. This indicates that PPOx- and PS-resistant channelized slides produce sperm samples of similar quality to those from conventional upstream development.

[0140] Example 5 - Mouse Sperm Selection The effectiveness of the channelized slide device in selecting optimal sperm in mammalian species other than humans was determined. Sperm recovered from mouse epididymis was evaluated using a protocol comparable to that proven effective for human sperm.

[0141] Materials and methods Mouse sperm separation Male C57BL / 6J mice (8–12 weeks old) were purchased from the Laboratory Animal Service (LAS) of The University of Adelaide and held for 5 weeks prior to use. Mice were placed in clean cages under controlled temperature (25 br 3°C), appropriate humidity (50 br 5%), and a 12-hour light / dark cycle. The procedure described below was approved by the Animal Ethics Committee of The University of Adelaide (Approval No. M-2022-041). Briefly, after cervical dislocation of the male mice, the entire epididymis and half of the vas deferens were carefully dissected, and residual fat was trimmed. The tissue was then transferred to a 15 mL round-bottom tube and incubated at 37°C with 1 mL of pre-warmed G-IVF. TM Plus medium. Then, puncture the tissue at 5 or more sites with sharp scissors and allow it to stand for 15 minutes (37°C and 5% CO2) to allow sperm to swim out. Remove the membrane-covered tissue from the medium and aspirate the remaining fluid up and down to recover the sperm suspension. Then, incubate the perm with PPOx and anti-PS-coated coverslips, or apply it to the inlet wells of PPOx and anti-PS-coated channelized slides (0.4 μm, prepared as described above) or uncoated channelized slides. After 45 minutes, recover unbound sperm from the coverslips or the outlet wells of the channelized slides and assess sperm motility, apoptosis markers (annexin V binding and PI uptake), and sperm surface attachment as described in previous sections. Assess sperm DNA fragmentation using TUNEL staining. Compare the data with control sperm that were not selected in the channelized device.

[0142] Sperm DNA fragmentation was assessed using TUNEL assay. Using Apo-Direct TM The TUNEL assay kit (Thermofisher) was used to assess sperm DNA fragmentation. The TUNEL assay was performed with minor modifications according to the manufacturer's protocol. Perm was measured at 2-4 × 10⁻⁴. 6The sperm were suspended in 100 μL of 1% (v / v) paraformaldehyde and incubated on ice for 15 min. After incubation, the sperm were washed twice with 5 mL PBS at 350 × g for 5 min, and then incubated overnight in 100 μL of ice-cold ethanol at -20 °C. The next day, the ethanol was aspirated and the sperm were washed twice with 1 mL of wash buffer at 350 × g for 5 min. The labeling reaction was performed by incubating in the dark at 37 °C with 50 μL of a labeling solution containing terminal deoxynucleotidyl transferase (TdT) for 2 h. After washing twice with 1 mL of wash buffer, the sperm sample was resuspended in 500 μL of propidium iodide / RNase A solution. The samples were then analyzed within 3 h of staining using a FACSCanto II flow cytometer (BD Biosciences). TUNEL-positive sperm (green fluorescence) were measured using a 530 ± 30 nm bandpass filter, and propidium iodide-labeled sperm (red fluorescence) were measured using a 620 nm longpass filter. A total of 10,000 events were analyzed per measurement, with a flow rate set to 200-300 cells / second. After gating out aggregates and cell debris, data were analyzed via FlowJo... TM 10. Software (BD Biosciences) is used to process the data.

[0143] result A representative example of flow cytometry analysis of mouse sperm anisein V binding and PI uptake is shown in Figure 5 In AD, the proportion of sperm binding annexin V and / or uptake of PI (evidence of apoptosis) decreased in sperm suspension after incubation with PPOx and anti-PS-coated channelized slides or with PPOx and anti-PS-coated coverslips, but not by incubation with uncoated channelized slides. Figure 5 E, 5F). Compared to the original sample, a ~50% reduction in the proportion of annexin V-positive sperm and a ~40% reduction in sperm DNA fragmentation were observed in the unattached sperm population. A higher percentage of motile sperm was observed in the unattached sperm subpopulation after culture with PPOx and anti-PS coated coverslips or channelized slides (but not uncoated channelized slides). Figure 5 G, 5H). After incubation with PPOx and anti-PS coated coverslips or channelized slides but not with uncoated channelized slides, the proportion of sperm with DNA fragmentation, as detected by TUNEL, was also reduced in the unligated sperm subpopulation. Figure 5 I).

[0144] Example 6 - Fabrication of a nano-gold monolayer (NGS) device The functional complexity of the PPOx and anti-PS-coated channel slides is further enhanced by incorporating additional features, including (1) gold nanospheres covalently bonded to the PPOX-coated glass substrate to increase the surface area available for anti-PS binding and sperm interaction with the anti-PS portion, and to mimic the labyrinthine complexity of the mammalian oviduct; (2) a non-covalently bonded progesterone gradient that exerts chemotactic and motility-enhancing effects on sperm to increase sperm motility and enhance the directionality and rate of sperm progression across the channel slide; and (3) functionalization of the spatial configuration of the channel slide surface to enable a two-stage selection process in which sperm delivered to the channel’s inlet pore first encounters anti-PS to remove suboptimal sperm, and then encounters the progesterone gradient to stimulate the progression of motile sperm across the channel and promote the motility of recovered sperm at the outlet pore.

[0145] Materials and methods Synthesis of Gold Nanoparticles (GNP) GNP synthesis was achieved using established protocols (Visalakshan et al., 2018; Goreham et al., 2011; Zhu et al., 2003) that utilize the reduction of tetrachloroauric acid (HAuCl4, Sigma-Aldrich, Australia) with trisodium citrate (Na3Ct, Sigma-Aldrich, Australia) to produce colloidal gold in aqueous solution in a controlled reflux system. HAuCl4 (0.01%) was dissolved in a continuously stirred solution and brought to a boil, followed by the addition of Na3Ct as a reducing agent and reboiling for 20 minutes. Limiting the amount of 1% Na3Ct solution added was used to control GNP size; the addition of 1.0, 0.5, and 0.3 mL of 1% Na3Ct yielded nanoparticles (NPs) of three different sizes with diameters of 16, 38, and 68 nm, respectively. Mercaptosuccinic acid (MSA, Sigma-Aldrich, Australia) and sodium hydroxide (chemically supplied) were then used as end-capping agents to modify the surface to provide enhanced stability to colloidal GNPs.

[0146] GNP fixation Next, COOH-functionalized GNPs were immobilized on the surface of a PPOx-coated 400 μm channelized slide. The channelized slide (400 μm) was PPOx-coated as described in Example 1. Immobilization of GNPs with diameters of 16, 38, and 68 nm was performed by immersing the PPOx-coated surface in a GNP solution overnight. The next day, before drying the surface under nitrogen, excess and loosely bound GNPs were removed by rinsing three times with MilliQ water.

