Method and device for sperm selection

Abstract

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

Description

METHOD AND DEVICE FOR SPERM SELECTIONPRIORITY DOCUMENTS

[0001] The present application claims priority from Australian Provisional Patent Application No. 2023902280 titled “SPERM SORTING DEVICE AND METHOD” and filed on 17 July 2023 and Australian Provisional Patent Application No. 2024900010 titled “METHOD AND DEVICE FOR SPERM SELECTION” and filed on 2 January 2024, the contents of which are hereby incorporated by reference in their entirety.TECHNICAL FIELD

[0002] The present disclosure relates generally to assisted reproductive technology and more specifically to sorting or selection of sperm (sperm cells, sperm) for in vitro fertilisation, intracytoplasmic sperm injection, and artificial insemination.BACKGROUND

[0003] In warm-blooded animal species, including humans, fertilisation occurs by sperm cells binding to and fusing with an oocyte. This fertilised oocyte then divides to form an embryo. Over the last several decades, the use of assisted reproduction techniques has allowed scientists and clinicians to assist reproductive success or treat poor fertility or to store sperm, oocytes, or embryos for use at other locations or times. Various assisted reproduction techniques (ART) exist for applications in human or animal reproduction. For example, in vitro fertilisation (IVF) involves collection of eggs, followed by mixing eggs and sperm and culture of the embryos before transferring them to the uterine cavity of the female to continue their development. Intracytoplasmic sperm injection (ICSI) involves injection of a single sperm into an oocyte to elicit fertilisation and embryo development. Artificial insemination (Al) techniques, including intra-uterine insemination (IUI) and intracervical insemination (ICI), are other forms of ART used to facilitate conception.

[0004] The sperm in an ejaculate are not all the same. Their physical features are variable, and so is their ability to fertilise an oocyte and enable an embryo to develop. It is difficult to discern good quality from poor quality sperm based on their appearance, and the physical features that distinguish them are not well understood. Often, but not always, reduced developmental quality in sperm is accompanied by altered morphological features (shape of the sperm head or tail, for example) or activity (strength and directionality of motility, for example). In standard IVF, oocytes are exposed to pools of motile sperm commonly prepared using crude purification or enrichment techniques based on physical features such as morphological features or motility. For example, in standard IVF, sperm are typically selected by ‘swim-up’ or ‘gradient centrifugation’ techniques. Not all sperm with reduced developmental quality are removed by these techniques, and this is thought to be a significant reason for fertilisation failure. In ICSI, individual sperm are usually selected for injection into the oocyte based solely on morphological features. There is concern about potential adverse health consequences in offspring due to the ICSI process. In Al, sperm may be delivered into the female reproductive tract as neat ejaculate (not purified or enriched), or sperm may be purified or enriched using a range of techniques as used for IVF.

[0005] Presently, the inability to clearly distinguish and enrich or select the most competent sperm for fertilisation and embryo development is a constraining factor in ART. Many sperm have suboptimal or impaired functional and developmental quality despite apparently normal physical features. This can include damage to nuclear or mitochondrial DNA or other critical cellular elements, that arises due to reactive oxygen species and other reasons. Damaged or suboptimal sperm often exhibit an apoptotic or pre-apoptotic state, but they may retain normal morphology and motility. If conception with damaged or suboptimal sperm occurs, the sperm can pass on undesirable qualities to the fertilised oocyte that in turn cause embryo demise, and / or cause problems that manifest later in development as foetal congenital abnormalities, placental anomalies, and / or pregnancy complications, and / or compromised health in offspring. In particular, suboptimal quality of sperm selected for injection is implicated in causing the adverse health impacts arising from ICSI.

[0006] Other techniques that have been used to date in sperm selection for ART. These include Annexin V magnetic activated cell sorting (AV-MACS) using paramagnetic Annexin V-conjugated microbeads to eliminate sperm with externalised phosphatidylserine, which is considered one of the features of apoptosis. More recently, a biomimetic microfluidic selection platform providing improved sperm quality metrics compared to swim-up has been proposed (Warkiani et al., 2022). The 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 the sperm. For example, it has been shown that magnetic fields increase the ROS expression in human cells. Additionally, sperm exposed to magnetic microbeads may carry the microbeads into the oocyte when used for fertilisation. Thus, the use of microbeads and magnetic fields may affect the fertilisation and developmental potential of sperm selected using these techniques and may impart adverse consequences to foetuses and offspring.

[0007] Another sperm screening technique that has been used is Hyaluronic Acid (HA) Binding - Sperm Slow or Physiological Intra-cytoplasmic Sperm Injection (PICSI) which involves sperm binding to substrates coated with hyaluronic acid, then physical removal of bound sperm for use in fertilisation. Hyaluronic acid-selected sperm have reduced levels of DNA damage and aneuploidy. Use of hyaluronic acid-based sperm selection for ICSI (so-called physiological ICSI (PICSI)) is reported to reduce the proportion of pregnancies that end in miscarriage. However, the effect of PICSI or similar positiveselection methods on sperm integrity and subsequent embryo and fetal development and livebirth rates is uncertain. In another technique, sperm with high negative surface electrical charge, or "Zeta potential", are selected for ICSI because they are mature and more likely to have intact chromatin. In addition, X- bearing sperm carry more negative charge. Sperm have also been separated by electrophoresis (see, for example, WO2018 / 071977). The effect on sperm integrity of exposure to electric fields and electrophoresis and subsequent embryo and fetal development is also uncertain.

[0008] Unfortunately, all of the current techniques for sperm selection for ART are limited by (a) not truly addressing the fertilization and developmental competence of the sperm, and / or (b) carrying risk of imparting sperm damage or impairment. Accordingly, there is a need for improved sperm preparation techniques in order to improve the success rates and offspring health outcomes of assisted reproduction interventions.

[0009] There is thus a need to provide an apparatus and / or method for sperm selection for ART that ameliorates or reduces one or more of the disadvantages of known apparatus and / or methods.Alternatively, or in addition, there is a need to provide an apparatus and / or method for sperm selection for ART that is a useful alternative to known apparatus and / or methods.SUMMARY

[0010] In a first aspect, provided herein is a sperm sorting device comprising a substrate having a functionalised surface comprising a sperm selection agent that is capable of sequestering sperm based on at least one biological or chemical property of the sperm when sperm is placed in contact with the functionalised surface.

[0011] A functional surface may also be considered a functionalised surface, in that the functionality has been applied or imparted to the surface.

[0012] In a second aspect, provided herein is a sperm sorting device for sequestering reduced competency sperm from a sperm sample to be used for ART, the device comprising a substrate having a functionalised surface comprising a sperm selection agent that is capable of preferentially sequestering reduced competency sperm based on at least one biological or chemical property of the reduced competency sperm when sperm is placed in contact with the functionalised surface.

[0013] The sperm sorting device of the first or second aspect may be particularly useful for sorting sperm based on fertilisation competency for use in ART.

[0014] In certain embodiments of the first and second aspects, the functionalised surface is present in one or more flow channel having an inlet for delivering sperm to 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 selection agent is a biological or chemical agent that is covalently bound, directly or indirectly, to the substrate. Covalent binding of the sperm selection agent to the substrate is advantageous because it prevents transmission of the sperm selection agent and / or associated materials and chemicals into the sperm that will be used for fertilisation.

[0016] In certain embodiments of the first and second aspects, the sperm selection agent is a biological or chemical agent that is non-covalently bound, directly or indirectly, to the substrate. Non-covalent binding of the sperm selection agent to the substrate may occur when chemical release into the soluble phase or interaction with the sperm that will be used for fertilisation is appropriate, for example when the chemical is a natural biochemical usually present in seminal fluid or the female reproductive tract.

[0017] In certain embodiments of the first and second aspects, the sperm selection agent comprises an agent that preferentially captures and / or sequesters apoptotic sperm, or sperm in a pre-apoptotic or damaged state. The agent may be biological agent. The biological agent may be selected from the group consisting of, but not limited to, anti-phosphatidyl serine antibodies, anti-fibronectin antibodies, Annexin V, and hyaluronic acid. These agents replicate aspects of the natural biological sequestration of abnormal sperm that exhibit altered membrane features subsequent to oxidative stress.

[0018] In certain other embodiments of the first and second aspects, the sperm selection agent comprises an agent that promotes sperm endurance and / or primes the sperm ready for fertilisation. The agent that promotes sperm endurance and / or primes the sperm ready for fertilisation may be progesterone. Progesterone acts as a sperm chemotactic and survival factor.

[0019] In certain embodiments of the first and second aspects, the sperm sorting device comprises a first functionalised surface comprising a sperm selection agent that preferentially captures apoptotic sperm and a second functionalised surface comprising a sperm selection agent that promotes sperm endurance and primes the sperm ready for fertilisation. In certain of these embodiments, the first functionalised surface and the second functionalised surface are spatially separated from one another on the substrate. In certain other of these embodiments, the first functionalised surface and the second functionalised surface are at least partially coincident with one another on the substrate.

[0020] In certain embodiments of the first and second aspects, the sperm that is placed in contact with the functionalised surface is in the form of liquified semen that is not mixed with any other material orreagent prior to being placed in contact with the functionalised surface. This advantageously minimises the risk of any kind of contamination and also simplifies the process because there is no need to mix the liquefied semen with a reagent and incubating the mixture at room temperature for 15 minutes, such as may be required in the prior art magnetic activated cell sorting (MACS) and AV-MACS techniques.

[0021] In a third aspect, provided herein is a method of preparing semen for fertilisation using the sperm sorting device of the first aspect or the second aspect, the method comprising: contacting sperm before selection with the functionalised surface; separating sperm that have not been sequestered on the functionalised surface from sperm that have been sequestered on the functionalised surface; and harvesting high competency sperm from the sperm sorting device.

[0022] In a fourth aspect, provided herein is a method of preparing a sperm sorting device of the first aspect or the second aspect, the method comprising: providing a substrate; depositing a functional film on a surface of the substate; and attaching to the functional film a sperm selection agent that is capable to interacting with sperm based on at least one biological characteristic of the sperm.

[0023] In certain embodiments, the sperm sorting device of the first aspect or the second aspect further comprises a sperm sorting zone comprising a surface architecture that interacts with sperm motility. Thus, in a fifth aspect, provided herein is a sperm sorting device comprising a substrate having one or more flow channel having an inlet for delivering sperm to 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 comprising at least one biological sperm sorting zone capable of sorting sperm based on a biological property of the sperm and at least one motility sperm sorting zone capable of sorting sperm based on a physical property of the sperm.

[0024] The sperm sorting device of the fifth aspect may be particularly useful for sorting sperm based on fertilisation competency for use in IVF.

[0025] In certain embodiments of the fifth aspect, the at least one biological sperm sorting zone comprises a surface that is functionalised with one or more biological agents that can interact with sperm. For example, the surface that is functionalised with one or more biological agents that can interact with sperm may be a surface that preferentially captures apoptotic sperm (including pre-apoptotic sperm and DNA-damaged sperm). The surface that preferentially captures apoptotic sperm may be functionalised with anti-phosphatidyl serine antibodies. The anti-phosphatidyl serine antibodies replicate the immune cell-mediated sequestration of abnormal sperm that exhibit altered membrane features subsequent tooxidative stress. In another example, the surface that is functionalised with one or more biological agents that can interact with sperm may be a surface that promotes sperm endurance and primes the sperm ready for fertilisation. The surface that promotes sperm endurance and primes the sperm ready for fertilisation may be functionalised with progesterone. Progesterone acts as a sperm chemotactic and survival factor.

[0026] In certain embodiments of the fifth aspect, the sperm sorting device comprises a first biological sperm sorting zone comprising a surface that preferentially captures apoptotic sperm, wherein the first biological sperm sorting zone is upstream (i.e. closer to the inlet of the channel) of a second biological sperm sorting zone comprising a surface that promotes sperm endurance and primes the sperm ready for fertilisation.

[0027] In certain embodiments of the fifth aspect, the motility sperm sorting zone comprises a surface architecture that interacts with sperm motility. The motility sperm sorting zone replicates aspects of the labyrinthine features of the mammalian oviduct.

[0028] In a sixth aspect, provided herein is a method of preparing semen for fertilisation using the sperm sorting device of the fifth aspect, the method comprising: a sperm adding step of adding sperm before selection to the inlet; a sperm sorting step of contacting sperm in the channel with the biological sperm sorting zone and the motility sperm sorting zone under conditions for the sperm to interact with the biological sperm sorting zone and the motility sperm sorting zone, and a harvesting step of recovering sperm that pass along the channel at the outlet.

[0029] In a seventh aspect, provided herein is a method of preparing a sperm sorting device of the fifth aspect, the method comprising: providing a substrate having one or more flow channel; depositing a functional film on a surface of the one or more flow channel; attaching to the functional film a biological agent that is capable to interacting with sperm under conditions to form at least one biological sperm sorting zone in the channel; and covalently attaching to the functional film nanoparticles under conditions to form at least one motility sperm sorting zone in the channel.BRIEF DESCRIPTION OF THE FIGURES

[0030] Embodiments of the present disclosure will be discussed with reference to the accompanying figures wherein:

[0031] Figure 1 shows the process of surface engineering and characterisation. (A) Schematic illustration of poly-oxazoline (PPOx) deposition on the surface using plasma polymerisation and its subsequent interaction with biomolecules. (B) PPOx interaction with COOH groups on biomolecules leads to the formation of a covalent amide bond. (C) XPS survey spectrum of clean glass surface. (D) XPS survey spectrum of a PPOx-coated glass surface. (E) High resolution Cis spectrum (50 W, 5 min). (F) Immunofluorescence staining images of immobilised anti-PS at increasing concentrations onto PPOx-coated glass. PPOx-coated glass without anti-PS coating was included as a control;

[0032] Figure 2 shows the results of an investigation of the biocompatibility of the PPOx-coated surface with sperm integrity and function. 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 h and 5 h, then their viability (A and B) and total motility (C and D) was assessed following 1 h (A and C) and 5 h (B and D) incubation. A fourth group contained sperm recovered by standard swim-up technique. Results for each of four donor semen samples are shown as different shaped symbols;

[0033] Figure 3 shows the effect of incubation with PPOx-coated surface-immobilised antiphosphatidyl serine (anti-PS) cover slip on sperm selection. (A) Representative light microscope images demonstrating the attachment of washed sperm to the surface immobilised-anti-PS antibody at increasing concentrations (50, 100, 200 and 400 ug / mL). (B) The proportion of sperm adhered to the surface immobilised anti-PS at various concentrations after 30-60 min incubation. Washed sperm at the density of 1 x 106were incubated in prewarmed and CCP-gasscd G-IVF™ Plus in contact with surface-modified coverslip at 37°C and 5% CO2. (C) Average sperm adhesion on surface-immobilised anti-PS at 200 pg / mL (using washed sperm prepared by swim-up from n=5 individual men). (D) The proportion of sperm undergoing early- or late stage apoptosis (apoptotic sperm) labelled with Annexin V was evaluated by flow cytometry. (E) The proportion of necrotic (dead) sperm was evaluated using propidium iodide (PI) staining by flow cytometry. Results for each of five sperm preparations are shown as different shaped symbols;

[0034] Figure 4 shows a comparison of performance of channelled slides and standard swim-up technique for sperm selection. (A) Schematic image of swim-up and channelled slide techniques. (B) Comparison of progressive motile sperm recovery after swim-up and using channelled slides at different channel heights (0.1, 0.2, 0.4, 0.6, and 0.8 pm). (C) Percentage of sperm DNA fragmentation (%SDF) as determined by Halosperm® analysis. (D, E) Sperm total motility and progressive motility values are shown. Results for each of five semen donors are shown as different shaped symbols. Channel height for C-E is 0.4 pm;