[0147] GNP characterization After the GNP suspension was dried on a carbon-coated copper grid (ProSciTech, Australia), the shape and size distribution of the GNPs were characterized using transmission electron microscopy (TEM, JEOL-2100F, Japan). The surface morphology of the GNPs was evaluated using atomic force microscopy (AFM) in a non-contact mode with gold-coated silicon nitride tips (NT-MDT, NSG03) having a resonant frequency of 65–100 kHz. Surface chemistry was characterized by XPS spectral measurements.

[0148] PPOx coating of surface-immobilized gold nanoparticles To covalently immobilize anti-PS onto gold nanoparticles on one half of the channelized slide surface, and to electrostatically adhere progesterone to gold nanoparticles on the other half of the slide, a second functionalization layer is then applied only to the immobilized gold nanoparticles on one half of the slide (the half closest to the inlet aperture). This is achieved during processing in the plasma reactor using a mask covering the other half of the slide (the half closest to the outlet aperture). Thus, the half of the slide closest to the inlet aperture is functionalized with PPOx groups to enable covalent immobilization of anti-PS onto the gold nanoparticles and the slide surface, while the other half retains its carboxylic acid groups to enable electrostatic adsorption of progesterone.

[0149] The intact oxazoline ring on the PPOx membrane surface promotes irreversible bonding of ligands with COOH- ions by forming amide bonds. To confirm the binding, the prepared slides were incubated with sodium dodecyl sulfate (SDS) detergent solution (1% v / v in 5M NaCl, pH 10) for 1 hour, followed by rinsing with MilliQ water to stimulate the binding of PPOx and COOH-GNP. XPS analysis was used after washing to observe and confirm the strong binding between PPOx and GNP.

[0150] result The unique chemical properties and reactivity of plasma-deposited PPOx make it an ideal coating material for biomaterial surfaces. Our data and published studies have shown that PPOx coating can be used to ensure the safe chemical and irreversible bonding of gold nanoparticles to glass substrates and other materials (Visalakshan et al., 2018). This example demonstrates the feasibility of sequentially coating a glass channel slide with PPOx, followed by gold nanospheres, and then further applying the PPOx coating to a proportion of the gold nanospheres. This method is designed to provide a complex surface structure that replicates the labyrinthine features of the mammalian oviduct and allows sperm to be exposed to different microenvironments in stages during their progression through the channelized slide.

[0151] These results demonstrate that it is possible to achieve spatially distinct regions of channelized slides coated with gold nanospheres, with one half of the slides subsequently covalently coated with anti-PS and the other half non-covalently coated with progesterone.

[0152] The channel slide coated with gold nanoparticles allows for the binding of anti-PS to first replicate the interception of morphologically abnormal sperm mediated by immune cells, and secondly to allow sperm to interact with progesterone to act as a sperm chemotaxis and survival factor.

[0153] Channel slides coated with gold nanoparticles containing anti-PS and progesterone-coated regions are called “Nano Gold Selective Devices” (“NGS Devices”) (Figure 6).

[0154] Example 7 - Using NGS devices to assess the effectiveness of human sperm selection The efficacy of the channelized slides prepared as described in Example 6 was evaluated. The NGS device (a 400 μm channelized slide coated with gold nanoparticles, and spatially distinct regions coated with anti-PS and progesterone) was evaluated for its ability to select high-quality sperm from raw liquefied semen samples (without any prior preparation procedures). The aim was to evaluate the utility of the NGS device as a single-step separation method to produce high-quality human sperm for IVF or ICSI.

[0155] Materials and methods Sperm separation in NGS device Semen from two donors was prepared as described in Example 4—a transient semen sample was liquefied and then added to an NGS device (prepared as described in Example 6) or an uncoated channelized slide without any further preparation steps. 100 μL of G-IVF was used. TM Carefully load the inlet orifice of the channelized slide, then load 60-100 μL of liquefied raw semen sample into the inlet orifice. The outlet orifice is filled with 40-60 μL of G-IVF. TM Plus medium. Incubate the samples at 37°C and 5% CO2 for 45–60 minutes to allow motile sperm to swim through the channels from the inlet pore to the outlet pore. Carefully collect the supernatant from the outlet pore and then assess sperm count and motility, annexin V positive staining and / or PI uptake ratio, and sperm DNA fragmentation as described in Examples 3 and 4. Compare sperm recovered from the NGS device with sperm prepared by incubation in uncoated channelized slides or by standard rapid techniques.

[0156] result A representative example of flow cytometry for human sperm anisein V binding and PI uptake is shown in Figure 7In AC, the percentage of annexin V+ sperm, as determined by flow cytometry, was significantly lower in sperm recovered from the NGS device compared to sperm applied to uncoated channelized slides or standard upstream techniques. Figure 7 D). A similar reduction in the proportion of sperm with DNA fragmentation was observed using an NGS device, which produced sperm with a very low rate of DNA fragmentation compared to sperm prepared by standard rapid techniques or uncoated channelized slides. Figure 7 E). Furthermore, after separation using an NGS device, a higher proportion of sperm exhibited a large halo compared to sperm prepared via spiral or uncoated channelized slides (E). Figure 7 F, 7I-K) indicate improved DNA integrity in sperm prepared in the NGS device. Sperm recovered from the NGS device showed comparable levels of motility and forward motility to sperm prepared via upstream or uncoated channelized slides. Figure 7 (G, 7H). These data show that the NGS device is more efficient than standard rapid procedures in producing optimal quality sperm, as determined by annexin V positivity and sperm DNA integrity. Furthermore, this example demonstrates that the NGS device can produce optimal quality sperm directly from liquefied human semen without any further preparation steps before the semen sample is applied to the device.

[0157] Example 8 - Evaluation of the quality of recovered sperm by the various selective elements of the NGS device The contribution of each component included in the NGS device to its selective efficacy was assessed. To this end, individual elements (anti-PS, progesterone, gold nanoparticles) were removed during device manufacturing, and their impact on the quality of recovered sperm was measured by annexin V and PI uptake.

[0158] Materials and methods As described in Example 6, channelized slides were coated with all components, or one or more individual components were removed from the coating sequence using a variant. Channelized slides (400 μm) were coated with all components described in Example 6 (e.g., antiphosphatidylserine (anti-PS), gold nanoparticles (GNP), and progesterone (P)), with GNP and P instead of anti-PS; with GNP and anti-PS instead of P; with anti-PS and P instead of GNP; with P instead of anti-PS or GNP; with anti-PS instead of P or GNP; with GNP instead of anti-PS or P). Liquefied semen was prepared from each of the two donors as described in Example 6 and added to the inlet well of each of the aforementioned channelized slides, followed by incubation for 45 minutes, after which selected semen was recovered from the outlet well (as described in Example 6). Unselected sperm and sperm selected using standard upstream techniques were included for comparison. The quality of the recovered sperm was measured as the proportion of sperm stained with annexin V and / or PI uptake.

[0159] result like Figure 8 As shown, when sperm were selected using an NGS device coated with all components (anti-PS, gold nanoparticles, and progesterone), the lowest values ​​of annexin V staining and PI uptake were observed. Figure 8 (A and B). These sperm quality parameters were again substantially lower than those of sperm recovered using standard upstream techniques, thus confirming the improved performance of the NGS device compared to standard upstream techniques. When any or more of these individual selective elements were removed from the NGS device, the values ​​of annexin V staining and PI uptake increased. These data indicate that all three components contribute to the quality of sperm recovered from the NGS device when included in the channelized slide coating. This result suggests that the efficacy of the NGS device in producing high-quality sperm is not entirely attributable to any single component of the channelized sliding coating, but rather depends on the unique combination of coating components within the device.

[0160] Biomolecules including recombinant annexin V protein (In Vitro Technologies), antiphosphatidylserine (anti-PS, Sigma-Aldrich), anti-fibronectin antibody (anti-Fn, rabbit monoclonal F14, Abcam), and sodium hyaluronate disaccharide ΔDiHA (ΔDiHA, Sigma-Aldrich) were immobilized on a plasma-deposited PPOx surface. Prior to biomolecule immobilization, the plasma-deposited PPOx surface was washed with PBS (Sigma Aldrich) to remove unattached PPOx groups. The concentration was 200 μg / mL. 2Each biomolecule was individually incubated with the plasma-coated surface overnight at 4°C. To avoid nonspecific protein adsorption and sperm attachment to the unreacted PPOx plasma array, the surface was backfilled with 5% human serum albumin (HSA) overnight at 4°C. The coated surface was then washed three times with sterile PBS for 1 hour each time to remove unfixed biomolecules before use.

[0161] Example 9 - Preparation and use of immobilized anti-fibronectin antibody (anti-Fn) and its effect on sperm selection The anti-fibronectin antibody (anti-Fn, Abcam) was immobilized on the PPOx plasma polymerization surface according to the scheme provided in Example 3.

[0162] Immunofluorescence (IF) staining was used to examine biomolecule fixation levels. After constructing the anti-Fn coating and backfilling with HSA, the coating surface was washed with PBS for 1 hour to remove unfixed proteins and incubated with a secondary antibody (goat anti-rabbit IgG (Alexa Fluor® 488), Abcam) at 4°C for 12 hours to detect the binding of anti-Fn (…). Figure 9 A). A clean glass surface coated with anti-Fn was used as a control. Figure 9 B).

[0163] Fibronectin (Fn) is a 440 kDa glycoprotein that is believed to help humans (Thys et al Sperm-oocyte interactions in sperm (Marta.nez.P.Lel.n et al., 2009). Typically, sperm express Fn on their surface after capacitation (Marta.nez.P.Lel.n et al., 2019), and the detection of Fn receptor integrin α5β1 on male and female human gametes suggests a role for Fn glycoproteins in sperm-oocyte interactions during fertilization. In the female reproductive tract, Fn may also act as an opsonin factor for damaged sperm cells, thereby labeling them for neutrophil-mediated binding and chelation (Thys. et al (Nair and Zingde 2001). Consistent with this, it has been reported that fibronectin (Fn) levels in seminal plasma are elevated in cases of male infertility and low sperm motility. We believe that the presence of fibronectin (Fn) on the sperm plasma membrane increases the likelihood of neutrophil-mediated chelation in the female reproductive tract.

[0164] This embodiment was conducted to evaluate the expression of Fn in sperm after rapid selection using flow cytometry and to assess the efficacy of anti-Fn coated surfaces in immobilizing damaged sperm.

[0165] To confirm the specificity of the anti-Fn coating, two controls were included: 1) a sample incubated with an isotype-matched rabbit IgG antibody prior to FITC-labeled secondary antibody treatment ( Figure 10 A) and 2) were samples treated without primary antibody before incubation with FITC-labeled secondary antibody. Human peripheral blood mononuclear cells were used as a positive control to confirm anti-Fn binding function. Figure 10 B).

[0166] Fn expression in motile sperm samples from five healthy men was examined by flow cytometry. The percentage of Fn-positive sperm varied from 2.7% to 15%. Figure 11 ).

[0167] Motile sperm were collected using rapid technology and the concentration was adjusted to 2.5 × 10⁻⁶. 6 ml -1 Before incubating sperm with the prepared anti-Fn coated surface, the surface was washed with sterile PBS for 1 hour to remove any excess non-covalently bound proteins and HSA. Each coverslip was placed in a 24-well plate (Nunc) and incubated at 15,000 sperm / mm². 2 (2.5×10 6 ml -1 Sperm were inoculated at a density of 100-120 min. First, sperm were incubated with the coated surface at 37°C and 5% CO2 for 60-120 min, then unconjugated sperm were collected for further analysis. PPOx surfaces coated with unrelated isotype-matched rabbit IgG antibodies and 5% HSA or PPOx alone were used as controls. After incubating sperm on the anti-Fn coated surface for 120 min, 2%-10% of the sperm population adhered to the surface. Figure 12 ), while no sperm adhered to the control surface.

[0168] Example 10 - Preparation and uses of immobilized annexin V protein and its effect on sperm selection. Annexin V is a surface protein previously used for sperm selection to improve ART success rates. Sperm with pre-apoptotic or apoptotic characteristics can maintain normal morphology (Sakkas et al., 2015) and retain the ability to fertilize oocytes, particularly during ICSI procedures where a single sperm is injected into the oocyte cytoplasm. This procedure adversely affects embryonic development and pregnancy rates and may pose risks to child health. Several studies have demonstrated that ejaculated sperm, particularly in infertile men, undergo apoptotic-like characteristics (Sakkas et al., 2015). et alChanges in phosphatidylserine (PS) occur in sperm cells. If apoptosis occurs, sperm cells reconstitute phosphatidylserine (PS) from the inner lobules to the outer lobules of the plasma membrane, externalizing PS (Marta.nez et al., 2018). PS has a high affinity for annexin V (a protein molecule with a molecular weight of 36.8 kDa), which cannot penetrate the intact sperm membrane (Sakkas et al., 2015). Therefore, eliminating sperm cells with externalized PS, as detected by positive annexin V binding, and further enriching the population of annexin V-negative sperm from which sperm are selected for fertilization, would provide a useful and important advance for ART.