[0035] Figure 5 shows the results of mouse sperm selection using functionalised surfaces. (A)Representative flow cytometry plot showing expression of Annexin V and PI of sperm after isolationfrom the epididymis. (B) Representative flow cytometry plot showing expression of Annexin V and PI of sperm remaining unattached following incubation with anti-PS coated coverslips. (C) Representative flow cytometry plot showing expression of Annexin V and PI of sperm following selection through channelled slides. (D) Representative flow cytometry plot showing expression of Annexin V and PI of sperm after selection through anti-PS coated channelled slides, with slide height of 0.4 mm. (E and F) Expression of Annexin V and PI in sperm recovered following incubation with coated coverslips or channelled slides. Evaluation of sperm total motility (G) and progressive motility (H) in sperm recovered following incubation with coated coverslips or channelled slides. (I) Percentage of sperm DNA fragmentation (%SDF) in sperm recovered following incubation with coated coverslips or channelled slides as determined through flow cytometry by TUNEL staining. Results for each of six sperm preparations are shown as different shaped symbols respectively;

[0036] Figure 6 shows a schematic illustration of the fabrication of gold nano-particle-coated channelled slides, containing areas of anti-PS and progesterone coatings (the ‘NGS Device’). (A) Surface-coated PPOx achieved covalent immobilisation of COOH-GNP was followed by a second functional layer of gold nano-particles using PPOx; (B) covalent immobilising of anti-PS antibody (200 pg mL ') onto the glass surface next to the inlet well and electrostatic adsorption of progesterone onto the half of the glass surface next to the outlet well. (C) TEM images of 68 nm gold nano-particles, and (D) atomic force microscopy (AFM) images of surface-bound 68 nm gold nano-particles after 6 h incubation;

[0037] Figure 7 shows results from the evaluation of human sperm quality following preparation using an NGS Device. (A) Representative flow cytometry plot demonstrating Annexin V positivity and PI uptake in sperm within the neat semen sample. (B) Representative flow cytometry plot showing levels of Annexin V positivity and PI uptake in sperm recovered via conventional swim-up techniques. (C) Representative flow cytometry plot demonstrating the expression of Annexin V positivity and PI uptake in sperm recovered after passage through an NGS Device. Comparison of (D) Annexin V expression, and (E and F) percentage of sperm with DNA fragmentation (%SDF) as measured via Halosperm® in sperm from neat semen, conventional swim-up, uncoated channelled slide, and NGS Device. (G) Total motility and (H) progressive motility of sperm prepared using each protocol were also compared. Visual representation of sperm DNA fragmentation as determined by Halosperm® staining, (I) Halosperm® staining of neat samples of liquified semen (leftmost circle = medium to large halo, indicating unfragmented sperm DNA; second circle from left = small halo, indicative of sperm DNA fragmentation; third circle from left = no halo, indicating extensive sperm DNA fragmentation; and right most circle = degraded sperm). (J) Halosperm® staining of sperm after preparation by standard swim-up technique (upper circle = small halo, indicative of sperm DNA fragmentation’ lower circle = no halo, indicating extensive sperm DNA fragmentation). (K) Halosperm® staining of sperm after passage through an NGS Device. Results for each of two different semen donors are shown as circles and squares respectively;

[0038] Figure 8 shows the results of investigations into the role of different selective components of the NGS Device in contributing to optimal sperm selection and quality. Channelled slides were coated with all components as described in Example 6 (‘NGS device’, eg. anti-phosphatidyl serine (anti-PS), gold nanoparticles (GNP), and progesterone (P)), with GNP and P but not anti-PS (‘Chan_GNP_P’); with GNP and anti-PS but not P (‘Chan_GNP_anti-PS’); with antiPS and P but not GNP (‘Chan_Anti-PS_P’), with P but not anti-PS or GNP (‘Chan P’), with anti-PS but not P or GNP (‘Chan_Anti-PS’), with GNP but not anti-PS or P (‘Chan_GNP’), or were uncoated (‘Chan’). Unselected sperm (‘Neat’) and sperm collected by standard swim-up technique (‘Swim-up’) were included for comparison. The quality of recovered sperm was measured as the proportion of sperm staining with Annexin V (A) and the proportion of sperm positive for PI uptake (B) following recovery after exposure to different sperm selection strategies. Results for each of two different semen donors are shown as circles and squares respectively;

[0039] Figure 9 shows IF staining images of anti-Fn coated at concentrations of 200 pg / mL on plasma polymerised PPOx surfaces after passivation (back-filling) with 5% HSA (12 h reaction time). The presence of functional anti-Fn molecules was revealed by interaction with FITC-labeled secondary antibody. A) IF staining of the coated anti-Fn on plasma polymerised PPOx surfaces B) IF staining of the coated anti-Fn on clean glass surface;

[0040] Figure 10 shows fluorescent images of sperm and monocytes labeled with rabbit anti human monoclonal to Fn combined with DAPI staining. A) Monocytes incubated with isotype-matched rabbit IgG antibody control prior to the FITC-labeled secondary antibody treatment. B) IF staining of monocytes with anti-Fn and relevant secondary antibody. C) IF staining of neat sperm (includes sperm and monocytes);

[0041] Figure 11 shows flow cytometry result of Fn expression on the sperm surface. A) isotype- matched rabbit IgG antibody control. B) Fn expression. C) The percentage of sperm expressing Fn on the plasma membrane in 5 participants;

[0042] Figure 12 shows sperm attachment on the anti-Fn coated surface. A) Bright field image of attached sperm on the anti-Fn coated surface. B) The percentage of attached sperm in 3 participants;

[0043] Figure 13 shows detection of apoptosis in sperm using Annexin V-FITC / PI staining. A) Florescence imaging of Annexin V and PI positive sperm population. B) Quantifying of Annexin V / PI positive sperm population by flow cytometry. C, D) Data from flow cytometry of neat sperm and swim up sperm population: "No dye" was considered as a control in which cells were not stained. "PI only", samples were stained with just PI. "Annexin V only", samples were stained with just Annexin V conjugated with Alexa Fluor™ 488. "Annexin V plus PI", samples were stained with both Annexin Vconjugated with Alexa Fluor™ 488 and PI. The x axis represents Annexin V fluorescence; the y axis represents PI fluorescence. In each panel, lower left quadrant displays cells which are negative for both Annexin V and PI, upper left displays PI positive Annexin negative sperm which are necrotic, lower right displays Annexin V positive PI negative sperm which are in early stage apoptosis, and upper right displays both Annexin V and PI positive, which are in late stage apoptosis or necrosis;

[0044] Figure 14 shows fluorescence imaging after bioconjugation of Annexin V-FITC on plasma polymerised PPOx surface (200 pg / ml concentration and 12 h reaction time). The surface was passivated with 5% HSA for overnight and was washed 3 times for 1 h before image capturing;

[0045] Figure 15 shows detection of apoptosis in sperm using Annexin V-FITC / PI staining. A, B) Bright field imaging of attached sperm from two different participants on the Annexin V coated surface. C) The percentage of attached sperm from two different participants on the Annexin V coated surface. D, E) Quantified Annexin V / PI positive sperm population by flow cytometry. C, D) Data from flow cytometry of unattached sperm population on Annexin V coated surface: “No dye” was considered as negative control in which cells were not stained positive. “PI only”, samples were stained with just PI. “Annexin V only”, samples were stained with just Annexin V conjugated with Alexa Fluor™ 488.“Annexin V plus PI”, samples were stained with both Annexin V conjugated with Alexa Fluor™ 488 and PI. The x axis represents Annexin V fluorescence; the y axis represents PI fluorescence. In each panel, lower left quadrant displays cells which are negative for both Annexin V and PI, upper left displays PI positive Annexin negative sperm which are necrotic, lower right displays Annexin V positive PI negative sperm which are in early stage apoptosis, and upper right displays both Annexin V and PI positive sperm, which are in late stage apoptosis or necrosis;

[0046] Figure 16 shows flow cytometry analysis of changes in production of ROS after neat ejaculation, swim up and incubation with Annexin V-coated surface. A) Control in which sperm were not stained. B) Neat ejaculated sample, C) Swim up sperm, D) Unattached sperm on Annexin V-coated surfaces, E) Percentages of ROS positive sperm population (n=l);

[0047] Figure 17 shows the impact of incubating sperm with Annexin V-coated glass coverslips on sperm selection. (A-D) representative images of attached sperm on coverslip surfaces coated by 10, 50, 100, 200 pg / ml Annexin V respectively. (E) The proportion of sperm attached to the surface-immobilised Annexin V was measured after an incubation period of 45-60 minutes. The experiments were conducted using washed human sperm at a density of 1-2 x 106incubated in prewarmed and CCF-gasscd G-IVF™ Plus medium in contact with the surface-modified glass coverslips. The incubation was performed at 37°C and 5% CO2. (F) The average sperm adhesion on the surface-immobilized Annexin V at a concentration of 200 pg / mL, utilizing swim-up sperm samples obtained from a total of six individual men. (G-I) The proportion of apoptotic or necrotic sperm in each treatment group was assessed usingFITC-labelled Annexin V and PI staining via flow cytometry. Representative flow cytometry histograms depicting Annexin V and PI staining in a neat sample (G), swim-up sample (H), and sample incubated with Annexin V-coated coverslip (at a concentration of 200 pg / ml)(I). The proportion of apoptotic or necrotic sperm staining with FITC-labelled Annexin V (J) and PI staining (K) was quantified. The 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;

[0048] Figure 18 shows the impact of incubating human sperm with HA- and Anti Fn-coated glass coverslips. The experiments were conducted using washed sperm (n=3 individual samples per experiment) at a density of 1-2 x 106incubated in prewarmed and CCF-gasscd G-IVF™ Plus medium. The incubation with coated coverslips was performed at 37°C and 5% CO2. (A) Representative image of attached sperm on coverslip surface coated by 300 pg / ml HA. (B) The proportion of sperm attached to the surface-immobilised HA was measured after an incubation period of 45-60 minutes. (C) Representative image of attached sperm on coverslip surface coated with 40 pg / ml Anti-Fn. (B) The proportion of sperm attached to the surface-immobilised Anti-Fn was measured after an incubation period of 45-60 minutes. Representative flow cytometry histograms depicting Annexin V staining in a neat sample (E), swim-up sample (F), and sample incubated with HA-coated coverslip (at a concentration of 300 pg / ml) (G). Representative flow cytometry histograms depicting Annexin V staining in a neat sample (H), swim-up sample (I), and sample incubated with anti-Fn-coated coverslip (at a concentration of 100 pg / ml) (J). (K) The proportion of sperm unattached to HA-coated coverslips labelled with Annexin V was calculated. (L) The proportion of sperm unattached to Anti-Fn-coated coverslips labelled with Annexin V was calculated;

[0049] Figure 19 shows evaluation of the effect of sperm interaction with a range of coatings on glass coverslips. Glass coverslips were treated to generate either a plasma-polymerized amine film or a PPOx film, then coated with either GNPs and anti-PS antibody, or with anti-PS antibody without GNPs, giving rise to 4 different surface coating variants (NH2, GNP, PPOx, Anti-PS; PPOx, GNP, PPOx, Anti-PS; NH2, Anti-PS, and PPOx, Anti-PS). Sperm prepared by swim-up were incubated with the coated coverslips and evaluated for apoptosis by Annexin V staining, and motility parameters, compared to a swim-up control. (A-G) Representative flow cytometry dot plots for each sperm preparation. (H, I) The proportion of sperm labelled with Annexin V and PI, indicating apoptotic and necrotic populations respectively. (J, K) Total and progressive sperm motility assessed following collection of recovered sperm from different groups;

[0050] Figure 20 shows the effect of prior coating with Protein-G on Anti-PS immobilisation and sperm interaction with the surface. (A) Schematic representation of immobilization of Protein-G on the surface and its interaction with Anti-PS, and subsequently with sperm. (B-E) Confirmation of Protein G availability on PPOx-coated surfaces detected using anti-Fn labelled with Alexa Fluor 488. Fluorescenceimages depict the interaction between different anti-Fn concentrations (2, 5, 20, and 50 pg / ml) and the Protein-G-coated surface. Corresponding control reactions are displayed in the upper left corner of each image. The scale bar measures 100 pm. (F-H) Assessment of Protein-G functionality in immobilisation of Anti-PS on PPOx-coated glass coverslip surfaces by mass photometry. Mass photometer images show the relative density of Anti-PS molecules available in solution, before and after incubation with the different surfaces, as indicated. A lower concentration of residual Anti-PS is evident in a solution incubated with a PPOx plus Protein-G coated surface (H), compared to PPOx alone (G), or control not incubated with a coated surface (F). (I) Mass photometry analysis of the relative density of Anti-PS in solution at a concentration of 50 pg / ml, before and after incubation with the different surfaces, as indicated in the legend. (J-K) Flow cytometry analysis of sperm recovered from channelled slides prepared with and without inclusion of Protein-G 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). The results from each of three different sperm preparations are shown using distinct symbols corresponding to each preparation. Data were analysed by ANOVA and post-hoc t-test. * P< 0.01;

[0051] Figure 21 shows a comparative analysis of glass and polystyrene substrates for surfacemodification and assembly into channelled slide devices for sperm selection. (A-D) Representative flow cytometry dot plots for (A) no dye; (B) neat sperm, or sperm preparations recovered from (C) tissue culture plastic channelled slide (TCP-NGS) or (D) glass channelled slide (Glass-NGS) devices. (E-F) Flow cytometry analysis showing the proportion of sperm in neat seminal fluid or recovered sperm preparations (from TCP or glass channelled slide devices) labelled with Annexin V (E) and PI (F), indicating apoptotic and necrotic populations respectively. (G, H) Total and progressive sperm motility assessed following collection of recovered sperm from different groups;

[0052] Figure 22 shows evaluation of channelled slides with functionalised surface for pig sperm selection. (A) Photomicrograph showing attached pig sperm captured by immobilized Anti-PS in functionalised surface channelled slides. (B-D) Representative flow cytometry dot plots of neat sperm (B), sperm prepared by swim-up (C) and sperm after recovery from functionalised surface channelled slides (D). (E-F) Flow cytometry analysis showing the proportion of sperm in neat seminal fluid or recovered sperm preparations labelled with Annexin V (E) and PI (F), indicating apoptotic and necrotic populations respectively. (G) Total sperm motility assessed following collection of recovered sperm from different groups; and

[0053] Figure 23 shows the effect of addition of microchannels to NGS Devices on sperm selection efficiency. (A) Stylized schematic showing addition of microchannels to glass slides prior to NGS assembly. (B) Photomicrograph image of the microchannels on glass slides. (C) Profilometry measurements of surface topography and depth profiles of the microchannels on glass slides. (D-G)Representative flow cytometry dot plots of neat sperm (D), sperm prepared by swim-up (E), and sperm after recovery from non-coated NGS Devices with microchannels (F), and from coated NGS Devices with microchannels (G). (H-I) Flow cytometry analysis showing the proportion of sperm in neat seminal fluid or recovered sperm preparations labelled with Annexin V (H) and PI (I), indicating apoptotic and necrotic populations respectively. (J, K) Total and progressive sperm motility assessed following collection of recovered sperm from different groups. (E) The proportion of sperm with DNA fragmentation determined by Halosperm assay. (M-O) Representative images of sperm analysed by Halosperm in neat seminal fluid (M), after swim-up (N), and after recovery from NGS Devices with added microchannels.DESCRIPTION OF EMBODIMENTS

[0054] The following definitions are provided for specific terms which are used in the following written description.

[0055] As used in the specification and claims, the singular form "a", "an" and "the", include plural references unless the context clearly dictates otherwise.