[0169] In this embodiment, the proportions of annexin V-positive and necrotic (PI-positive) sperm in the population were determined by incubation with an annexin V-coated surface before and after sperm sorting, and compared with raw ejaculate and sperm prepared via upstream technology. The proportion of annexin V-positive sperm in the raw semen did not differ significantly from that in the upstream process (25% ± 11 and 21% ± 15, respectively, n=4). Figure 13 To determine whether there was any reduction in annexin-positive sperm after interaction with a surface coated with annexin V, raw semen was incubated on a surface prepared by immobilizing annexin V on a coverslip coated with plasma-polymerized PPOx. The availability of annexin V on the surface was confirmed by fluorescence imaging after HSA passivation and PBS washing. Figure 14 ).

[0170] Then, a density of 15,000 sperm / mm 2 (2.5×10 6 ml -1 The motile sperm were incubated with the prepared Annexin V-coated surface at 37°C and 5% CO2 for 60–120 minutes. Unattached sperm were collected for further analysis.

[0171] After 120 minutes of incubation, the sperm attachment rates of the two semen samples (n=2) were 12% and 20%, respectively. Figure 15 Compared to upstream sperm in each individual, the number of annexin V-positive sperm in unattached sperm was reduced by 44.4% and 62.5%, respectively.

[0172] Compare the levels of reactive oxygen species (ROS) recovered from surfaces coated with annexin V, including those from virgin ejaculate, upstream sperm, and unattached sperm. Figure 16The results showed that the ROS content of motile sperm collected via the upstream method was higher than that of the original sperm (52% and 35%, respectively). There was almost no difference in ROS content between sperm prepared via the upstream method and the unconnected sperm population recovered from the functionalized surface.

[0173] Example 11 - Sperm attachment on annexin V coated surfaces and its effect on sperm recovery The effect of different concentrations of surface-fixed annexin V on sperm attachment ratios was examined in a comprehensive study using sperm samples. Sperm collected from upstream samples were exposed to coverslips coated with different concentrations of annexin V (Vitro Technologies, 10, 50, 100, and 200 μg / mL). Sperm binding patterns were assessed using optical microscopy by counting the number of unattached and attached sperm. Figure 17 The proportion of attached sperm was determined by dividing the count of attached sperm across the entire surface by the initial sperm inoculation density and then multiplying the result by 100.

[0174] There is a significant correlation between annexin V concentration and sperm cell adhesion. Figure 17 E). A concentration-dependent pattern of sperm attachment was observed, with attachment peaks observed at 100 and 200 μg / mL annexin V.

[0175] To assess the applicability of annexin V-coated coverslips to sperm selection in different individuals and to evaluate inter-individual differences, the proportion of sperm adhering to coverslips coated with 200 μg / mL annexin V was examined in sperm samples from six individuals. The findings revealed that the percentage of sperm adhering to surfaces treated with 100 μg / mL annexin V ranged from 5% (minimum) to 23% (maximum). Figure 17 F). This experiment ultimately demonstrated that sperm binding to surfaces coated with annexin V depends on the annexin V coating, as minimal sperm attachment was observed when coverslips were coated with PPOx alone or with PPOx and 2% HSA.

[0176] To examine the effectiveness of incubating sperm with surface-fixed annexin V, apoptotic sperm populations in unattached sperm were investigated. The fraction of sperm displaying phosphatidylserine (PS) (a marker of early or late apoptosis) on the outer leaflets of the membrane was determined using annexin V and PI staining, assessed by flow cytometry. Analysis showed a significantly decreased proportion of sperm exhibiting positive binding to annexin V compared to untreated initial samples and sperm isolated via conventional upstream methods. Figure 17GK). The percentages (mean ± SEM) of annexin V-positive sperm subgroups, upstream separated sperm, and unattached sperm remaining after incubation with the annexin V-coated surface (concentration 200 μg / ml) were (mean ± SEM) 36.5% ± 8.6%, 34.9% ± 12.8%, and 20.8% ± 12.1%, respectively. Figure 17 J). This indicates that the annexin V positive population was significantly reduced after exposure to surfaces coated with annexin V (P≤0.01).

[0177] Similarly, for the original sample, upstream isolated sperm, and sperm incubated with surfaces coated with annexin V, the percentages of sperm showing PI-positive staining (indicating damaged membrane integrity typically associated with late-stage apoptotic or necrotic sperm) were 29.8% ± 10.3%, 19.4% ± 9.8%, and 20.1% ± 12.1%, respectively. Figure 17 This indicates that the PI-positive population was significantly reduced after exposure to PPOx-coated or annexin V-coated surfaces (P≤0.01).

[0178] Example 12 - Sperm attachment on hyaluronic acid (HA) coated surfaces and its effect on recovered sperm. The study investigated the effects of hyaluronic acid (HA) on the quality of upstream sperm attachment and unattached sperm populations, such as... Figure 18 As depicted in the text. Incubation was performed in preheated and CO2-ventilated G-IVFTM Plus medium in contact with surface-modified glass coverslips at a density of 1–2 × 10⁻⁶. 6 Experiments were conducted using washed human sperm.

[0179] The degree to which HA binds to sperm is believed to be related to sperm maturity and motility. HA is a linear polysaccharide composed of repeating disaccharide units containing carboxylic acid groups in its molecular structure. The D-glucuronic acid component of HA includes carboxylic acid groups (-COOH), which mediate the covalent bonding between HA and PPOx groups. HA (sodium hyaluronate, Sigma Aldrich) was immobilized at a concentration of 200-300 μg / ml on PPOx-coated glass coverslips. Incubation was performed overnight at 4°C. Subsequently, the surface was washed, and the remaining PPOx groups were blocked with 2% HSA at room temperature for 30-60 minutes.

[0180] To assess whether HA-coated coverslips could facilitate sperm binding and selection, the proportion of sperm adhering to HA-coated coverslips was examined in sperm samples from three individuals. The results revealed that the percentage of sperm adhering to the surface treated with 100 μg / mL annexin V ranged from 14% (minimum) to 38% (maximum). Figure 18A, 18B). The number of sperm bound to the HA-coated surface depends on the HA coating, as minimal sperm attachment is observed when the coverslip is uncoated or coated with PPOx and 2% HSA. Figure 18 B).

[0181] To examine the effectiveness of surface-fixed HA incubation on sperm, the proportion of apoptotic sperm in unconnected sperm was investigated. The fraction of sperm showing annexin V binding positivity was comparable to that of primitive sperm and higher than that of sperm collected upstream. Figure 18 (EG, 18K). This indicates that exposure to HA-coated surfaces does not improve the ability of unattached populations as measured by annexin V positivity, and is instead consistent with the selective binding of capable sperm to the surface.