[0056] In the specification and claims, an item, object, device, apparatus, system, method or process can "comprise" (open ended) or "consist essentially of" the listed elements as well as other ingredients or elements described herein. As used herein, "comprising" means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. The terms "having" and "including" are also to be construed as open ended unless the context suggests otherwise. As used herein, "consisting essentially of" means that the invention may include ingredients in addition to those recited in the claim, but only if the additional ingredients do not materially alter the basic and novel characteristics of the claimed invention.

[0057] The term "about" or "approximately" means within an acceptable range for the particular value as determined by those skilled in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Unless otherwise stated, the term "about' means within an acceptable error range for the particular value, such as ± 1-20%, preferably ± 1-10% and more preferably ± 1-5%. In even further embodiments, "about" should be understood to mean + / -5%.

[0058] Where a range of values is provided, it is understood that each intervening value, between the upper and lower limits of that range and any other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0059] All percentages and ratios used herein are by weight of the total composition unless otherwise indicated herein. All temperatures are in degrees Celsius unless specified otherwise. All measurements made are at 25 °C and normal pressure unless otherwise designated.

[0060] All ranges recited herein include the endpoints, including those that recite a range "between" two values. Terms such as "about", "generally", "substantially", "approximately" and the like are to be construed as modifying a term or value such that it is not an absolute, but does not read on the prior art. Such terms will be defined by the circumstances and the terms that they modify as those terms are understood by those skilled in the art. This includes, at very least, the degree of expected experimental error, technique error and instrument error for a given technique used to measure a value.

[0061] Where used herein, the term "and / or" when used in a list of two or more items means that any one of the listed characteristics can be present, or any combination of two or more of the listed characteristics can be present. For example, if a composition of the present invention is described as containing characteristics A, B, and / or C, the composition can contain A feature alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0062] Disclosed herein is a sperm sorting device 10, an embodiment of which is shown in Figure 6 A and B. The device 10 comprises a substrate 12 having a functionalised surface 14 comprising a sperm selection agent that is capable of sorting sperm based on at least one biological or chemical property of the sperm when sperm is placed in contact with the functionalised surface 14.

[0063] Also disclosed herein is a sperm sorting device 10 for sequestering reduced competency sperm from a sperm sample to be used for ART. The device 10 comprises a substrate 12 having a functionalised surface 14 comprising a sperm selection agent that is capable of preferentially sequestering reduced competency sperm based on at least one biological or chemical property of the reduced competency sperm when sperm is placed in contact with the functionalised surface 14.

[0064] In the female reproductive tract, there are several biological mechanisms that work collectively to select sperm with optimal features, and to ensure that sub-standard sperm do not have access to fertilise an oocyte. In effect, sperm must navigate a form of biological ‘obstacle course’ as they pass through the cervical canal, the lumen of the uterus, and the fallopian tube. Along the way, they will interact withimmune cells and epithelial cells, and many are sequestered (physically restrained) or eliminated by the actions of these cells or their secretions, so that only a small proportion reach the ampulla region of the upper fallopian tube where fertilisation takes place. Along the way, the most competent and ‘fit’ sperm improve their ability to pass the final test of penetrating the oocyte zona pellucida. The sperm sorting device 10 disclosed herein integrates molecular features to provide a simple, reliable, and effective selection of the best quality sperm in an ejaculate, in a format that is suitable for applications in human and animal clinical ART laboratories.

[0065] The device 10 provides for a relatively simple sperm sorting technique. Many prior art sperm sorting techniques involve multiple steps, such as incubation and magnetic field application. These steps could potentially lead to sample loss, and the overall efficiency of the method may be affected. Mixing of liquefied semen with a reagent and incubating the mixture may also have clinical considerations. In contrast, the devices and methods disclosed herein use immobilised sperm selection agents (e.g. immobilised antibodies) to sequester sperm without mixing any reagents with the semen. This reduces the risk of sample contamination. Furthermore, the devices and methods disclosed herein do not require the use any magnetic field which may affect the fertilisation potential of sperm.

[0066] In addition, some prior art devices, such as the one disclosed in Warkiani et al, require the use of strong magnets and, as a result, are inappropriate for 'at home' use. The devices and methods disclosed herein are simpler to use and can be used for home preparation of sperm for Al by self-administration.

[0067] The sperm selection agent that can interact with sperm can be any molecule that has a molecular interaction with sperm. The agents that can interact with sperm may be biomolecules. The biomolecules may for example be selected from amino acids, peptides, proteins, steroid hormones, aptamers, nucleic acids, DNA molecules, RNA molecules, antibodies, growth factors, antimicrobial agents, antithrombogenic agents, and cell attachment proteins. The biomolecules may be in the form of particles or nanoparticles comprising the biomolecule.

[0068] The sperm selection agent may be an agent that preferentially captures and / or sequesters apoptotic sperm, or sperm in a pre-apoptotic or damaged state. The agent may be a biological agent such as anti-phosphatidyl serine antibodies, anti-fibronectin antibodies, Annexin V, and hyaluronic acid. These agents replicate aspects of the immune cell-mediated sequestration of abnormal sperm that exhibit altered membrane features subsequent to oxidative stress. Alternatively, or in addition, the sperm selection agent can be any agent that promotes sperm endurance and / or primes sperm ready for fertilisation. One suitable agent that promotes sperm endurance and / or primes the sperm ready for fertilisation is progesterone. Progesterone acts as a sperm chemotactic and survival factor.

[0069] The sperm selection agent may include secreted and surface bound proteins and biological agents, including cytokines and / or other immune -regulatory molecules, that are involved in the naturally- occurring sequestration and / or killing of sperm by female immune cells or epithelial cells within the reproductive tract after intromission of seminal fluid.

[0070] The sperm selection agent may be covalently attached to substrate surface or it may be non- covalently attached to the substrate surface. Covalent attachment to the substrate surface may be by way of a functional film and facilitated by reaction between one or more functional groups on the functional film and the sperm selection agent. For example, amino groups in a functional film may react with carboxylic, carboxylate or aldehyde groups of the sperm selection agent. If required, reaction between the one or more functional groups on the functional film and the sperm selection agent may be facilitated or catalysed. For example, reaction between the one or more functional groups on the functional film and the sperm selection agent may be facilitated by a coupling agent such as a carbodiimide coupling agent (for example EDC, DCC, DIC), a triaminophosphonium coupling agent (for example BOP, PyBOP, PyBrOP) or a tetramethylaminium / tetramethyluronium coupling agent (for example HATU, HBTU, HCTU). If required, a cross linker moiety may be used between the functional film and the sperm selection agent.

[0071] There is a considerable benefit associated with covalent attachment of the sperm selection agent to the surface of the substrate and this ensures there will be no transmission of chemicals into the sperm that will be used for fertilisation.

[0072] The functionalised surface 14 may be present in any suitable form on the substate 12. Advantageously, the functionalised surface 14 is configured on the substrate 12 so that a sperm sample is able to pass over the functionalised surface 14 under conditions for lower competency sperm to be sequestered on the functionalised surface 14 and for higher competency sperm to pass over the functionalised 14 without being sequestered. This can be done in a number of ways, including positioning the functionalised surface 14 in a channel into which the sperm can be introduced. In certain embodiments, the functionalised surface 14 is present in one or more flow channel 16 having an inlet 18 for delivering sperm to the flow channel 16 and an outlet 20 for harvesting sorted sperm from the flow channel 16. As such, 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 comprises a glass microscope slide 30 featuring a 500 pm x 5 cm flow channel 16 and 100 microlitre reservoirs at the inlet 18 and outlet 20 of the flow channel 16. The lower glass surface of the flow channel 16 is functionalised with (1) covalently bound anti-phosphatidyl serine (anti-PS) antibodies and (2) ionic (non-covalent) bound progesterone on the substrate surface. The anti-phosphatidyl serine forms a first functionalised surface 14a that replicates the immune cell-mediated sequestration of abnormal sperm (that exhibit altered membrane features subsequent to oxidative stress), and the progesterone forms a second functionalised surface 14b whereinthe progesterone is a survival factor that promotes sperm endurance and primes the sperm ready for fertilisation.

[0073] The biological agents, such as anti-phosphatidyl serine antibodies, anti-fibronectin antibodies, Annexin V, and hyaluronic acid, are covalently bound to the surface of the flow channel 14 by functionalising the surface with a functional film that is capable of covalently bonding to the anti-PS antibodies.

[0074] Any suitable substrate can be used, provided a functional film can be formed on the surface thereof and can be retained on the surface thereof under typical operating conditions. Suitable substrate materials include glass, silicon, ceramics, metals, plastics, polymeric materials, paper, paper laminates, cellulose, carbon fibre, biomaterials, surfaces comprising biological molecules, surfaces comprising small organic molecules, surfaces comprising inorganic molecules, etc. The plastic may be selected from the group consisting of: polycarbonate, polyethylene, polypropylene, polystyrene, polytetrafluoroethylene, polyethylene terephthalate; polyethylene naphthalene dicarboxylate, tetrafluoroethylene - hexafluoropropylene copolymers, polyvinyl-difluoride, nylon, polyvinylchloride, copolymers of the aforementioned, and mixtures of the aforementioned. In embodiments, the substrate is glass. In other embodiments, the substrate is polystyrene.

[0075] The substrate has one or more flow channel 16. The one or more flow channel 16 can be any suitable size, geometry or configuration. The one or more flow channel 16 can be open or enclosed, such as channels commonly used in "flow through" type diagnostic devices. In some embodiments, the substrate contains one or more microfluidic flow channel or microchannel 16. As used herein, the terms “microfluidic”, “microchannel” and variants thereof, means that the chip, device, apparatus, substrate, channel or related apparatus contains fluid control features that have at least one dimension that is submillimetre.

[0076] Optionally, an inner surface of the one or more flow channel 16 may be coated with a material having different properties to the substrate material, such as for example poly(dimethylsiloxane) (PDMS) or epoxy.

[0077] Optionally, the one or more flow channel 16 can be defined with three dimensional (3D) patterns or elements to alter surface topography and enhance interaction between the sperm and the functionalised surface 14. By way of non-limiting examples, the functionalised surface 14 (such as the flow channel 16) may comprise nanoparticles, such as gold nanoparticles, that are bound covalently to the surface. Alternatively, or in addition, the functionalised surface 14 (such as the flow channel 16) may comprise grooves or etching onto which a functional layer is applied. 3D printing may be used tofabricate a range of surfaces having some form of 3D patterning or topography and onto which a functional layer can be added.

[0078] The sperm sorting device 10 may be particularly useful for sorting sperm for competence in ART in humans and other mammals, including rodents (rats, mice, hamsters, guinea pigs, etc.), primates, simians (including monkeys, apes, etc.), monkeys (including marmosets, baboons, etc), apes (including gorillas, chimpanzees, orangutans, gibbons, etc.), farm and livestock animals (including sheep, goats, pigs, cows, horses, etc.), sport animals, endangered species of animals (including panda bears, etc), and pets, e.g., (dogs, cats, etc.).

[0079] The functional film can comprise any inorganic, organic and / or biological material, molecule or mixture of molecules that can be attached to the surface of the substrate by non-ionic bonding.

[0080] Alternatively, the functional film can comprise any inorganic, organic and / or biological material, molecule or mixture of molecules that can be attached to the surface of the substrate by covalent or ionic bonding and contain one or more functional groups available for covalent or ionic bonding to at least one sperm selection agent used functionalised surface 14. The one or more functional groups of the functional film are preferably capable of bonding to carboxylic acid groups, carboxylate groups, amino groups or amido groups on a biological molecule.

[0081] Examples of chemistries suitable for covalent attachment of biological sperm selection agents include oxazolines, amines, epoxies, aldehydes, anhydrides, thiols, EDC / NHS related chemistries, click chemistries, isocyanates, nitriles and imines.

[0082] In certain embodiments, the functional film is a polymer. The polymer may be formed by classical polymerisation techniques. Thus, polymers can be formed on a surface of the substrate by polymerisation of suitable starting monomers or pre-polymers using suitable polymerisation agents, as is known in the polymer art.

[0083] In other certain embodiments, the functional film may be formed by be formed by plasma polymerisation. Thus, in certain embodiments, the functional film is a plasma polymer formed by plasma polymerisation of one or more functional starting materials. Oxazoline, amine, epoxy, aldehyde, anhydride, thiol, isocyanate, nitrile and imine containing starting materials can be plasma polymerised using the conditions described herein to form plasma polymers containing oxazoline, amine, epoxy, aldehyde, anhydride, thiol, isocyanate, nitrile and imine groups that can then be reacted with the biological agents that can interact with sperm to form covalent bonds therewith.

[0084] Non-limiting examples of starting materials that can 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. propanal), glycidyl methacrylate, and ally glycidyl ether.

[0085] In certain embodiments, the functional film is a plasma polymerized polyoxazoline ("PPOx"). As used herein, the term "polyoxazoline" means a homopolymer or copolymer formed from at least one oxazoline starting material or monomer. The polyoxazoline polymer may or may not comprise intact oxazoline moieties. The polyoxazoline polymer may be a copolymer formed by plasma polymerisation of at least one oxazoline starting material or monomer and at least one comonomer. The comonomer may be chosen based on the desired properties it may provide to the polyoxazoline polymer and / or its suitability for plasma polymerisation (e.g. its vapour pressure or volatility). The comonomer may be selected from the group consisting of but not limited to: silanes, siloxanes, fluorocarbons, hydrocarbons, reactive functional monomers, organo-based monomers, and unsaturated monomers such as N-vinylpyrrolidone, hydroxyethylmethacrylate, acrylamide, dimethylacrylamide, dimethylaminoethylmethacrylate, acrylic acid, methacrylic acid, a vinyl substituted polyethylene or polypropylene glycol, a vinylpyridine, and a vinylsulfonic acid.

[0086] The plasma polymerised polyoxazoline polymer and functional film on the surface of the substrate can be prepared by exposing the surface of a substrate to a plasma comprising an oxazoline monomer vapour under conditions to polymerise the oxazoline monomer to form the plasma polymerised polyoxazoline polymer on the surface of the substrate.

[0087] The conditions required to polymerise the oxazoline monomer to form the plasma polymerised polyoxazoline polymer may comprise a power of from about 10W to about 50W, a deposition time of from about 1 minute to about 7 minutes, and / or a monomer pressure of from about 0.5 to about 3 x 101mbar. A power of about 50W for a time of about 2 minutes are particularly suitable conditions to polymerise the oxazoline monomer to form the plasma polymerised polyoxazoline polymer because they provide stable plasma polymerised polyoxazoline polymer films having a thickness of greater than about 20 nm, such as about 20 nm to 40 nm or 20 nm to 30 nm. A range of other deposition conditions would be applicable depending on the plasma deposition equipment design and power coupling efficacy.

[0088] The plasma comprising an oxazoline monomer vapour is formed at reduced pressure in a vacuum chamber. Thus, the step of exposing the surface of a substrate to a plasma comprising an oxazoline monomer vapour may include placing the substrate in a chamber, sealing the chamber, forming a plasma in the chamber, introducing a vapour containing the oxazoline monomer into the chamber, and maintaining the substrate at a temperature suitable for polymerisation of the oxazoline monomer so as to form a polymer film on the surface. Persons skilled in the art will understand that a plasma is anelectrically excited ionised gas or gases, that, upon excitation (eg. ignition), forms a highly reactive environment that can modify materials directly exposed to the plasma discharge. The plasma deposition step can be operated over a wide range of pressures (for example, from 10 mTorr to above atmospheric pressure (eg. lOx atmosphere or higher)). The plasma may consist of a combination of an inert gas (eg. helium, neon, argon, krypton, xenon, radon) and the oxazoline monomer (or other suitable monomer as required depending on the chemistry of functional film). 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).

[0089] The above plasma polymerization conditions can also be used to form plasma polymers from other functional starting materials as required.