[0182] Example 13 - Sperm attachment on antifibronectin-coated surfaces and its effect on sperm recovery The effects of anti-fibronectin (anti-Fn) biomolecules on upstream sperm attachment and the quality of the unattached sperm population were investigated. Figure 18 As shown. Incubation was performed using preheated and CO2-ventilated G-IVFTM Plus medium in contact with surface-modified glass coverslips at a density of 1–2 × 10⁻⁶. 6 Experiments were conducted using washed human sperm.

[0183] Fibronectin is a glycoprotein present on a certain percentage of sperm surfaces. Its importance in sperm motility is not yet well understood. Following the same protocol as HA, anti-Fn (Abcam) was immobilized on PPOx-coated surfaces at concentrations of 40-100 μg / mL. To assess whether Fn-coated coverslips could facilitate sperm binding and selection, the proportion of sperm adhering to anti-Fn-coated coverslips was examined in sperm samples from three individuals. The results revealed that the percentage of sperm adhering to surfaces treated with 100 μg / mL anti-Fn ranged from 10% (minimum) to 28% (maximum). Figure 18 C Figure 18 D). Sperm binding to anti-Fn-coated surfaces depends on the anti-Fn coating, as minimal sperm attachment is observed when the coverslip is uncoated, or coated with PPOx and 2% HSA, or with an allotype-matched unrelated monoclonal antibody. Figure 18 D).

[0184] To examine the effectiveness of surface-fixed anti-Fn incubation of sperm, the proportion of apoptotic sperm in unconnected sperm was investigated. The results showed that the fraction of annexin V-binding positive sperm was comparable to that of primitive sperm, and sperm were collected upstream (…). Figure 18 HJ, 18L). This indicates that exposure to anti-Fn coated surfaces does not enhance the ability of unattached populations to be measured positive by annexin V.

[0185] in conclusion Examples show that glass coverslips coated with PPOx using each of annexin V, hyaluronic acid, and antifibronectin can provide a surface capable of selectively binding sperm. Annexin V, such as anti-PS in the examples, chelates less capable sperm (measured by annexin V and PI binding). Hyaluronic acid appears to isolate more capable sperm. Anti-Fn traps a subset of sperm, but the percentage of annexin V staining does not change, and therefore may have no effect on the competence of unbound sperm.

[0186] Example 14 - Alternative methods for sperm isolation using surface activation and surface conjugation of biomolecules and gold nanoparticles The effects of different chemical approaches on combining biomaterials and gold nanoparticles were investigated. Specifically, we compared the use of plasma polymerization to generate films with reactive amine groups (-NH2, referred to as "plasmopolymerized amine films") with the use of PPOx films with reactive -COOH groups (used in previous examples). Gold nanoparticles (GNPs) and anti-PS antibodies were coupled to the surface of glass coverslips using two alternative coating methods, and the effects on sperm behavior upon incubation with different functionalized surfaces were examined. The plasmapolymerized amine groups (-NH2) interact non-covalently with carboxyl groups (-COOH), such as -NH3, on biomolecules. + and -COO - Electrostatic attraction between ions, and hydrogen bonding between the hydrogen atoms of the amine and the oxygen atoms of the carboxyl group. These interactions are not expected to be as strong as the covalent bonds provided by PPOx treatment. However, they can be used in sperm separation devices that require alternative binding chemistry.

[0187] Materials and methods In this embodiment, according to an established protocol, allylamine (AAm) is used as a precursor to generate a plasma-polymerized amine film on a glass coverslip (Liu et al., 2014). As described in previous embodiments, 2-methyl-2-oxazoline is used as a precursor to coat an additional glass coverslip to generate a PPOx film.

[0188] Then, glass coverslips were coated with 68 nm gold nanoparticles (GNP) and anti-PS antibody or with anti-PS antibody without GNP to produce four different surface coating variants.

[0189] For coatings without GNP, anti-PS at a concentration of 200 μg / ml was directly applied to the surface of coverslips with plasma-polymerized amine or PPOx films to generate coated NH2-anti-PS' and PPOx-anti-PS', respectively.

[0190] For coatings containing GNPs, 68 nm GNPs are first immobilized on the surface of a coverslip using a plasma-polymerized amine film or PPOx film. Then, both GNP surfaces are treated with plasma polymerization to achieve PPOx coating. Anti-PS at a concentration of 200 μg / ml is then applied to the surfaces as described in the previous example to produce coatings referred to as 'NH2-GNP-PPOx-anti-PS' and 'NH2-GNP-PPOx-anti-PS', respectively.

[0191] Then the selection efficacy of these different sperm coatings was compared. In short, semen was collected from healthy donors. Standard upstream methods were used in preheated and aerated G-IVF. TM Motile sperm were isolated in Plus medium. Within 2 hours of collection, sperm were added to coated glass coverslips in 1 ml culture wells.

[0192] Human sperm prepared by buoyancy (200 μl) (n=1) was incubated for 45 minutes on a modified coverslip surface at 37 °C and 5% CO2. Sperm were then collected, and the incidence of apoptotic and necrotic populations was analyzed by flow cytometry using annexin V staining and PI uptake, as described in previous examples. Sperm exposed to the coated coverslip were compared with control sperm not exposed to the coated coverslip.

[0193] result result( Figure 19 The efficacy of all four coated surfaces was demonstrated. In sperm exposed to NH2-GNP-PPOx-anti-PS and NH2-anti-PS surfaces (15.0% and 20.0%, respectively), the apoptotic sperm population was reduced, compared to 34.9% in the clearance control. This reduction was comparable to that observed on PPOx-coated surfaces, where the rates of apoptotic sperm on PPOx-GNP-PPOx-anti-PS and PPOx-anti-PS-coated surfaces were 13.6% and 17.9%, respectively. For each different coverslip treatment, the percentage of sperm with PI uptake was comparable to that of upstream sperm.

[0194] These findings demonstrate the utility of alternative coating chemistry, including plasma-polymerized amines alone or in combination with PPOx coatings and / or gold nanoparticles, prior to combining biomaterials (e.g., anti-PS) to isolate apoptotic populations and improve the quality of recovered sperm.

[0195] Example 20 - Surface conjugation of biomolecules using protein-G to trap sperm G proteins, due to their strong affinity for the Fc region of immunoglobulin (antibody) proteins, can be used for antibody attachment to surfaces, enabling the immunoglobulin Fab region to be stably oriented and effectively approach homologous epitopes, thus offering benefits for various biomedical applications.

[0196] method G protein-mediated binding of antifibronectin to the surface of PPOx coating In this embodiment, G protein was bound to a PPOx membrane on a glass coverslip before incubation with antifibronectin or anti-PS, and the effect of sperm chelation on efficacy was evaluated.

[0197] Initially, to investigate the ability of G proteins to attach to PPOx-coated surfaces and facilitate subsequent antibody binding, an anti-fibronectin (anti-Fn) antibody conjugated with Alexa Fluor 488 dye was used as an indirect detection method. Figure 20 PPOx-coated glass coverslips treated with or without G protein (diluted in PBS, at 4°C) o The surfaces were incubated overnight at 4°C, followed by passivation with 2% HSA for 30–60 minutes at room temperature. The control group consisted of coverslips coated with uncoated or passivated PPOx without protein-G. The surfaces were then exposed to different concentrations of anti-Fn labeled with Alexa Fluor 488 and incubated overnight at 4°C. Anti-Fn fixation was analyzed by comparing the fluorescence signals of bare glass, passivated PPOx (without Protein-G), and Protein-G coated surfaces incubated with different concentrations of anti-Fn-Alexa Fluor 488.

[0198] In the second experiment, anti-PS antibodies of various concentrations (20-200 μg / mL) were incubated with glass coverslips coated with PPOx and G protein-PPOx, and the binding efficiency under different conditions was evaluated by mass spectrometry.

[0199] In the third experiment, channelized slides were coated with PPOx and G protein, or not coated with G protein, prior to anti-PS coating. The apoptotic sperm retention efficacy of the channelized slides was then evaluated using sperm from n=3 human donors, as described in previous embodiments.

[0200] result G protein-mediated binding of antifibronectin to the surface of PPOx coating The strong fluorescence observed on the G protein-coated surface confirmed the availability of the G protein and its efficacy in anti-Fn binding; the fluorescence intensity reflected the amount of anti-Fn binding. The G protein-coated surface exhibited significantly stronger fluorescence at higher antibody concentrations (50 μg / mL), indicating effective binding, while the control showed negligible signal, confirming specific binding to the G protein. Figure 20 BE).

[0201] G protein-mediated binding of anti-PS to the PPOx coating surface Mass spectrophotometric analysis demonstrated that the binding affinity of the anti-PS antibody was stronger when the PPOx-coated surface was first incubated with the G protein. Higher affinity was consistently observed at all antibody concentrations, indicating enhanced anti-PS fixation due to the G protein. Figure 20 FI).

[0202] G protein-mediated binding of anti-PS to PPOx-coated channelized slides Further experiments evaluated the effect of protein-G on sperm-anti-PS interaction. Channel slides coated with PPOx and G proteins were used, and sperm interaction was assessed based on apoptotic and necrotic sperm populations. Figure 20 J, 20K). Compared to surfaces coated with the same concentration (50 μg / mL) of anti-PS without G protein, surfaces coated with G protein plus anti-PS (50 μg / mL) showed significantly lower numbers of annexin V-positive and PI-positive sperm. Compared to surfaces coated with a higher concentration of anti-PS (200 μg / mL) without G protein, sperm recovered from channelized slides coated with G protein plus anti-PS (50 μg / mL) showed comparable numbers of annexin V-positive and PI-positive sperm.

[0203] These experiments demonstrate that G proteins can effectively immobilize anti-PS within channelization devices, and that lower concentrations of anti-PS can be used within the devices to achieve the same or better chelation efficiency of apoptotic sperm. Therefore, protein-G can be a useful coating additive to improve the quality of recovered sperm.

[0204] Example 21 - Use of polystyrene plastics or glass for functionalized surface coatings and surface conjugation of biomolecules to isolate sperm. Functionalized surfaces can be applied to a range of materials for various applications in biology and medicine. In this embodiment, we investigated the effects of surface modification and subsequent sperm selection efficacy using two different substrates: glass and polystyrene plastic (tissue culture plastic, TCP).

[0205] Materials and methods Glass and polystyrene tissue culture plastic (TCP) slides were treated to apply surface modifications as described in previous embodiments, including applying PPOx coating, gold nanoparticles (GNP, 68 nm), and additional PPOx coating. The slides were assembled into channelized slides (0.4 μm in height). Anti-PS antibody was immobilized on the coated surface at a concentration of 200 μg / ml, and progesterone was applied to the outlet well at a concentration of 10 μM, as described in previous embodiments. The apoptotic sperm retention efficacy of the channelized slides was then evaluated using sperm from n=1 human donors, as described in previous examples.

[0206] result The reduction in apoptotic and necrotic sperm populations after recovery from the channelized slide was assessed using flow cytometry and compared with upstream and original samples. The results showed no significant difference in the reduction of apoptotic and necrotic populations between devices constructed with glass or plastic substrates. Figure 21 ).

[0207] This experiment demonstrates that the functionalized surfaces retain their effectiveness in isolating apoptotic sperm regardless of the substrate material. We found that functionalized surfaces on plastic or glass substrates can be used to improve the quality of recovered sperm.

[0208] Example 22 - Selection of boar sperm The effectiveness of the channelized slide device in selecting optimal sperm in a second mammalian species other than humans was determined. Sperm recovered from ejaculating pigs was evaluated using a protocol comparable to that proven effective for human sperm.

[0209] Materials and methods Bovine semen purchased from a commercial supplier was applied to the inlet well (0.4 μm) of a channelized slide and prepared by coating with PPOx, gold nanoparticles (68 nm), anti-PS, and progesterone (prepared as described in previous examples). Unbound sperm were then recovered from the outlet well of the channelized slide after 45 minutes, and markers of sperm motility, apoptosis, and subsequent action (annexin V binding and PI uptake) were assessed as described in previous sections. Data were compared with untreated or control sperm prepared using standard upstream techniques.

[0210] result The results showed that, compared with raw sperm or sperm prepared by standard upstream techniques, the porcine sperm recovered from the channelized sliding device method had a reduced population of apoptotic and necrotic individuals. Figure 22 Specifically, compared with the upstream method ( Figure 22Compared to 18.5% in E), the proportion of apoptotic sperm decreased to 10.4% after recovery from the channelized slide. Similarly, the necrotic population decreased to 5.0% after recovery from the channelized slide, while the proportion of sperm recovered upstream was 25.3%. Figure 22 F). These findings suggest that channelized slides are effective for preparing high-quality porcine sperm with low levels of apoptosis and necrosis.

[0211] Example 23 - Enhancing the selection of high-quality sperm by adding microchannels to the functionalized surface of an NGS device Applying microchannels to functionalized surfaces is considered a means to increase the efficiency of separating high-quality sperm. Microchannels are evaluated considering their potential to increase the surface area for sperm interaction with functionalized surfaces, providing easy access to surfaces and corners that may improve sperm propulsion, and offering a labyrinthine structure that mimics the natural microenvironment of the female reproductive tract.