[0090] The oxazoline monomer may be a substituted oxazoline with a substituent at any 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 the plasma polymerised polyoxazoline polymer provided they are a vapour under the plasma deposition conditions used. In embodiments, the oxazoline monomer is selected from the group consisting of 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 substituent(s) on the oxazoline ring may be selected from the group consisting of: halogen, OH, NO2, CN, NH2, optionally substituted Ci-C alkyl, optionally substituted C2- Ci2alkenyl, optionally substituted C2-Ci2alkynyl, optionally substituted C2-Ci2heteroalkyl, optionally substituted Cs-C cycloalkyl, optionally substituted C2-Ci2heterocycloalkyl, optionally substituted C2- Ci2heterocycloalkenyl, optionally substituted CT-Cis aryl, optionally substituted Ci-Cisheteroaryl, optionally substituted Ci-Ci2alkyloxy, optionally substituted C2-Ci2alkenyloxy, optionally substituted C2- Ci2alkynyloxy, optionally substituted C2-Ci2heteroalkyloxy, optionally substituted Cs-C cycloalkyloxy, optionally substituted Cs-C^cycloalkenyloxy, optionally substituted Ci-Ci2heterocycloalkyloxy, optionally substituted C2-Ci2heterocycloalkenyloxy, optionally substituted Ce-Cixaryloxy, optionally substituted Ci-Cisheteroaryloxy, optionally substituted Ci-C alkylamino, SO3H, SO2NH2, SO2R, SONH2, SOR, COR, COOH, COOR, CON RR, NRCOR , NRCOOR , NRSO2R, N RCON RR", and NRR. In some embodiments, the oxazoline monomer comprises a 2-substituted oxazoline. In specific embodiments, the oxazoline monomer is a 2-alkyl-2-oxazoline. The alkyl substituent may be a C1-C12 alkyl, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, pentyl, etc.

[0091] In specific embodiments, the oxazoline monomer is selected from the group consisting of 2- alkyl-2-oxazolines and 2-aryl-2-oxazolines. The alkyl substituent of the 2-alkyl-2-oxazolines may be a C1-C10 alkyl, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, pentyl, and the like. The alkyl substituent may be optionally substituted. The aryl substituent of the 2-aryl-2-oxazolines may be aC5-C10 aryl, such as optionally substituted phenyl, optionally substituted naphthyl, optionally substituted thienyl, optionally substituted indolyl, and the like.

[0092] In certain other embodiments, the functional film is a plasma polymerized amine. The amine polymer may be formed by plasma polymerisation of at least one unsaturated amine monomer, such as allylamine.

[0093] The surface of the substrate may be treated prior to deposition of the plasma polymerised polyoxazoline polymer. For example, the surface may be treated by cleaning with a detergent, water or a suitable solvent. Alternatively, or in addition, the surface may be treated by exposing the surface to air in a plasma chamber in order to activate the surface.

[0094] The reactivity of the oxazoline ring present on the co-terminus of polyoxazolines has been used for conjugation with protein and drugs in solution. The reactivity of the oxazoline ring is believed to lead to the formation of a covalent amide bond by reaction with carboxylic acid functional groups. Plasma deposited coatings allow for the retention of intact oxazoline rings at the surface, which is typically not the case when other techniques for surface preparation are used. Retention of such reactive chemical functionalities then allows convenient and rapid covalent coupling of proteins, antibodies and the like.

[0095] Optionally, the functional film may further comprise an additional binding moiety that is capable of covalently binding to the functional film, such as plasma polymerised polyoxazoline or amine polymer, and also capable of binding to a sperm selection agent of interest. The additional binding moiety may comprise any inorganic, organic and / or biological material, molecule or mixture of molecules. For example, Protein G can be utilised for antibody attachment to surfaces due to its strong affinity for the Fc region of immunoglobulin (antibody) proteins.

[0096] The sperm may be delivered to the inlet 18 (and hence the channel 16) in any suitable form, including as semen sample, sperm and fluid, etc. The semen sample used may be a fresh semen sample or a stored semen sample. Any storing methods may be used for storing the semen sample, for example freezing the semen sample.

[0097] To sort sperm in the sperm sorting device 10 a sperm preparation medium is introduced into the channel. Any suitable sperm preparation medium comprising a medium of fluid which supports cell culture such as IVF medium, sperm preparation medium, and / or isotonic salt solutions can be used. The sperm preparation medium may contain substances to further protect sperm such as substances protecting the sperm from free oxygen radicals, etc. The substances to protect the sperm can be, for example, EDTA, albumin, and / or proteins. Examples of liquid media which may be used are described in PCT / EP97 / 06721 which is hereby incorporated by reference. A range of suitable sperm preparation media are also availablecommercially. For example, commercially available G-IVF™ Plus medium available from Vitrolife may be used. In use, sperm delivered to the inlet 18 are able to swim in the sperm preparation medium from the inlet 18 to the outlet 20 and, in doing so, must interact with the functionalised surface 14 (which may comprise a single functionalised surface 14 or it may comprise a first functionalised surface 14a and a second functionalised surface 14b) before reaching the outlet 20 where they can be recovered.

[0098] Optionally, the sperm to be sorted in the sorting device 10 may be subjected to a pre-screen beforehand. Any suitable known sperm sorting method can be used as a pre-screen such as, for example, standard swim-up methods to isolate motile sperm.

[0099] To use the sperm sorting device 10, seminal fluid can be introduced to a reservoir at the inlet 18 and a commercial sperm preparation medium can be introduced to the reservoir at the outlet 20 and the flow channel 16. The sperm sorting device 10 can then be incubated under suitable conditions before sperm are recovered from the reservoir at the outlet 20. Any suitable incubation conditions can be used and can be determined by the skilled person. For example, the incubation conditions may be 45-90 minutes at 37°C and 6% CO2. The sperm recovered from the outlet 20 are ‘ready to use’ in ART and have been removed of their seminal plasma component (without requirement for further swim -up or washing by centrifugation, etc.).

[0100] Also disclosed herein is a method of preparing semen for fertilisation using the sperm sorting device disclosed herein. The method comprises contacting sperm before selection with the functionalised surface. Sperm that have not been sequestered on the functionalised surface from sperm that have been sequestered on the functionalised surface are then separated and high competency sperm are harvested from the sperm sorting device.

[0101] Also disclosed herein is a method of preparing a sperm sorting device as disclosed herein. The method comprises providing a substrate, depositing a functional film on a surface of the substate and attaching to the functional film a sperm selection agent that is capable to interacting with sperm based on at least one biological characteristic of the sperm.

[0102] Also disclosed herein is a sperm sorting device 10, an embodiment of which is shown in Figure 6B. The device 10 comprises a substrate 12 having one or more flow channel 14 having an inlet 16 for delivering sperm to 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 comprises at least one biological sperm sorting zone 22 capable of sorting sperm based on a biological property of the sperm and at least one motility sperm sorting zone 24 capable of sorting sperm based on a physical property of the sperm.

[0103] Advantageously, the sperm sorting device 10 disclosed in Figure 6B and described herein replicates the naturally occurring processes of sperm selection in the human female reproductive tract after coitus. In the female reproductive tract, there are several biological mechanisms that work collectively to select sperm with optimal features, and to ensure that sub-standard sperm do not have access to fertilise an oocyte. In effect, sperm must navigate a form of biological ‘obstacle course’ as they pass through the cervical canal, the lumen of the uterus, and the tortured labyrinth of the fallopian tube. Along the way, they will interact with immune cells and epithelial cells, and many are sequestered (physically restrained) or eliminated by the actions of these cells or their secretions, or their inability to traverse the physical barriers of the cervix, uterus and lower fallopian tube, so that only a small proportion reach the ampulla region of the upper fallopian tube where fertilisation takes place. Along the way, the most competent and ‘fit’ sperm access survival factors and become primed or ‘licensed’ to increase their competitive advantage over other sperm, improving their physical ability to pass the final test of penetrating the oocyte zona pellucida. The sperm sorting device 10 disclosed herein integrates a combination of physical and molecular features to provide a simple, reliable, and effective selection of the best quality sperm in an ejaculate, in a format that is suitable for applications in human and animal clinical IVF laboratories.

[0104] In the embodiment illustrated in Figure 6B, the sperm sorting device 10 comprises a glass microscope slide 30 featuring a 500 pm x 5 cm flow channel 14 and 100 microlitre reservoirs at the inlet 16 and outlet 20 of the flow channel 14. The lower glass surface of the flow channel 14 is functionalised with (1) gold nanospheres and (2) covalently bound anti-phosphatidyl serine (anti-PS) antibodies and ionic (non-covalent) bound progesterone on the glass and gold surfaces. The flow channel 14 and the gold nanospheres provide a motility sperm sorting zone 24 having a complex architecture that replicates the labyrinth of the oviduct. The anti-phosphatidyl serine forms a first biological sperm sorting zone 22a that replicates the immune cell-mediated sequestration of abnormal sperm (that exhibit altered membrane features subsequent to oxidative stress), and the progesterone forms a second biological sperm sorting zone 22b wherein the progesterone is a survival factor that promotes sperm endurance and primes the sperm ready for fertilisation.

[0105] The gold nanospheres and anti-phosphatidyl serine (anti-PS) antibodies are covalently bound to a surface of the channel by functionalising the surface with a functional film that is capable of covalently bonding to the gold nanospheres and anti-PS antibodies.

[0106] Also disclosed herein is a method of preparing semen for fertilisation using the sperm sorting device 10 as disclosed in Figure 6B. The method comprises a sperm adding step of adding sperm before selection to the inlet followed by a sperm sorting step of contacting sperm in the channel with the biological sperm sorting zone and the motility sperm sorting zone under conditions for the sperm tointeract with the biological sperm sorting zone the motility sperm sorting zone. Sperm that pass along the channel are recovered at the outlet in a harvesting step.

[0107] Also disclosed herein is a method of preparing a sperm sorting device 10 as disclosed in Figure 6B. The method comprises providing a substrate having one or more flow channel and depositing a functional film on a surface of the one or more flow channel. A biological agent that is capable of interacting with sperm is attached to the functional film under conditions to form at least one biological sperm sorting zone in the channel. Nanoparticles are then covalently attached to the functional film under conditions to form at least one motility sperm sorting zone in the channel.EXAMPLES

[0108] Example 1 - Preparation of antibody-coated, plasma-polymerised polyoxazoline (PPOx) thin films on glass channel slides

[0109] This example demonstrates (1) the feasibility of applying PPOx thin films to functionalise the surface of glass channel slides, and (2) the suitability of the PPOx-coated channel slides for coating with antibody, in a biologically active form that would allow development of a sperm selection device.

[0110] Anti-phosphatidyl serine (anti-PS) antibody was chosen for coating studies in order to mimic the surface properties of neutrophils that act in the female reproductive tract in vivo to sequester sub- optimal sperm from the ejaculate. Neutrophils bind sperm that are undergoing early or late-stage apoptosis, which causes loss of sperm plasma membrane integrity and externalisation of phosphatidyl serine (PS) moieties. Neutrophils recognise surface PS through their expression of Annexin V, which binds sperm membrane-associated PS and causes neutrophils to sequester sperm in neutrophil extracellular traps (NETs).

[0111] Materials and methods

[0112] Plasma polymerisation of channelled slides

[0113] Plasma polymerisation was carried out by deposition of 2-methyl-2-oxazoline (Sigma- Aldrich, Australia) onto oxygen plasma-etched Sticky-Slide I Luer (Ibidi®) and standard glass slides in a capacitively-coupled bell chamber reactor, using established standard methods (Macgregor-Ramiasa et al., 2017, Ramiasa et al., 2015). Plasma deposition was performed using a plasma power of 50 W and 0.8 x 101mbar pressure for deposition of oxazoline precursor for 2 min. The resultant plasma deposited polyoxazoline (PPOx) surface coating generates an irreversible covalent amide bond upon reacting with carboxylic acid groups on proteins.

[0114] Surface characterisation

[0115] X-ray photoelectron spectroscopy (XPS) (SPECS SAGE, Germany, monochromatic Mg Ka radiation source, 15 kV, 10 mA) was used to define the atomic composition of the PPOx film. Survey spectra were obtained at the pass energy of 40 eV with a resolution of 1 eV and measurement angle of 54° between the X-ray irradiation and the analyser. Atomic concentrations were determined by analysis of the spectrum peak areas using Casa XPS software version 2.3.10 (Casa Software).

[0116] Water contact angles were measured after PPOx plasma polymerization of glass slides. Oxygen plasma etched glass was used as a control. After surface polymerization, 5 pL Milli-Q water (Eppendorf) drops were formed by motor-driven microliter syringe on the surface. Next, three points along the surface were evaluated in triplicate and the process was monitored by video camera. The contact angles were measured from images of the drops using ImageJ software.

[0117] The thickness of the PPOx films was measured by ellipsometry (Variable Angle Spectroscopic Ellipsometer, J.A Woolam Co. Inc., USA) and data were analysed using WVASE32 software (NIH) with the Drop Analysis plugin.

[0118] Antibody immobilisation onto the PPOx-coated surface

[0119] Next, the suitability of the PPOx-coated channelled slides for coating with antiphosphatidylserine (anti-PS) antibody was evaluated. The PPOx-coated channelled slide surface was washed with sterile Dulbecco’s phosphate-buffered saline solution (PBS, pH 7, Sigma Aldrich) three times for 15 min each. Anti-PS antibody (clone 1H6, Catalog number: 05-719, Sigma Aldrich) was diluted in PBS to concentrations ranging from 10 to 400 pg / ml. Depending on the surface area to be coated, 20-70 pl of anti-PS was gently pipetted 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 h at 4°C.

[0120] Immunofluorescence analysis was performed to detect anti-PS antibody immobilised at different concentrations onto the PPOx-coated surface. After passivation with 2% HSA for 5 h the coated surfaces were washed with sterile PBS prior to use. Slides were then incubated with fluoroscein isothiocyanate (FITC)-conjugated anti-mouse antibody (Abeam, diluted 1 / 500 in PBS for 30 mins at room temperature). Incubation of secondary antibody with a PPOx-coated channelled slide without prior anti-PS antibody coating served as a negative control.

[0121] Results

[0122] Plasma-based systems have the advantage of being substrate independent, solvent-free, reproducible, and scalable compared to alternative techniques for binding antibodies to surfaces (Sainz- Garcia et al., 2022, Macgregor-Ramiasa et al., 2017). The oxazoline-based plasma deposited coating is biocompatible and facilitates the irreversible covalent binding of biomolecules via their COOH groups (Macgregor-Ramiasa et al., 2017) (Figures 1A and B).

[0123] In this example, 2-methyl-2-oxazoline was used as a precursor to produce a PPOx film on glass substrates, with the resultant PPOx film forming a hydrophilic surface with a water contact angle of 56.3 ± 4.8 (Table 1).

[0124] Table 1 - Effect of PPOx coating on surface wettability measured by water contact angle

[0125] The thickness of the plasma deposited PPOx films was measured by ellipsometry and found to be approximately 26 nm in thickness. The stability of the film was confirmed by re-measuring the thickness of the film following incubation with media used for sperm capture (G-IVF™ Plus, Vitrolife) for 24 hours. No significant change in film thickness was observed (Table 2), supporting stability of the coating upon incubation with sperm biocompatible solutions.

[0126] Table 2 - Ellipsometry measurement of PPOx film thickness before and after incubating with G-IVF™ Plus media

[0127] The atomic composition of the PPOx film was determined using the typical XPS survey spectra consisting of oxygen (8.5%), carbon (73%) and nitrogen (18%) (Figure ID), with the reference XPS survey spectra for the uncoated glass surface shown in Figure 1C. Silicon was not detected in the XPS spectra, indicating that the glass substrate was covered with a conformal, pinhole-free PPOx coating. Based on the high resolution Cis XPS spectra, it was determined that approximately 30% of the carbon was in the C-0 environment, while 10% was in the C=O environment (Figure IE). These reactivechemical functions are formed due to structural fragmentation and recombination of the oxazoline precursor.