[0212] Materials and methods For this example, we used a dicing technique to create microchannels along the glass slide before assembling them into an NGS device. The microchannels were 45-50 μm deep, 100 μm wide, and 50 μm apart. This setup produced a total of 24-28 microchannels. Figure 23 A, B). Profilometry measurements reveal the detailed surface morphology and depth profile of the microchannels on the slide, demonstrating the uniformity and precision of their fabrication. Figure 23 C). Surface modification was performed as previously described, with the surface coated using PPOx via plasma polymerization. Subsequently, 68 nm diameter gold nanoparticles (GNPs) were covalently linked and coated with PPOx to achieve a covalent bioconjugation of anti-PS at a concentration of 200 μg / ml, and progesterone (10 μM) was applied to the outlet chamber as described in the previous example. After blocking unreacted groups with 2% HSA and washing with PBS, all liquefied semen (n=2) was loaded into the inlet chamber. The samples were then incubated at 37 °C and 5% CO2 for 45 min. The annexin V and PI-positive populations of sperm recovered from the outlet chamber were examined by flow cytometry to assess the efficacy of the modified surface microchannels, and the incidence of DNA-damaged sperm was determined by Hlosperm assay.

[0213] result Analysis showed a significant reduction in annexin V-positive (apoptotic) sperm, observed in only 3.5% of sperm recovered from the NGS device with microchannels. This is a significant improvement compared to the 13.0% and 11.0% apoptotic sperm found in the upstream method and the uncoated channelized device with microchannels, respectively. Furthermore, analysis by DNA fragmentation assays showed that less than 1% of sperm recovered from the NGS device with coated microchannels exhibited DNA fragmentation. In contrast, sperm prepared by the standard upstream method had 15.0% DNA fragmentation, and the uncoated microchannels produced sperm with a DNA fragmentation rate of 10.0%. These results highlight the superior performance of the NGS device modified to add surface microchannels. The modified NGS device selects high-quality non-apoptotic sperm with minimal DNA damage. This low DNA damage rate offers great potential for improving results in assisted reproductive technology applications.

[0214] Those skilled in the art will understand that this disclosure is not limited in its use to the one or more specific applications described. Preferred embodiments of this disclosure are also not limited to the specific elements and / or features described or depicted herein. It should be understood that this disclosure is not limited to the one or more disclosed embodiments, but is capable of many rearrangements, modifications, and substitutions without departing from the scope set forth and defined by the appended claims.

[0215] References GOREHAM RV, SHORT RD, VASILEV K. 2011. METHOD FOR THE GENERATION OFSURFACE-BOUND NANOPARTICLE DENSITY GRADIENTS. THE JOURNAL OF PHYSICALCHEMISTRY C 115 (8), 3429-3433. LIU MACGREGOR-RAMIASA, M., MCNICHOLAS, K., OSTRIKOV, K., LI, J., MICHAEL,M., GLEADLE, J. M.& VASILEV, K. 2017. A PLATFORM FOR SELECTIVE IMMUNO-CAPTURE OF CANCER CELLS FROM URINE. BIOSENSORS AND BIOELECTRONICS, 96, 373-380. MARTÍNEZ‐LEÓN, E., ET AL., FIBRONECTIN MODULATES THE ENDOCANNABINOIDSYSTEM THROUGH THE CAMP / PKA PATHWAY DURING HUMAN SPERM CAPACITATION.MOLECULAR REPRODUCTION AND DEVELOPMENT, 2019. 86(2): P. 224-238. MARTÍNEZ, M.G., ET AL., MAGNETIC-ACTIVATED CELL SORTING IS NOTCOMPLETELY EFFECTIVE AT REDUCING SPERM DNA FRAGMENTATION. JOURNAL OF ASSISTEDREPRODUCTION AND GENETICS, 2018. 35(12): P. 2215-2221. NAIR, K.S. AND S.M. ZINGDE, ADHESION OF NEUTROPHILS TO FIBRONECTIN:ROLE OF THE CD66 ANTIGENS. CELLULAR IMMUNOLOGY, 2001. 208(2): P. 96-106 RAMIASA, M., CAVALLARO, A., MIERCZYNSKA, A., CHRISTO, S., GLEADLE,J., HAYBALL, J.& VASILEV, K. 2015. PLASMA POLYMERISED POLYOXAZOLINE THINFILMS FOR BIOMEDICAL APPLICATIONS. CHEMICAL COMMUNICATIONS, 51, 4279-4282. SAINZ-GARCIA, E., LOPEZ, M., MUGICA-VIDAL, R., ROJO-BEZARES, B.,LOZANO, C., GONZALEZ-MARCOS, A., TOLEDANO, P., MURO-FRAGUAS, I., SAINZ-GARCIA, A.& SAENZ, Y. 2022. PROMOTION OF BIOFILM PRODUCTION VIA ATMOSPHERIC-PRESSURE PLASMA POLYMERIZATION FOR BIOMEDICAL APPLICATIONS. APPLIED SURFACE SCIENCE, 581,152350. SAKKAS, D., ET AL., SPERM SELECTION IN NATURAL CONCEPTION: WHAT CANWE LEARN FROM MOTHER NATURE TO IMPROVE ASSISTED REPRODUCTION OUTCOMES? HUMANREPRODUCTION UPDATE, 2015. 21(6): P. 711-726. SCHJENKEN, JE AND SA ROBERTSON, THE FEMALE RESPONSE TO SEMINAL FLUID. PHYSIOLOGICAL REVIEWS, 2020. 100(3): P. 1077-1117. THYS, M., ET AL., EXPRESSION AND PUTATIVE FUNCTION OF FIBRONECTIN ANDITS RECEPTOR (INTEGRIN Α5Β1) IN MALE AND FEMALE GAMETES DURING BOVINEFERTILIZATION IN VITRO. REPRODUCTION, 2009. 138(3): P. 471-482. VISALAKSHAN RM, MACGREGOR MN, CAVALLARO AA, SASIDHARAN S, BACHHUKA A,MIERCZYNSKA-VASILEV AM, HAYBALL JD, VASILEV K. 2018. CREATING NANO-ENGINEEREDBIOMATERIALS WITH WELL-DEFINED SURFACE DESCRIPTORS. ACS APPLIED NANOMATERIALS 1 (6), 2796-2807. WARKIANI EM, VASILESCU S, DING L, YAZDAN PARAST F, NOSRATI R. 2022. ABIOMIMETIC MICROFLUIDIC SELECTION PLATFORM PROVIDING IMPROVED SPERM QUALITYMETRICS COMPARED TO SWIM-UP. DOI: 10.21203 / RS.3.RS-1993459 / V1. ZHU T, VASILEV K, KREITER M, MITTLER S, KNOLL W. 2003. SURFACEMODIFICATION OF CITRATE-REDUCED COLLOIDAL GOLD NANOPARTICLES WITH 2-MERCAPTOSUCCINIC ACID. LANGMUIR 19 (22), 9518-9525. THE REFERENCE TO ANY PRIOR ART IN THIS SPECIFICATION IS NOT, ANDSHOULD NOT BE TAKEN AS, AN ACKNOWLEDGEMENT OR ANY FORM OF SUGGESTION THATSUCH PRIOR ART FORMS PART OF THE COMMON GENERAL KNOWLEDGE. Any reference to prior art in this specification is not and should not be construed as any form of recommendation confirming or indicating that such prior art is part of common general knowledge.