[0128] Immunofluorescence analysis confirmed successful deposition of anti-PS antibody onto the functionalised glass surface with increasing absorption in a concentration-dependent manner (Figure IF). No fluorescent signal was detected on negative control glass. Immunofluorescence staining showed an anti-PS concentration-dependent increase in staining intensity and homogeneous fluorescence distribution.

[0129] Example 2 - Assessment of biocompatibility of PPOx-coated glass with human sperm

[0130] In this example, the biocompatibility of PPOx-coated glass with human sperm was determined and the absence of adverse effects of the PPOx coating on sperm viability parameters was confirmed.

[0131] Materials and methods

[0132] Semen preparation

[0133] Briefly, ejaculated human semen was collected from four healthy donors by masturbation following a period of 2-4 days of ejaculatory abstinence. Sperm were applied to glass coverslips within 2 h of collection. Standard swim-up methods were used to isolate motile sperm in prewarmed and gassed G-IVF™ Plus media which were then added to PPOx-coated glass coverslips in 1 ml culture wells.

[0134] Incubation of sperm with PPOx-coated surfaces

[0135] To evaluate the biocompatibility of the PPOx-coated surface, washed sperm at a concentration of 1 x 106in 100 pL G-IVF™ Plus media were added to PPOx-coated glass coverslips in polystyrene tissue culture-grade petri dishes. PPOx-coated and uncoated standard glass coverslips were placed in 4- well plates (Nunc™, Thermo Scientific) and washed three times with copious sterile PBS for 15 min each time to remove any excess unreacted PPOx groups on the surface. The surface was then cleansed of potential microbial contaminants by incubation with 200 pg / mL streptomycin (Thermofisher), 200 U / mL penicillin (Thermofisher), and 500 ng / mL amphotericin B (Thermofisher) in sterile PBS for 2-3 h. Sperm were incubated at a density of 2 x 106per well on the coated or non-coated coverslip surfaces for 1 h and 5 h with G-IVF™ Plus (Vitrolife) at 37°C and 5% CO2. Additional sperm were incubated in petri dishes (TCP) without coverslips. Sperm viability and motility in all groups was assessed 1 h and 5 h postincubation (at 37°C and 5% CO2).

[0136] Evaluation of sperm viability

[0137] Staining with propidium iodide (PI), which is taken up by cells with impaired plasma membrane integrity, was used to quantify necrotic sperm. Following incubation for 1 and 5 h, sperm were collected into 5 mL flow cytometry (FACS) tubes. Next, 4 pL of propidium iodide (PI)-RNAase A staining solution (Thermofisher) was added to the sperm preparation, mixed gently and incubated for 5 min in the dark. To calibrate the flow cytometer settings, a control tube of unstained sperm cells was used. The viability of sperm was analysed on a FACSCanto II flow cytometer (BD Biosciences) using excitation wavelengths between 400 and 600 nm and emission wavelengths between 600 and 700 nm.

[0138] Evaluation of sperm motility

[0139] Sperm motility was assessed following incubation for 1 and 5 h, according to WHO V guidelines (World Health Organization, Geneva, 2010). Evaluation of sperm motility was measured on the CASA® semi-automatic semen analyser (Microptic, Spain, Barcelona). Briefly, 2 pL of sample was added to a Leja 10 slide and scanned using light microscopy (lOx objective). A pre-set human count / motility program was used to calculate the proportion of progressive (STR >80%, where STR = straight linear velocity / average path velocity* 100), non-progressive (STR <80%) and immotile sperm. Total motility was calculated by the proportion of progressive and non-progressive sperm. At least 200 sperm from at least five fields of view were evaluated.

[0140] Results

[0141] No overt detrimental or cytotoxic effects of the PPOx coating was observed on functional measures of sperm quality including sperm viability (PI uptake) and total motility after short or long-term incubation (Figure 2). This result demonstrates that the plasma polymerised PPOx film is biocompatible with sperm, with no loss of sperm viability or motility after contacting PPOX-coated glass. This indicates this coating is appropriate for sperm selection and related biomedical applications.

[0142] Example 3 - Preparation and use of immobilised anti PS device and effect of immobilised anti PS on sperm selection

[0143] The ability of anti-PS coated coverslip to sequester sub-optimal sperm expressing surface membrane PS, in order to remove sperm in early or late-stage apoptosis and generate a sperm population enriched for sperm with maximum viability and fertilisation competence was investigated. The proportion of sperm expressing the surface membrane PS was determined by incubating sperm with Annexin V, the natural ligand of surface membrane PS, in a method commonly used to identify cells in early or late-stageapoptosis. In addition, unselected and selected sperm were evaluated using PI, which is taken up by cells in late-stage apoptosis and necrotic cells.

[0144] Materials and methods

[0145] Immobilization of anti PS onto PPOx plasma polymerized surface

[0146] PPOx-coated glass coverslips (prepared as above) were washed with sterile PBS to remove any contamination and not cross-linked, soluble, low molecular weight fractions from the surface of the plasma polymer before anti-PS was next immobilised on the PPOx-coated surface. Anti-PS was diluted to concentrations of 10, 50, 100, 200, and 400 pg / mL (in PBS) then incubated with PPOx-coated channelled slides overnight at 4°C. Following incubation, the antibody solution was aspirated, and the remaining active polymer surface blocked by the addition of 2% HSA solution for 5 h at 4°C. The coverslips were then thoroughly rinsed three times with PBS for 15 min each to remove any unbound HSA and anti-PS. Immobilisation of the anti-PS antibody onto the PPOx substrate was confirmed using immunofluorescence (IF) staining (as above).

[0147] Sperm attachment

[0148] The effect of incubating sperm with coverslip with different densities of surface immobilised anti-PS (10, 50, 100, 200, and 400 ug / mL) on sperm attachment was then evaluated. Briefly, ejaculated human semen was collected from six healthy donors by masturbation following a period of 2-4 days of ejaculatory abstinence. Standard swim-up methods were used to isolate motile sperm, then washed sperm (1 x 106per mL) were briefly held in prewarmed and gassed G-IVF™ Plus media before addition of 1 x 106sperm in 100 pL onto coverslip. Samples were applied to slides within 2 h of collection. Once sperm were added, channelled slides were incubated for 30-60 min at 37°C and 5% CO2. Sperm were then removed by washing with G-IVF™ Plus. The number of attached sperm on the surface was quantified using light microscopy and the percentage of sperm attachment was calculated by dividing the number of attached sperm by the total number of sperm added on the coverslip.

[0149] Annexin V / PI Assay

[0150] The proportion of sperm labelled with the early-stage apoptotic marker Annexin V and cell viability marker PI was then assessed in the neat ejaculate, sperm obtained using conventional swim-up techniques, and residual unbound sperm populations collected following incubation with the immobilised anti-PS surface, using Annexin V / PI immunofluorescence staining and flow cytometry. To do this, Annexin V-Alexa Fluor™ 488 (Thermofisher) and PI / RNase A solution were used to detect sperm in which translocation of PS from the inner to the outer leaflet of the plasma membrane has occurred(Annexin V+sperm) and apoptotic / necrotic sperm (PI+sperm). Briefly, harvested sperm were washed firstly with 1 mF PBS (350 x g, 5 min, 25°C), then samples were washed with 500 pL Annexin Binding Buffer (ABB) comprised of 50 m HEPES, 700 mM NaCl, 12.5 mM CaCh, pH 7.4 (Thermofisher). Sperm pellets were resuspended in ABB and aliquoted in 200 uL ABB at a final sperm density of 2 x 106in FACS tubes. Sperm samples were then gently mixed and 5 F of Annexin V-FITC at a final concentration of 2.5 pg / mL and 4 pL PI at a final concentration of 2 pg / mL was added, followed by incubation for 5 min at room temperature. After incubation, sperm samples were washed in 500 pL ABB and then resuspended in ABB and analysed within 10 min by flow cytometry.

[0151] Results

[0152] To investigate the effect of incubating sperm with a range of concentrations of surface immobilised anti-PS antibody on the proportion of sperm attachment to the coated surface, sperm were incubated onto coverslips coated with either 50, 100, 200 or 400 ug / mL anti-PS. The proportion of sperm bound to the surface was then visualised using light microscopy (Figure 3A) and quantified manually. The concentration of adhered anti-PS antibody was found to have a significant influence on sperm attachment (Figure 3B), with a concentration-dependent effect on sperm sequestration. Maximal sperm attachment was observed using an anti-PS concentration of 200 pg / mL, and no significant increase in sperm attachment density was detected using anti-PS concentrations above this.

[0153] The proportion of sperm attached on coverslips coated with 200 ug / mL anti-PS was then evaluated in 6 individuals to determine whether anti-PS coated slides are efficacious for selection of sperm in samples from a range of men, and to evaluate differences between individuals. The proportion of sperm attached to the anti-PS coated surface was found to range from approximately 12% (minimum) to 22% (maximum) (Figure 3C). Furthermore, the attachment of sperm to the anti-PS antibody coated surface was shown to be specific to the anti-PS reagent, as negligible sperm binding was observed following incubation with coverslip coated with either PPOx alone, an irrelevant isotype-matched control antibody, or bovine serum albumin (BSA) (Figure 3C).

[0154] To investigate the effects of incubating sperm with surface-immobilised anti-PS on the proportion of sperm expressing PS (taken to designate the population of sperm in early or late stage apoptosis), unattached sperm were stained with Annexin V and PI and evaluated by flow cytometry. The Annexin V positive sperm population was found to be significantly reduced compared to neat semen and sperm isolated using conventional swim-up techniques (Figure 3D). This was also the case for the proportion of PI positive sperm (taken to designate the population of sperm with compromised surface membrane integrity, indicative of late-stage apoptosis or necrotic sperm) (Figure 3E).

[0155] Example 4 - Comparison of characteristics of sperm isolated from channelled slides versus conventional swim-up techniques

[0156] Sperm quality after selection using the PPOx- and anti-PS coated channelled slide was compared to standard swim-up techniques. Quality was measured as %total motility and %progressive motility and DNA fragmentation (as determined by Halosperm® analysis). A second aim was to investigate whether channelled slides with different channel heights exhibit differential efficacy in selection of optimal quality sperm.

[0157] Materials and methods

[0158] Evaluation of sperm recovery from slides with varying channel heights

[0159] To compare the performance of the channelled slide and swim-up selection techniques and to assess the effect of channel 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, ejaculated human semen was collected from five healthy donors by masturbation following a period of 2-4 days of ejaculatory abstinence, with samples applied to slides within 2 h of collection. Semen samples were kept at 37°C for 30-60 min to liquefy. Channelled slides were then prepared for use, by firstly loading the channels with an appropriate volume of G-IVF™ Plus. The volume of G-IVF™ Plus added was dependent on channel height as shown in Table 3. The loading of the channels was performed slowly and gently, particularly in slides with low channel height (0.1 and 0.2 mm) to avoid trapping air inside the channel. Any trapped air acted to impede or completely prevent progressive sperm movement through the channel. Next, 60-100 pL of neat liquified semen was loaded into the inlet well, and the outlet well was filled with 60-100 pL G-IVF™ Plus media. Slides were then incubated for 45-60 min at 37°C and 5% CO2 to allow progressively motile sperm to swim through the channel from the inlet well to the outlet well. Following incubation, the upper layer of the outlet well was carefully collected and both sperm count and motility were assessed. A second fraction of liquefied semen underwent preparation using standard swim-up methodology. The proportion of sperm recovered and the proportion of recovered sperm categorised as progressive motile was then compared with sperm isolated using conventional swim-up technique.

[0160] Table 3 - Channel loading volume according to channel height

[0161] Evaluation of Sperm DNA Fragmentation ( Halosperm )

[0162] DNA fragmentation level was evaluated using Halosperm G2 kit (Halotech®) according to the manufacturer's protocol. In brief, 100 pL agarose was placed in a 90 °C water bath for 30 min and was then incubated at 37°C for at least 5 min before use. Then, 50 pL of semen sample was gently mixed with the gel avoiding formation of bubbles. Then, 8 pL of the mixed sample was pipetted onto an agarose- coated slide and covered with a coverslip. To solidify the gel, the slides were placed at 4 °C for 5 mins. The coverslip was then removed gently, and the slide was vertically submerged in solution 1 for 7 min at room temperature. Then, the slide was immersed in solution 2 for 20 min and washed with distilled water for 5 min. A 70% and 100% ethanol solution were applied to the slide for two minutes each to dehydrate it. After air drying, the slides were incubated in solution 3 and 4 for 7 min each step. To evaluate the percentage of sperm DNA fragmentation, the slides were imaged using a light microscope at x20 magnification. At least 100 sperm per slide were scanned to measure the ‘halo’ size using sperm class analyser (SCA) software (Microptic L.S.). The size of the halo is known to correlate with the degree of sperm DNA fragmentation, with a large halo indicating intact DNA and a small or absent halo indicating fragmented DNA. The percentage of sperm DNA fragmentation (%SDF) was calculated as the number of sperm with fragmented DNA over the total number of analysed sperm.

[0163] Results

[0164] The recovery of progressive motile sperm after selection using channelled slides was comparable to the recovery of sperm obtained from a conventional swim-up, regardless of whether 0.1 pM, 0.2 pM, 0.4 pM or 0.8 pM channels were utilised, although there was a trend towards lower recovery with shallower channels (Figure 4B). The sperm recovered from channelled slides were also comparable with those from conventional swim-up when markers of sperm quality were assessed on the basis of DNA fragmentation (Figure 4C) and motility (Figures 4D and E). This suggested that the PPOx- and anti-PS coated channelled slides produce a similar quality of sperm sample to that of conventional swim-up.

[0165] Example 5 - Mouse sperm selection

[0166] Effectiveness of the channelled slide device for selection of optimal sperm in a mammalian species other than human was determined. Sperm recovered from mouse epididymis were evaluated in a protocol comparable to that demonstrated to be effective for human sperm.

[0167] Materials and methods

[0168] Mouse sperm isolation

[0169] C57BL / 6J male mice (8-12 weeks of age) were purchased from Laboratory Animal Services (LAS) at The University of Adelaide and kept for 5 weeks before being used. Mice were housed in clean cages under controlled temperature (25 ± 3°C), proper humidity (50 ± 5%) and a 12 h light / dark cycle. The procedure described below was approved by the University of Adelaide Animal Ethics Committee (approval number M-2022-041). Briefly, after cervical dislocation of male mice, the whole epididymis and half of the vas deferens was carefully dissected and trimmed of residual fat, before being transferred to 15 mL round bottom tube with 1 ml of pre-warmed G-IVF™ Plus media at 37°C. The tissue was then perforated with sharp scissors in 5 or more places, and then let sit for 15 minutes (37 °C and 5% CO2) to allow sperm to swim out. The epididymal tissue was removed from culture media and the remaining fluid was pipetted up and down to recover sperm suspensions. Sperm were then either incubated with PPOx- and anti-PS -coated coverslips, or applied to the inlet wells of PPOx- and anti-PS -coated channelled slides (0.4 pm, prepared as described above), or uncoated channelled slides. Unbound sperm were then recovered from co ver slips, or from the outlet wells of channelled slides, 45 mins later and evaluated for sperm motility, markers of apoptosis (Annexin V binding and PI uptake), and sperm surface attachment, as described in previous sections. Sperm DNA fragmentation was assessed using TUNEL staining. Data were compared to control sperm that was not selected in a channelled device.

[0170] Evaluation of sperm DNA fragmentation by TUNEL

[0171] Sperm DNA fragmentation was evaluated using the Apo-Direct™ TUNEL assay kit (Thermofisher). The TUNEL assay was performed according to the manufacturer's protocol with some minor modifications. Sperm was suspended in 1% (v / v) paraformaldehyde at a density of 2-4 x 106in 100 pL and incubated on ice for 15 min. Following incubation, sperm were washed twice with 5 mL of PBS for 5 min at 350 x g and then incubated in 100 uL of ice-cold ethanol at -20°C overnight. The next day, the ethanol was aspirated, and sperm washed twice with 1 mL Wash Buffer for 5 min at 350 x g. The labelling reaction was carried out by incubation with 50 pL of labelling solution containing terminal deoxynucleotidyl transferase (TdT) for 2 h at 37 °C in the dark. After washing twice with 1 mL Rinse Buffer, sperm samples were resuspended in 500 pL of propidium iodide / RNase A solution. Then, the sample was analysed within 3 h of staining using the FACSCanto II flow cytometer (BD Biosciences). TUNEL-positive sperm (green fluorescence) were measured using a 530 ± 30 nm band-pass filter, and propidium iodide-labelled sperm (red fluorescence) using a 620 nm long-pass filter. A total of 10 000 events was analysed for each measurement and the flow rate was set to 200-300 cells / second. Data were processed by FlowJo™ 10 software (BD Biosciences) after gating out aggregates and cellular debris.

[0172] Results

[0173] Representative examples of flow cytometry for mouse sperm Annexin V binding and PI uptake are shown in Figures 5A-D. The proportion of sperm binding Annexin V and / or taking up PI (indicating evidence of apoptosis) was reduced in sperm suspensions after passage of sperm through PPOx- and anti- PS-coated channelled slides, or incubation with PPOx- and anti-PS-coated coverslips, but was not reduced by incubation in uncoated channelled slides (Figures 5E, 5F). A -50% reduction in the proportion of the Annexin V positive sperm subpopulation and -40% reduction in sperm DNA fragmentation was observed in the unattached sperm subpopulation compared with neat samples. A higher percentage of motile sperm was observed in the unattached sperm subpopulation after incubation with PPOx- and anti-PS coated coverslips or channelled slides, but not uncoated channelled slides (Figures 5G, 5H). The proportion of sperm with DNA fragmentation detected by TUNEL was also reduced in the unattached sperm subpopulation after incubation with PPOx- and anti-PS coated coverslips or channelled slides, but not uncoated channelled slides (Figure 51).

[0174] Example 6 - Fabrication of a Nano-Gold Sperm (NGS) device

[0175] The functional complexity of the PPOx- and anti-PS-coated channel slides was further increased, to incorporate additional features including (1) gold nanospheres covalently bound to the PPOX-coated glass substrate, in order to increase the surface area available for anti-PS binding and sperm interaction with anti-PS moieties, and mimic the labyrinthine complexity of the mammalian oviduct; (2) a gradient of non-covalently-bound progesterone, which exerts chemotactic and pro-viability effects on sperm, to increase sperm viability and enhance directionality and rate of sperm progression across the channel slide, and (3) a spatially configured functionalisation of the channel slide surface to enable a 2- stage selection process, wherein sperm delivered to the input well of the channel first encounter anti-PS to remove sub-optimal sperm, and secondly encounter the progesterone gradient, to stimulate progression of motile sperm across the channel, and to promote viability of recovered sperm at the outlet well.

[0176] Materials and methods

[0177] Gold Nanoparticle ( GNP) Synthesis

[0178] GNP synthesis was achieved using an established protocol (Visalakshan et al., 2018; Goreham et al., 2011; Zhu et al., 2003) that utilises reduction of tetrachloroauric acid (HAuCU, Sigma- Aldrich, Australia) with trisodium citrate (NasCt, Sigma- Aldrich, Australia) to fabricate colloidal gold in aqueous solution in a controlled reflux system. HAuCU (0.01%) was dissolved in a continuously stirring solution and allowed to boil, before Na?Ct was added as the reducing agent, and allowed to boil for an additional 20 min. Restricting the amount of 1% Na?Ct solution added was used to control GNP size, with theaddition of 1.0, 0.5 and 0.3 mL of 1% Na?Ct producing three different sized nanoparticles (NPs) with diameters of 16, 38, and 68 nm respectively. Mercaptosuccinic acid (MSA, Sigma-Aldrich, Australia) and sodium hydroxide (Chem-supply) were then used as a capping agent, thereby modifying the surface to offer enhanced stability to the colloidal GNPs.

[0179] Immobilization of GNPs

[0180] Next, the COOH-functionalised GNPs were immobilized on the surface of PPOx -coated 400 pm channelled slides. Channelled slides (400 pm) were 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 GNP solution overnight. The next day, excess and loosely bound GNPs were removed by rinsing 3 times with Milli-Q water before the surface was dried under nitrogen gas.

[0181] GNP characterisation

[0182] The shape and size distribution of GNPs were characterized using transmission electron microscopy (TEM, JEOL-2100F, Japan) after a droplet of GNP suspension was dried on a carbon-coated copper grid (ProSciTech, Australia). Surface topography of GNPs was evaluated using atomic force microscopy (AFM), through a noncontact mode using a gold-coated silicon nitride tip (NT-MDT, NSG03) with 65-100 kHz resonance frequencies. Surface chemistry was characterised by XPS survey spectra.

[0183] PPOx coating of the surface immobilised gold nanoparticles

[0184] To covalently immobilise anti-PS onto gold nanoparticles on one half of the channelled glass slide surface, and to electrostatically adhere progesterone onto gold nanoparticles on the other half of the slide, a second layer of functionalisation was then applied to the immobilised gold nanoparticles only on one half of the glass slide (the half closest to the inlet well). This was achieved using a mask to cover the other half of the slide (the half closest to the outlet well) during treatment in the plasma reactor. As a result, the half of the slide closest to the inlet well acquired functionalisation with PPOx groups to enable covalent immobilising of anti-PS to the gold nanoparticle and slide surface, while the other half retained their carboxylic acid group to enable electrostatic adsorption of progesterone.

[0185] Through formation of amide bonds the intact oxazoline rings on the surface of the PPOx film facilitate irreversible bonding of COOH-bearing ligands. To confirm binding, slides prepared as above were incubated with sodium dodecyl sulfate (SDS) detergent solution (1% v / v in 5M NaCl, pH 10) for one hour, then flushed with Milli-Q water, to challenge the PPOx- and COOH-GNP binding. Strong binding between PPOx and GNP was observed and confirmed using XPS analysis after washing.

[0186] Results

[0187] The unique chemical functionality and reactivity of plasma-deposited PPOx make it an ideal coating material for biomaterial surfaces. Our data and published studies show that the PPOx coating can be utilised to ensure secure chemical and irreversible binding of gold nanoparticles to a glass substrate and other materials (Visalakshan et al., 2018). The example showed that sequential coating of glass channel slides with PPOx, then gold nanospheres, then a further application of PPOx coating to a proportion of the gold nanospheres, is feasible. This approach was devised to provide a complex surface architecture that replicates the labyrinthine features of the mammalian oviduct and allows staged exposure of sperm to different microenvironments during their progression through the channelled slide.

[0188] These results show that it is possible to achieve spatially distinct areas of the gold nanosphere- coated channelled slide, with one half of the slide subsequently covalently coated with anti-PS, and the other half non-covalently coated with progesterone.

[0189] The gold nanoparticle-coated channel slides allowed binding of anti-PS to firstly replicate immune cell-mediated sequestration of morphologically abnormal sperm, and secondly allow sperm interaction with progesterone to act as a sperm chemotactic and survival factor.

[0190] The gold nanoparticle-coated channel slides, containing areas of anti-PS and progesterone coatings, were termed ‘Nano Gold Selection’ Devices (‘NGS Devices’) (Figure 6).

[0191] Example 7 - Evaluation of human sperm selection effectiveness using the NGS Device

[0192] The efficacy of channelled slides prepared as described in Example 6 was evaluated. NGS Device (400 pm channelled slides coated with gold nanoparticles, and spatially distinct areas of anti-PS and progesterone coatings) were evaluated for selection of high quality sperm from neat liquified semen samples (without any prior preparation procedure). The intent was to evaluate the utility of the NGS Device as a single step separation method to generate high quality human sperm for use in IVF or ICSI.

[0193] Materials and methods

[0194] Sperm separation in NGS Device

[0195] Semen from two donors was prepared as described in Example 4 - briefly semen samples were allowed to liquify and were then added to NGS Devices (prepared as described in Example 6), or uncoated channelled slides, without any further preparation steps. The input wells of channelled slides were carefully loaded with 100 pL of G-IVF™ Plus, then 60-100 pL of the liquified neat semen sample was loaded into the inlet well. The outlet well was filled with 40-60 pL of G-IVF™ Plus media. Thesample was incubated at 37°C and 5% CO2 for 45-60 min to allow motile sperm to swim through the channel from the inlet well to the outlet well. The upper layer of the outlet well was collected carefully, then sperm count and motility, the proportions of Annexin V positive staining and / or PI uptake, and sperm DNA fragmentation, were assessed as described in Examples 3 and 4. Sperm recovered from NGS Devices were compared with sperm prepared by incubation in uncoated channelled slides or by standard swim-up technique.

[0196] Results

[0197] Representative examples of flow cytometry for human sperm Annexin V binding and PI uptake are shown in Figures 7A-C. The percentage of Annexin V+ sperm as determined by flow cytometry was substantially reduced in sperm recovered from NGS Devices, compared to sperm applied to channelled slides without coating, or standard swim-up technique (Figure 7D). A similar decrease in the proportion of sperm with DNA fragmentation was seen using the NGS Device, which yielded sperm with very low rates of DNA fragmentation compared with sperm prepared by standard swim-up technique or uncoated channelled slides (Figure 7E). Furthermore, a higher proportion of sperm exhibited large halos after separation using the NGS Device, compared with sperm prepared by swim-up or uncoated channelled slides (Figures 7F, 7I-K), indicating improved DNA integrity in sperm prepared in NGS Devices. Sperm recovered from the NGS Device showed levels of motility and progressive motility comparable to sperm prepared by swim-up or uncoated channelled slides (Figures 7G, 7H). These data show that the NGS Device has higher efficacy in generation of optimal quality sperm, as judged by Annexin V positivity and by sperm DNA integrity, than a standard swim up procedure. Furthermore, this example shows that the NGS device can yield optimal quality sperm directly from liquefied human semen, without any further preparation steps prior to application of the semen sample to the Device.

[0198] Example 8 - Evaluation of individual selective elements of the NGS Device on recovered sperm quality

[0199] The degree to which the individual elements included in the NGS device contribute to its selective efficacy was evaluated. To do this, individual elements (anti-PS, progesterone, gold nanoparticles) were removed during device fabrication, and the effect on quality of sperm recovered was measured by Annexin V and PI uptake.

[0200] Materials and methods

[0201] Channelled slides were coated with all components as described in Example 6, or with variations to remove one or more individual components from the coating sequence. Channelled slides (400 um) were coated with all components as described in Example 6 (eg. anti-phosphatidyl serine (anti-PS), gold nanoparticles (GNP), and progesterone (P)), with GNP and P but not anti-PS; with GNP and anti-PS but not P; with anti-PS and P but not GNP, with P but not anti-PS or GNP, with anti-PS but not P or GNP, with GNP but not anti-PS or P, or were uncoated. Liquified semen was prepared from each of two donors as described in Example 6 and was added to the input wells of each of the above channelled slides, then incubated for 45 mins, after which selected sperm were recovered from output wells (as described in Example 6). Unselected sperm and sperm selected by standard swim-up technique were included for comparison. The quality of recovered sperm was measured as the proportion of sperm staining with Annexin V and / or PI uptake.

[0202] Results

[0203] As shown in Figure 8, the lowest values for Annexin V staining and PI uptake were seen when sperm were selected using the NGS device coated with all components (anti-PS, gold nanoparticles, and progesterone) (Figures 8A and B). These parameters of sperm quality were again substantially lower than values for sperm recovered by standard swim-up technique, confirming the improved performance of the NGS device compared to the standard swim-up technique. Values for Annexin V staining and PI uptake were increased when any one or more of these individual selective elements were removed from the NGS device. These data show that all three components, when included in the channelled slide coating, contribute to the quality of sperm recovered from the NGS device. This result shows that the efficacy of the NGS device in generating high quality sperm is not fully attributable to any single component of the channelled slide coating, but instead depends on the unique combination of coating components in the device.

[0204] Biomolecules including recombinant Annexin V protein (In Vitro Technologies), anti- Phosphatidylserine (anti-PS, Sigma Aldrich), anti-Fibronectin antibody (anti-Fn, rabbit monoclonal F14, Abeam), and hyaluronic acid disaccharide ADiHA sodium salt (ADiHA, Sigma Aldrich) were immobilised on the plasma PPOx deposited surface. Before immobilization of biomolecules, the plasma deposited PPOx surfaces were washed with PBS (Sigma Alderich) to remove unattached PPOx groups. Biomolecules at a concentration of 200 pg / L2were each individually incubated with plasma-coated surfaces at 4°C overnight. To avoid nonspecific protein adsorption and sperm attachment to unreacted PPOx plasma groups, the surface was backfilled with 5% human serum albumin (HSA) at 4°C for overnight. The coated surfaces were then washed with sterile PBS three times for 1 h to remove nonimmobilised biomolecules prior to use.

[0205] Example 9 - Preparation and use of immobilised anti-fibronectin antibody (anti-Fn) and effect of immobilised anti-Fn on sperm selection

[0206] Anti-Fibronectin antibody (anti-Fn, Abeam) was immobilized onto PPOx plasma polymerized surface by following the protocol provided in Example 3.

[0207] The level of biomolecule immobilisation was examined using immunofluorescence (IF) staining. After construction of the anti-Fn coating and backfill with HSA, the coated surfaces were washed with PBS for 1 h to remove non-immobilized proteins and were incubated with secondary antibody (goat anti-rabbit IgG (Alexa Fluor® 488), Abeam) for 12 h at 4 °C to detect bound anti-Fn (Figure 9A). A clean glass surface coated with anti-Fn was used as a control (Figure 9B).

[0208] Fibronectin (Fn) is a 440 kDa glycoprotein that is thought to contribute to sperm-oocyte interaction in human (Thys et al, 2009). Generally, sperm express Fn on their surface after capacitation (Martinez-Leon et al, 2019), and detection of the Fn receptor integrin a5[31 on both male and female human gametes suggests a role for Fn glycoprotein in sperm-oocyte interaction at fertilization. In the female reproductive tract, Fn might also act as an opsonin factor on damaged sperm cells, thus marking them for neutrophil-mediated binding and sequestration (Thys et al, 2009; Nair and Zingde 2001). Consistent with this, it has been reported that the level of Fn in seminal plasma is elevated in the case of male infertility and low sperm motility. We considered that the presence of fibronectin (Fn) on the sperm plasma membrane increases the likelihood of neutrophil-mediated sequestration in the female reproductive tract.

[0209] The present example was performed to assess the expression of Fn on sperm after swim up selection using flow cytometry techniques and to evaluate the efficacy of anti-Fn coated surfaces in immobilisation of damaged sperm.

[0210] 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 the FITC-labelled secondary antibody treatment (Figure 10A), and 2) a sample processed without primary antibody prior to the incubation with the FITC-labelled secondary antibody. As a positive control, human peripheral blood monocytes were used to confirm the anti-Fn binding functionality (Figure 10B).

[0211] Fn expression by motile sperm samples from 5 healthy men was examined by flow cytometry.The percentage of Fn positive sperm varied from 2.7% to 15% (Figure 11).