Claims

1. A sperm sorting device comprising a substrate having a functionalized surface, the functionalized surface containing a sperm selector, wherein when sperm come into contact with the functionalized surface, the sperm selector is capable of sorting sperm based on at least one biological or chemical property of the sperm.

2. A sperm sorting device for retaining sperm with reduced sperm capacity from a sperm sample used in an ART method including IVF, ICSI, and AI, the device comprising a substrate having a functionalized surface containing a sperm selector, wherein when sperm contacts the functionalized surface, the sperm selector is capable of preferentially retaining sperm with reduced sperm capacity based on at least one biological or chemical property of the sperm with reduced sperm capacity.

3. The sperm sorting apparatus according to claim 1 or claim 2, wherein the functionalized surface is present in one or more flow channels having an inlet for delivering sperm into the flow channel and an outlet for harvesting sorted sperm from the flow channel, the flow channel providing a flow path for sperm from the inlet to the outlet.

4. The sperm sorting device as described in claim 3, wherein the flow channel is a microchannel.

5. The sperm sorting apparatus according to any one of claims 1 to 4, wherein the sperm selector is a biological or chemical reagent that is directly or indirectly covalently bound to the substrate.

6. The sperm sorting apparatus according to claim 5, wherein the sperm selector is covalently bonded to the substrate directly or indirectly via a functional membrane.

7. The sperm sorting device according to claim 6, wherein the functional membrane is selected from polyoxazoline polymers and polyamine polymers.

8. The sperm sorting apparatus according to any one of claims 1 to 7, wherein the sperm selector comprises a reagent that preferentially captures apoptotic sperm.

9. The sperm sorting apparatus according to claim 8, wherein the reagent for preferentially capturing apoptotic sperm comprises an antiphosphatidylserine antibody.

10. The sperm sorting apparatus according to any one of claims 1 to 7, wherein the sperm selector comprises a reagent that promotes sperm endurance (motility and developmental capacity) and / or enables the sperm to be capacitated and ready for fertilization.

11. The sperm sorting apparatus of claim 10, wherein the agent for promoting sperm endurance and / or enabling the sperm to be capacitated and ready for fertilization comprises progesterone.

12. The sperm sorting apparatus according to any one of claims 1 to 7, wherein the sperm sorting apparatus comprises a first functionalized surface and a second functionalized surface, the first functionalized surface comprising a sperm selector that preferentially captures apoptotic sperm, and the second functionalized surface comprising a sperm selector that promotes sperm endurance and enables the sperm to be capacitated and ready for fertilization.

13. The sperm sorting apparatus of claim 12, wherein the first functionalized surface and the second functionalized surface are spatially separated from each other on the substrate.

14. The sperm sorting apparatus of claim 12, wherein the first functionalized surface and the second functionalized surface at least partially overlap each other on the substrate.

15. A method for preparing semen for fertilization using the sperm sorting apparatus according to any one of claims 1 to 14, the method comprising: Sperm are brought into contact with the functionalized surface before selection; Separate the sperm that are not trapped on the functionalized surface from the sperm that are trapped on the functionalized surface; as well as High-potency sperm are harvested from the sperm sorting device.

16. A method for preparing a sperm sorting device as described in any one of claims 1 to 14, the method comprising: Provide base materials; A functional film is deposited on the surface of the substrate; as well as A sperm selector is attached to the functional membrane, the sperm selector being able to interact with the sperm based on at least one biological characteristic of the sperm.

17. A sperm sorting apparatus comprising a substrate having one or more flow channels, the flow channels having an inlet for delivering sperm into the flow channel and an outlet for harvesting sorted sperm from the flow channel, the flow channels providing a flow path for sperm from the inlet to the outlet, the flow path including at least one biological sperm sorting region capable of sorting sperm based on biological properties and at least one motile sperm sorting region capable of sorting sperm based on physical properties.

18. The sperm sorting apparatus of claim 17, wherein the at least one biological sperm sorting region comprises a surface functionalized with one or more biological reagents capable of interacting with sperm.

19. The sperm sorting device of claim 18, wherein the surface functionalized with one or more biological reagents capable of interacting with sperm is a surface that preferentially captures apoptotic sperm.

20. The sperm sorting apparatus of claim 19, wherein the surface that preferentially captures apoptotic sperm is functionalized with an antiphosphatidylserine antibody.

21. The sperm sorting apparatus of any one of claims 17 to 20, wherein the surface functionalized with one or more biological reagents that can interact with sperm is a surface that promotes sperm endurance and enables sperm capacitation in preparation for fertilization.

22. The sperm sorting apparatus of claim 21, wherein the surface that promotes sperm endurance and enables sperm capacitation in preparation for fertilization is functionalized with progesterone.

23. The sperm sorting apparatus according to any one of claims 21 to 22, wherein the sperm sorting apparatus includes a first biological sperm sorting region, the first biological sperm sorting region including a surface that preferentially captures apoptotic sperm, wherein the first biological sperm sorting region is upstream of a second biological sperm sorting region, the second biological sperm sorting region including a surface that promotes sperm endurance and enables sperm capacitation to prepare for fertilization.

24. The sperm sorting apparatus of any one of claims 17 to 23, wherein the motile sperm sorting zone comprises a surface structure that interferes with sperm motility.

25. The sperm sorting apparatus of claim 24, wherein the surface architecture that interferes with sperm motility is a particle array on the surface of the channel.

26. The sperm sorting apparatus of claim 25, wherein the particles are functionalized with antiphosphatidylserine antibody and / or progesterone.

27. The sperm sorting device according to any one of claims 17 to 26, when used for sorting sperm based on fertilization capacity, is used for IVF.

28. A method for preparing semen for fertilization using the sperm sorting apparatus according to any one of claims 17 to 27, the method comprising: The sperm addition step involves adding sperm to the entry point before selection; The sperm sorting step involves, under conditions where the sperm interact with the biological sperm sorting area and the motile sperm sorting area, bringing the sperm in the channel into contact with the biological sperm sorting area and the motile sperm sorting area, and... The harvesting step involves collecting the sperm that have traveled along the channel at the outlet.

29. A method for preparing a sperm sorting device as described in any one of claims 17 to 27, the method comprising: Provide a substrate having one or more flow channels; A functional film is deposited on the surface of one or more flow channels; A biological reagent capable of interacting with sperm is attached to the functional membrane under the condition that at least one biological sperm sorting zone is formed in the channel; as well as Nanoparticles are covalently attached to the functional membrane under the condition that at least one motile sperm sorting zone is formed in the channel.

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