[0212] The motile sperm were collected via swim up technique and the concentration was adjusted to2.5 x 106ml1. Prior to incubation of sperm with the prepared anti-Fn coated surfaces, the surfaces werewashed with sterile PBS for 1 h to remove any excess and noncovalently bound proteins and HSA. Each coverslip was placed in a 24-well plate (Nunc) and seeded with sperm at a density of 15000 sperm / mm2(2.5 x 106ml1). Sperm were initially incubated in contact with the coated surface for 60-120 min at 37°C and 5% CO2 then unattached sperm were collected for further analysis. PPOx surfaces coated with irrelevant isotype-matched rabbit IgG antibody and 5% HSA or PPOx alone were used as controls. After incubation of sperm on the anti-Fn coated surface for 120 min, 2% - 10% of the sperm population were adhered to the surface (Figure 12) while no sperm were adhered to control surfaces.

[0213] Example 10 - Preparation and use of immobilised Annexin V protein and effect of immobilised Annexin V protein on sperm selection

[0214] Annexin V is a surface protein that has been previously utilized in sperm selection for improving success rates of ART. Sperm with pre-apoptotic or apoptotic features can maintain normal morphology (Sakkas et al, 2015) and may retain capacity to fertilize an oocyte, especially during an ICSI procedure wherein a single sperm is injected into the oocyte cytoplasm. This procedure would adversely impact embryo development and pregnancy rate, and may pose a risk for child health. Several studies have demonstrated that ejaculated sperm particularly in infertile males undergo changes similar to apoptotic features (Sakkas et al, 2015; Schjenken and Robertson, 2020). If apoptosis occurs, sperm reconstruct and externalise phospholipid phosphatidylserine (PS) from the inner leaflet to the outer leaflet of the plasma membrane (Martinez et al, 2018). PS has high affinity for Annexin V, a protein molecule with molecular mass of 36.8 kDa, that is unable to penetrate through intact sperm membrane (Sakkas et al, 2015). Therefore, the capacity to eliminate sperm with externalised PS, as detected by positivity for Annexin V binding, and in turn enrich the population of Annexin V negative sperm from which to select sperm for fertilisation, will provide a useful and important advance for ART.

[0215] In this example, the proportion of Annexin V positive and necrotic (PI positive) sperm in the population was determined prior to and after sperm sorting by incubation with the Annexin V coated surface, and compared with neat ejaculate and sperm prepared by swim-up technique. There was no significant difference in the proportion of Annexin V positive sperm in neat semen versus swim-up (25% ± 11, 21% ± 15 respectively, n=4) (Figure 13). To determine whether there is any reduction of Annexin positive sperm after interacting with an Annexin V coated surface, neat semen was incubated on surfaces prepared by Annexin V immobilisation on plasma polymerised PPOx-coated coverslips. The availability of Annexin V on the surface after HSA passivation and PBS washing was confirmed by fluorescence imaging (Figure 14).

[0216] Then the motile sperm at a density of 15000 sperm / mm2(2.5 x 106ml1) were incubated with the prepared Annexin V coated surfaces for 60-120 min at 37 °C and 5% CO2. The unattached sperm were collected for further analysis.

[0217] The sperm attachment rate was 12% and 20% for two semen samples (n=2) after 120 min incubation (Figure 15). There was a 44.4% and 62.5% reduction in the Annexin V positive sperm population in the unattached sperm compared with the swim up sperm for each individual respectively.

[0218] The levels of reactive oxygen species (ROS) were compared between neat ejaculate, swim up sperm and unattached sperm recovered from Annexin V coated surfaces (Figure 16). The result showed that the ROS content of motile sperm collected by swim up method was higher than neat sperm (52% and 35% respectively). There was little difference in ROS content between sperm prepared by swim up and the unattached sperm population recovered from the functionalised surface.

[0219] Example 11 - Sperm attachment on Annexin V-coated surface and effect on recovered sperm

[0220] The effect of surface-immobilised Annexin V at different concentrations on proportion of sperm attachment was examined in a comprehensive study employing swim-up sperm samples. Sperm collected from swim-up specimens were exposed to coverslips coated with varying concentrations of Annexin V (In Vitro Technologies, 10, 50, 100, and 200 pg / mL). Light microscopy was used to evaluate sperm binding patterns (Figures 17A-D), by counting the number of unattached and attached sperm. The proportion of attached sperm was determined by dividing the count of attached sperm on the entire surface by the initial sperm seeding density, then multiplying the result by 100.

[0221] There was a significant correlation between concentration of Annexin V and sperm cell adherence (Figure 17E). A concentration-dependent pattern of sperm attachment was observed, with peaks of attachment observed at 100 and 200 pg / mL of Annexin V.

[0222] To evaluate the suitability of Annexin V-coated coverslips for sperm selection across different individuals and to gauge inter-individual variations, the proportion of sperm attached to co ver slips coated with 200 pg / mL of Annexin V was examined in sperm samples from six individuals. The findings revealed a range spanning 5% (minimum) to 23% (maximum) of sperm adhering to the surface treated with 100 pg / mL of Annexin V (Figure 17F). The experiment conclusively demonstrated that sperm binding to the Annexin V-coated surface was dependent on Annexin V coating, as minimal sperm attachment was seen when the coverslips were coated with PPOx alone or PPOx and 2% HSA.

[0223] In order to examine the effect of incubating sperm with surface-immobilised Annexin V, the apoptotic sperm population among unattached sperm was studied. The fraction of sperm displaying phosphatidylserine (PS), a marker for early or late-stage apoptosis, on the outer leaflet of the membrane was determined by Annexin V and PI staining assessed using flow cytometry. The analysis showed a significant decline in the proportion of sperm exhibiting positivity for Annexin V binding, in comparison to both the initial untreated sample and sperm isolated through conventional swim-up methods (Figures17G-K). The subset of Annexin V-positive sperm within neat sample, swim-up isolated sperm, and sperm left unattached after incubation with Annexin V-coated surfaces (at a concentration of 200 pg / ml) were (mean ± SEM) 36.5% ± 8.6%, 34.9% ± 12.8%, and 20.8% ± 12.1%, respectively (Figure 17J). This demonstrates a substantial decrease in the Annexin V-positive population after exposure to Annexin V- coated surfaces (P < 0.01).

[0224] Similarly, the fraction of sperm showing positivity for PI staining, which indicates compromised membrane integrity typically linked to late-stage apoptosis or necrotic sperm, were 29.8% ± 10.3%, 19.4% ± 9.8%, and 20.1% ± 12.1% for neat samples, swim-up isolated sperm, and sperm incubated with Annexin V-coated surfaces, respectively (Figure 17K). This demonstrates a substantial decrease in the Pl-positive population after exposure to PPOx-coated or Annexin V-coated surfaces (P < 0.01).

[0225] Example 12 - Sperm attachment on hyaluronic acid (HA) coated surface and effect on recovered sperm

[0226] The impact of hyaluronic acid (HA) on swim-up sperm attachment and the quality of the unattached sperm population was investigated, as depicted in Figure 18. The experiments were conducted using washed human sperm at a density of 1-2 x 106incubated in prewarmed and CCE-gasscd G-IVFTM Plus medium in contact with the surface-modified glass coverslips.

[0227] The degree of HA binding to sperm is thought to relate to the maturity and competence of sperm. HA is a linear polysaccharide composed of repeating disaccharide units that contains carboxylic acid groups in its molecular structure. The D-glucuronic acid component of HA includes a carboxylic acid group (-COOH), which mediates HA covalent binding to PPOx groups. HA (hyaluronic acid disaccharide sodium salt, Sigma Aldrich) was immobilised on PPOx-coated glass coverslips at a concentration of 200-300 pg / ml. The incubation was conducted overnight at 4°C. Subsequently, the surface was washed, and remaining PPOx groups were blocked by 2% HSA for 30-60 minutes at room temperature.

[0228] To evaluate whether HA-coated coverslips enable sperm binding and selection, the proportion of sperm attached to coverslips coated with HA was examined in sperm samples from three individuals. The findings revealed a range spanning 14% (minimum) to 38% (maximum) of sperm adhering to the surface treated with 100 pg / mL of Annexin V (Figures 18A, 18B). Sperm binding to the HA-coated surface was dependent on HA coating, as minimal sperm attachment was seen when the coverslips were uncoated, or coated with PPOx and 2% HSA (Figure 18B).

[0229] To examine the effect of incubating sperm with surface-immobilised HA, the proportion of apoptotic sperm population among unattached sperm was studied. The fraction of sperm exhibiting positivity for Annexin V binding was comparable to neat sperm, and higher than in sperm collected by swim-up (Figures 18E-G, 18K). This demonstrates that exposure to HA-coated surfaces does not improve the competency of the unattached population as measured by Annexin V-positivity, and conversely is consistent with selective binding of competent sperm to the surface.

[0230] Example 13 - Sperm attachment on anti-fibronectin coated surface and effect on recovered sperm

[0231] The impact of anti-fibronectin (anti-Fn) biomolecules on swim-up sperm attachment and the quality of the unattached sperm population was investigated, as depicted in Figure 18. The experiments were conducted using washed human sperm at a density of 1-2 x 106incubated in prewarmed and CO2- gassed G-IVFTM Plus medium in contact with the surface-modified glass coverslips.

[0232] Fibronectin is a glycoprotein present on the surface of a proportion of sperm. Its significance in sperm competence is not well understood. Anti-Fn (Abeam) was immobilised on the PPOx-coated surface at a concentration of 40-100 pg / ml, following the same protocol as HA. To evaluate whether Fn-coated coverslips enable sperm binding and selection, the proportion of sperm attached to coverslips coated with anti-Fn was examined in sperm samples from three individuals. The findings revealed a range spanning 10% (minimum) to 28% (maximum) of sperm adhering to the surface treated with 100 pg / mL of anti-Fn (Figures 18C, 18D). Sperm binding to the anti-Fn-coated surface was dependent on anti-Fn coating, as minimal sperm attachment was seen when the co ver slips were uncoated, or coated with PPOx and 2% HSA, or with an isotype-matched irrelevant monoclonal antibody (Figure 18D).

[0233] To examine the effect of incubating sperm with surface-immobilised anti-Fn, the proportion of apoptotic sperm population among unattached sperm was studied. The fraction of sperm exhibiting positivity for Annexin V binding was comparable to neat sperm, and sperm collected by swim-up (Figures 18H-J, 18L). This demonstrates that exposure to anti-Fn-coated surfaces does not improve the competency of the unattached population as measured by Annexin V-positivity.

[0234] Conclusion

[0235] The examples show that coating PPOx-coated glass coverslips with each of Annexin V, hyaluronic acid and anti-fibronectin are all able to provide a surface that enables selective binding of sperm. Annexin V, like anti-PS in the examples, sequesters the less competent sperm (as measured by Annexin V and PI binding). Hyaluronic acid seems to sequester more competent sperm. Anti-Fnsequesters a subset of sperm but there is no shift in % staining for Annexin V so it is likely no impact on competency of unbound sperm.

[0236] Example 14 - Use of alternative methods for surface activation and surface conjugation of biomolecules and gold nanoparticles to sequester sperm

[0237] The effect of different chemical methods to bind biomaterials and gold nanoparticles was investigated. Specifically, we compared the use of plasma polymerization to generate a film with reactive amine groups (-NH2, termed ‘plasma-polymerized amine film’), versus the PPOx film with reactive - COOH groups (used in prior examples). The two alternative coating methods were used to couple gold nanoparticles (GNP) and anti-PS antibody onto the surface of glass cover slips, and the effects on sperm behaviour when incubated with the different functionalised surfaces was examined. Plasma polymerized amine groups (-NH2) interact with carboxyl groups (-COOH) on biomolecules through non-covalent bonds, such as electrostatic attraction between -NH3+and -COO ions, and hydrogen bonding between the amine's hydrogen and the carboxyl's oxygen. These interactions are expected to be less strong than the covalent bonds provided by PPOx treatment. However, they may have utility in sperm separation devices where alternative binding chemistry is desired.

[0238] Materials and Methods

[0239] In this example, allylamine (AAm) was used as a precursor to produce a plasma-polymerised amine film on glass coverslips, according to established protocols (Liu et al, 2014). Additional glass coverslips were coated as described in previous examples, using 2-methyl-2-oxazoline as a precursor to produce a PPOx film.

[0240] Glass coverslips were then coated with either 68 nm gold nanoparticles (GNPs) and anti-PS antibody, or with anti-PS antibody without GNPs, giving rise to 4 different surface coating variants.

[0241] For coatings without GNPs, anti-PS at a concentration of 200 pg / ml was applied directly to the surface of coverslips with plasma-polymerized amine film or PPOx film, to generate coatings ‘NH2- Anti- PS’ and ‘PPOx- Anti-PS’ respectively.

[0242] For coatings with GNPs, 68 nm GNPs were first immobilised on the surface of coverslips with plasma-polymerized amine film or PPOx film, then both GNP surfaces were treated with plasma polymerization to achieve a PPOx coating. Anti-PS, at a concentration of 200 pg / ml, was then applied to the surface as described in previous examples, to generate coatings termed ‘NH2-GNP-PPOX- Anti-PS’ and ‘PPOx-GNP-PPOx- Anti-PS’ respectively.

[0243] We then compared the sperm selection efficacy of these different coatings. Briefly, ejaculated human semen was collected from a healthy donor. Standard swim-up methods were used to isolate motile sperm in prewarmed and gassed G-IVF™ Plus media. Sperm were added to coated glass coverslips in 1 ml culture wells within 2 h of collection.

[0244] Human sperm prepared by swim-up (200 pl) (n= 1 ) was incubated on modified coverslip surfaces for 45 minutes at 37°C and 5% CO2. The sperm were then collected and the incidence of apoptotic populations and necrotic populations was analysed by flow cytometry using Annexin V staining and PI uptake respectively as described in previous examples. Sperm exposed to coated coverslips were compared to control swim-up sperm not exposed to coated co ver slips.

[0245] Results

[0246] The results (Figure 19) indicate efficacy of all four coated surfaces. There was a reduction in the apoptotic sperm population in sperm exposed to NHz-GNP-PPOx- Anti-PS and NH2- Anti-PS surfaces (15.0% and 20.0% respectively), compared to 34.9% in the swim-up control. This reduction was comparable to that observed on PPOx-coated surfaces, which exhibited rates of apoptotic sperm of 13.6% and 17.9% for PPOx-GNP-PPOx-Anti-PS and PPOx-Anti-PS coated surfaces, respectively. The percentage of sperm with PI uptake was comparable to swim-up sperm for each of the different coverslip treatments.

[0247] These findings demonstrate the utility of alternative coating chemistry, including plasma polymerized amine groups either alone or in conjunction with PPOx coatings and / or gold nanoparticles, before binding of biomaterials such as anti-PS in order to sequester the apoptotic population and enhance the quality of recovered sperm.

[0248] Example 20 - Use of Protein-G for surface conjugation of biomolecules to sequester sperm

[0249] Protein G can be utilised for antibody attachment to surfaces due to its strong affinity for the Fc region of immunoglobulin (antibody) proteins, enabling stable orientation and efficient access of the immunoglobulin Fab region to cognate epitopes, with benefits for various biomedical applications.

[0250] Methods

[0251] Protein-G mediated binding of anti-fibronectin to PPOx-coated surfaces

[0252] In this example, Protein-G was bound to PPOx films on glass coverslips prior to incubation with anti-fibronectin or anti-PS, and the effect on efficacy of sperm sequestration was evaluated.

[0253] Initially, to investigate the ability of Protein G to attach to PPOx-coated surfaces and facilitate subsequent antibody binding, anti-fibronectin (Anti-Fn) antibodies conjugated to Alexa Fluor 488 dye were used as an indirect detection method (Figure 20). PPOx-coated glass coverslips were treated with or without Protein-G (diluted in PBS, overnight at 4°C), then subjected to passivation using 2% HSA passivation for 30-60 minutes at room temperature. Control groups consisted of uncoated or passivated PPOx-coated coverslips without Protein-G. Subsequently, surfaces were exposed to varying concentrations of anti-Fn labeled with Alexa Fluor 488 and incubated overnight at 4°C. Anti-Fn immobilization was analyzed by comparing the fluorescence signals of bare glass, passivated PPOx (no Protein-G), and Protein-G-coated surfaces after incubation with different concentrations of Anti-Fn- Alexa Fluor 488.

[0254] In a second experiment, anti-PS antibody at various concentrations (20-200 pg / mL) was incubated with PPOx and Protein-G-PPOx coated glass coverslips, and mass photometry was used to evaluate the binding efficiency under the different conditions.

[0255] In a third experiment, channelled slides were coated with PPOx and Protein-G, or without Protein-G, prior to Anti-PS coating. Channelled slides were then evaluated for efficacy of sequestration of apoptotic sperm, using sperm from n=3 human donors, as described in previous examples.

[0256] Results

[0257] Protein-G mediated binding of anti-fibronectin to PPOx-coated surfaces

[0258] The strong fluorescence observed on the Protein-G-coated surface confirms Protein-G availability and efficacy of anti-Fn binding, with fluorescence intensity reflecting the amount of anti-Fn binding. The Protein-G-coated surface exhibited significantly more intense fluorescence at higher antibody concentrations (50 pg / mL) indicating effective binding, while controls showed negligible signals, confirming specific binding to Protein-G (Figures 20B-E).

[0259] Protein-G mediated binding of anti-PS to PPOx-coated surfaces

[0260] The results of mass photometry analysis demonstrated a stronger binding affinity of Anti-PS antibodies when PPOx-coated surfaces were first incubated with Protein G. There was a consistently higher affinity across all antibody concentrations, indicating enhanced Anti-PS immobilization due to Protein G (Figures 20F-I).

[0261] Protein-G mediated binding ofanti-PS to PPOx-coated channelled slides

[0262] Further experiments assessed the impact of Protein-G on sperm interaction with Anti-PS. Channel slides with PPOx and Protein-G coatings were used, and sperm interaction was evaluated based on apoptotic and necrotic sperm populations (Figures 20J, 20K). Surfaces coated with Protein-G plus Anti-PS (50 pg / mL) showed significantly lower Annexin V positive sperm and Pl-positive sperm compared to surfaces coated with Anti-PS at the same concentration (50 pg / mL) without Protein G. The quality of sperm recovered from channelled slides coated with Protein-G plus Anti-PS (50 pg / mL) showed comparable Annexin V-positive and Pl-positive sperm compared to surfaces coated with Anti-PS at a higher concentration (200 pg / mL) without Protein G.

[0263] These experiments demonstrate that use of Protein-G can effectively immobilize Anti-PS in channelled devices, and that lower concentrations of Anti-PS can be used in devices to achieve the same or better efficiency of sequestration of apoptotic sperm. Protein-G can therefore be a useful addition to coatings to enhance the quality of recovered sperm.

[0264] Example 21 - Use of polystyrene plastic or glass for functionalised surface coatings and surface conjugation of biomolecules to sequester sperm

[0265] Functionalised surfaces can be applied to a range of materials for various applications in biology and medicine. In this example, we examined the effects of utilising two different substrates, glass and polystyrene plastic (tissue culture plastic, TCP), for surface modification and subsequent efficacy of sperm selection.

[0266] Materials and Methods

[0267] Glass and polystyrene tissue culture plastic (TCP) slides were treated as described in previous examples to apply surface modification, involving application of PPOx coating, gold nanoparticles (GNP, 68 nm), and an additional PPOx overcoat. The slides were assembled into channelled slides (with a height of 0.4 pm). Anti-PS antibody was immobilized on the coated surface at a concentration of 200 pg / ml, and progesterone was applied to outlet wells at a concentration of 10 pM, as described in previous examples. Channelled slides were then evaluated for efficacy of sequestration of apoptotic sperm, using sperm from n= I human donors, as described in previous examples.

[0268] Results

[0269] The reduction in apoptotic and necrotic sperm populations after recovery from channelled slides was assessed using flow cytometry and compared to swim-up and neat samples. The results demonstratedno significant difference in the reduction of apoptotic and necrotic populations between devices constructed with glass or plastic substrates (Figure 21).

[0270] This experiment demonstrates that the efficacy of functionalised surfaces to sequester apoptotic sperm is retained regardless of the substrate material. We find functionalised surfaces on plastic or glass substrates can be employed to enhance the quality of recovered sperm.

[0271] Example 22 - Pig sperm selection

[0272] Effectiveness of the channelled slide device for selection of optimal sperm in a second mammalian species other than human was determined. Sperm recovered from pig ejaculate were evaluated in a protocol comparable to that demonstrated to be effective for human sperm.

[0273] Materials and Methods

[0274] Pig seminal fluid purchased from a commercial supplier were applied to the inlet wells of channelled slides (0.4 pm), prepared by coating with PPOx, gold nano-particles (68 nm), anti-PS and progesterone (prepared as described in previous examples). Unbound sperm were then recovered from the outlet wells of channelled slides 45 mins later and evaluated for sperm motility, markers of apoptosis and nexrosis (Annexin V binding and PI uptake), as described in previous sections. Data were compared to control sperm that was untreated, or prepared by standard swim-up technique.

[0275] Results

[0276] The results demonstrate a reduction in the apoptotic and necrotic populations of pig sperm recovered from the channelled slide device method, compared to neat sperm or sperm prepared by standard swim-up technique (Figure 22). Specifically, the proportion of apoptotic sperm was reduced to 10.4% after recovery from channelled slides, compared to 18.5% in the swim-up method (Figure 22E). Similarly, the necrotic population decreased to 5.0% after recovery from channelled slides, whereas it was 25.3% in sperm recovered by swim-up (Figure 22F). These findings indicate that the channelled slide is effective for preparation of high quality pig sperm with low levels of apoptosis and necrosis.

[0277] Example 23 - Enhanced selection of high quality sperm by addition of microchannels to the functionalised surface ofNGS Devices

[0278] The application of microchannels to the functionalised surface in was considered as a means of adding efficacy to separating high quality sperm. Microchannels were evaluated given their potential to add to the surface area for sperm interaction with the functionalised surface, to provide ready access tosurfaces and corners that might improve sperm propulsion, and to provide a labyrinthine structure that mimics elements of the natural microenvironment of the female reproductive tract.

[0279] Materials and methods

[0280] For this example, we employed a dicing technique to create microchannels along glass slides prior to assembly into NGS Devices. Microchannels had a depth of 45-50 pm and a width of 100 pm, with 50 pm spacing between each microchannel. This setup yielded a total of 24-28 microchannels (Figure 23A,B). The profilometry measurements show detailed surface topography and depth profiles of the microchannels on the glass slide, revealing uniformity and precision in their fabrication (Figure 23C). Surface modification was conducted as previously described, using plasma polymerization to coat the surface with PPOx. Subsequently, gold nanoparticles (GNPs) with a diameter of 68 nm were covalently attached and overcoated with PPOx to enable the covalent bioconjugation of Anti-PS at a concentration of 200 pg / ml, and progesterone (10 pM) was applied to the outlet chamber, as described in previous examples. After blocking the non-reacted groups with 2% HSA and washing with PBS, whole liquefied semen (n=2) was loaded into the inlet chamber. The samples were then incubated for 45 minutes at 37°C and 5% CO2. The recovered sperm from the outlet chamber were examined for Annexin V and PI positive populations using flow cytometry to assess the efficacy of the modified surface microchannels, and by Hlosperm Assay to determine the incidence of DNA damaged sperm.

[0281] Results

[0282] The analysis revealed a significant reduction in Annexin V positive (apoptotic) sperm, with only 3.5% observed in sperm recovered from the NGS Devices with microchannels. This is a marked improvement compared to the 13.0% and 11.0% apoptotic sperm found in the swim-up method and the non-coated channelled devices with microchannels, respectively. Additionally, analysis by DNA fragmentation assay demonstrated that less than 1 % of the sperm recovered from NGS Devices with coated microchannels exhibited DNA fragmentation. In contrast, sperm prepared by standard swim-up method had 15.0% DNA fragmentation, and the non-coated microchannels yielded sperm with 10.0% DNA fragmentation rate. These results highlight the superior performance of the NGS Devices modified to add surface microchannels. The modified NGS Devices selected high-quality, non-apoptotic sperm with minimal DNA damage. This low rate of DNA damage offers substantial potential to improve outcomes in assisted reproductive technology applications.

[0283] It will be appreciated by those skilled in the art that the disclosure is not restricted in its use to the particular application or applications described. Neither is the present disclosure restricted in its preferred embodiment with regard to the particular elements and / or features described or depicted herein. It will be appreciated that the disclosure is not limited to the embodiment or embodiments disclosed, butis capable of numerous rearrangements, modifications and substitutions without departing from the scope as set forth and defined by the following claims.REFERENCES

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[0285] LIU X, FENG Q, BACHHUKA A, VASILEV K. 2014. Surface modification by allylamine plasma polymerization promotes osteogenic differentiation of human adipose-derived stem cells. ACS' applied materials & interfaces .6(\ 2), 9733-41.

[0286] 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.

[0287] MARTINEZ-LEON, E., et al., Fibronectin modulates the endocannabinoid system through the cAMP / PKA pathway during human sperm capacitation. Molecular reproduction and development, 2019. 86(2): p. 224-238.

[0288] MARTINEZ, M.G., et al., Magnetic-activated cell sorting is not completely effective at reducing sperm DNA fragmentation. Journal of assisted reproduction and genetics, 2018. 35(12): p. 2215- 2221.

[0289] 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

[0290] RAMIASA, M„ CAVALLARO, A., MIERCZYNSKA, A., CHRISTO, S., GLEADLE, J., HAYBALL, J. & VASILEV, K. 2015. Plasma polymerised polyoxazoline thin films for biomedical applications. Chemical Communications, 51, 4279-4282.

[0291] 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.

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[0298] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that such prior art forms part of the common general knowledge.

Claims

CLAIMS1. A sperm sorting device comprising a substrate having a functionalised surface comprising a sperm selection agent that is capable of sorting sperm based on at least one biological or chemical property of the sperm when sperm is placed in contact with the functionalised surface.

2. A sperm sorting device for sequestering reduced competency sperm from a sperm sample to be used for ART methods including IVF, ICSI and Al, the device comprising a substrate having a functionalised surface comprising a sperm selection agent that is capable of preferentially sequestering reduced competency sperm based on at least one biological or chemical property of the reduced competency sperm when sperm is placed in contact with the functionalised surface.

3. The sperm sorting device according to either claim 1 or claim 2, wherein the functionalised surface is present in one or more flow channel having an inlet for delivering sperm to 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 according to claim 3, wherein the flow channel is a microchannel.

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

6. The sperm sorting device according to claim 5, wherein the sperm selection agent covalently bound, directly or indirectly, to the substrate via a functional film.

7. The sperm sorting device according to claim 6, wherein the functional film is selected from polyoxazaline polymer and polyamine polymer.

8. The sperm sorting device according to any one of claims 1 to 7, wherein the sperm selection agent comprises an agent that preferentially captures apoptotic sperm.

9. The sperm sorting device according to claim 8, wherein the agent that preferentially captures apoptotic sperm comprises anti-phosphatidyl serine antibodies.

10. The sperm sorting device according to any one of claims 1 to 7, wherein the sperm selection agent comprises an agent that promotes sperm endurance (viability and developmental competence) and / or primes the sperm ready for fertilisation.

11. The sperm sorting device according to claim 10, wherein the agent that promotes sperm endurance and / or primes the sperm ready for fertilisation comprises progesterone.

12. The sperm sorting device according to any one of claims 1 to 7, wherein the sperm sorting device comprises a first functionalised surface comprising a sperm selection agent that preferentially captures apoptotic sperm and a second functionalised surface comprising a sperm selection agent that promotes sperm endurance and primes the sperm ready for fertilisation.

13. The sperm sorting device according to claim 12, wherein the first functionalised surface and the second functionalised surface are spatially separated from one another on the substrate.

14. The sperm sorting device according to claim 12, wherein the first functionalised surface and the second functionalised surface are at least partially coincident with one another on the substrate.

15. A method of preparing semen for fertilisation using the sperm sorting device according to any one of claims 1 to 14, the method comprising: contacting sperm before selection with the functionalised surface; separating sperm that have not been sequestered on the functionalised surface from sperm that have been sequestered on the functionalised surface; and harvesting high competency sperm from the sperm sorting device.

16. A method of preparing a sperm sorting device according to any one of claims 1 to 14, the method comprising: providing a substrate; depositing a functional film on a surface of the substate; and attaching to the functional film a sperm selection agent that is capable to interacting with sperm based on at least one biological characteristic of the sperm.

17. A sperm sorting device comprising a substrate having one or more flow channel having an inlet for delivering sperm to 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 comprising at least one biological sperm sorting zone capable of sorting sperm based on a biological property of the sperm and at least one motility sperm sorting zone capable of sorting sperm based on a physical property of the sperm.

18. The sperm sorting device according to claim 17, wherein the at least one biological sperm sorting zone comprises a surface that is functionalised with one or more biological agent that can interact with sperm.

19. The sperm sorting device according to claim 18, wherein the surface that is functionalised with one or more biological agent that can interact with sperm is a surface that preferentially captures apoptotic sperm.

20. The sperm sorting device according to claim 19, wherein the surface that preferentially captures apoptotic sperm is functionalised with anti-phosphatidyl serine antibodies.

21. The sperm sorting device according to any one of claims 17 to 20, wherein the surface that is functionalised with one or more biological agent that can interact with sperm is a surface that promotes sperm endurance and primes sperm ready for fertilisation.

22. The sperm sorting device according to claim 21, wherein the surface that promotes sperm endurance and primes sperm ready for fertilisation is functionalised with progesterone.

23. The sperm sorting device according to any one of claims 21 to 22 wherein the sperm sorting device comprises a first biological sperm sorting zone comprising a surface that preferentially captures apoptotic sperm, wherein the first biological sperm sorting zone is upstream of a second biological sperm sorting zone comprising a surface that promotes sperm endurance and primes sperm ready for fertilisation.

24. The sperm sorting device according to any one of claims 17 to 23, wherein the motility sperm sorting zone comprises a surface architecture that interferes with sperm motility.

25. The sperm sorting device according to claim 24, wherein the surface architecture that interferes with sperm motility is an array of particles on a surface of the channel.

26. The sperm sorting device according to claim 25, wherein the particles are functionalised with anti-phosphatidyl serine antibodies and / or progesterone.

27. The sperm sorting device according to any one of claims 17 to 26 when used for sorting sperm based on fertilisation competency for use in IVF.

28. A method of preparing semen for fertilisation using the sperm sorting device according to any one of claims 17 to 27, the method comprising: a sperm adding step of adding sperm before selection to the inlet; a sperm sorting step of contacting sperm in the channel with the biological sperm sorting zone the motility sperm sorting zone under conditions for the sperm to interact with the biological sperm sorting zone the motility sperm sorting zone, anda harvesting step of recovering sperm that pass along the channel at the outlet.

29. A method of preparing a sperm sorting device according to any one of claims 17 to 27, the method comprising: providing a substrate having one or more flow channel; depositing a functional film on a surface of the one or more flow channel; attaching to the functional film a biological agent that is capable to interacting with sperm under conditions to form at least one biological sperm sorting zone in the channel; and covalently attaching to the functional film nanoparticles under conditions to form at least one motility sperm sorting zone in the channel.

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