Methods for identifying, differentiating and / or sorting cells

Abstract

The present disclosure relates to a method for identifying, differentiating, and / or sorting sperm cells in a sperm sample, more particularly, X-chromosome-bearing sperm (X-CBS) cells and Y-chromosome-bearing sperm (Y-CBS) cells. The method comprises determining a level of reactive oxidant species and / or cellular changes mediated thereby (ROSCC) in each of a plurality of sperm cells in the sperm sample, and identifying, differentiating, and / or sorting X-CBS cells and Y-CBS cells in at least a portion of the plurality of sperm cells based on the determined ROSCC level. Enriched or purified X-CBS cells and Y-CBS cells and uses thereof are also described.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of Australian Provisional Patent Application No. 2022903552, filed on 23 November 2022, the entire contents of which are incorporated herein by reference.

[0002] The disclosure herein relates to methods for identifying, differentiating, and / or sorting cells. More specifically, the disclosure herein relates to methods for identifying, differentiating, sorting, separating, and / or isolating sex cells and producing enriched or purified populations of such cells. [Background technology]

[0003] In many areas of animal production, the ability to reliably select the sex of offspring is desirable. For example, in the dairy industry, female calves provide economic benefits, while male calves are an economic burden. In research, sex-specific diseases such as testicular or ovarian cancer limit studies to subjects of a single sex. In endangered species conservation, optimizing the sex ratio of a population—for example, increasing the number of females relative to males—maximizes population growth. However, best-practice breeding protocols employed in animal management often result in an even sex ratio, resulting in animal wastage and hindering the success of conservation programs. Despite comprehensive efforts to develop inexpensive, reliable, and safe technologies for selecting the sex of offspring, a suitable solution has yet to be found.

[0004] In research, and to a lesser extent in agriculture, offspring sex ratio is managed postnatally by culling individuals of the unwanted sex. This strategy raises ethical questions and is costly in terms of time and money. In dairy cows, for example, gestation lasts an average of nine months; farmers therefore lose nine months of investment in half of their herds. In Australia alone, postnatal sex ratio control results in the culling of approximately 400,000 male dairy calves each year, causing distress to calves and cows and increasing the incidence of retained placenta and mastitis.

[0005] Despite strict regulations on animals used in research, accurate figures for animal disposal are difficult to obtain. Data collated from four Australian states in 2018 reported that 5.5 million laboratory mammals were used for scientific and educational purposes. The number of animals culled after birth due to excess demand is not reported because regulators do not require this information, but the number of animals needlessly killed after birth could be in the millions.

[0006] Agricultural companies avoid equal offspring sex ratios by utilizing specialized techniques to separate sperm into subpopulations of X- and Y-chromosome-bearing sperm (CBS) for assisted reproduction. Current sperm sorting techniques rely on the use of variants of the fluorescent dye Hoechst, most commonly 33342, which binds to the nucleotide bases adenine and thymine in the minor groove of DNA. Following activation of Hoechst-stained DNA under intensely focused ultraviolet light, X- and Y-CBS are identified based on differences in fluorescence intensity, depending on nucleotide binding. X-CBS emit more light than Y-CBS because their total DNA content is greater than that of Y-CBS, a difference ranging from 1 to 7.5% in mammals studied to date. The larger X chromosome provides more binding sites for Hoechst. The measured difference in fluorescence intensity between the two sperm types is the basis for their separation via specialized flow cytometry and microfluidic sorting systems. The purity of the X- or Y-CBS, which ranges from 70-90%, correlates with sorting time and estimated cost.

[0007] Although sperm preselection is theoretically preferable to postnatal culling, Hoechst as a tool poses practical risks to sperm and offspring. To allow Hoechst dye to bind adequately to DNA, sperm must first be exposed to Hoechst dye at testicular temperatures for extended periods, typically 45–90 minutes. Following the staining process, sperm undergo a lengthy sorting protocol that damages the sperm. Long treatment times can affect viability, sometimes dropping viable sperm to as low as 30%. Hoechst sorting is notoriously difficult; therefore, sperm cell sorting equipment is specialized and can be prohibitively expensive. Furthermore, the nature of Hoechst-nucleotide interactions is mutagenic in some cells, and ultraviolet light is well known to damage cells and their DNA in particular. This is further complicated by the lack of efficient DNA repair in sperm cells, resulting in the introduction of DNA mutations into the germline of offspring. While Hoechst technology is the only available option, technical difficulties and associated risks prevent its widespread use.

[0008] Therefore, improved methods of identifying and / or sorting cells, such as sorting sperm into X- and Y-chromosome-bearing sperm, that address one or more of the above-mentioned problems or at least provide a useful alternative are desirable. In particular, improved, rapid and accurate methods of identifying and / or sorting sperm that reduce the risk of mutagenicity, avoid exposing the sperm to ultraviolet light, and / or increase sperm viability are desirable.

[0009] The invention described herein provides a novel method for sorting sperm cells that overcomes the need to use time-consuming and highly sensitive sorting procedures that are prone to error as a result of orientation and hydrodynamic effects, which are required to distinguish differences in total DNA content in X- and Y-chromosome-bearing sperm cells. The novel method desirably aids in preserving sperm health and the genetic integrity of sperm DNA by providing a faster and more robust platform for distinguishing between sperm cells that is also non-mutagenic in preferred embodiments.

[0010] Reference to a patent document or other matter provided herein as prior art should not be construed as an admission that such document or matter was publicly known or that the information it contains was part of common general knowledge at the priority date of any of the claims. Summary of the Invention

[0011] Unless the context requires otherwise, when used in this specification (including the claims) the words "comprise", "comprises" and "comprising" are to be interpreted as specifying stated features, integers, steps or components but not as excluding the presence of one or more other features, integers, steps or components or groups thereof.

[0012] According to a first aspect of the present invention, there is provided a method of sorting sperm cells, the method comprising the steps of: determining a level of reactive oxidant species and / or cellular changes mediated thereby (ROSCC) in each of a plurality of sperm cells; and sorting at least a portion of the plurality of sperm cells into X chromosome-bearing sperm (X-CBS) cells and / or Y chromosome-bearing sperm (Y-CBS) cells based on the determined ROSCC level.

[0013] The method may include sorting at least a portion of the plurality of sperm cells into X-CBS cells and Y-CBS cells. In some embodiments, the plurality of sperm cells may include a mixture of X-CBS and Y-CBS. ROSCC may be selected from reactive oxidant species selected from reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS), or a combination of two or more thereof, and / or cellular changes mediated by reactive oxidant species selected from ROS-induced oxidative changes, RNS-induced oxidative changes, and RSS-induced oxidative changes, or a combination of two or more thereof. Identifying the level of ROSCC may include treating the plurality of sperm cells with a ROSCC detection agent, and in some embodiments, the detection agent includes a dye, such as a fluorescent dye. When a fluorescent dye is used as the ROSCC detection agent, fluorescence may be activated by reaction of the dye with one or more ROSCCs in the cells. ROSCC may be detectable in a component of a sperm cell selected from lipid components such as the plasma membrane or mitochondrial membrane, protein components such as chromatin or enzymes, organelles such as mitochondria, extracellular components such as ROSCC generated by cell surface enzymes, cytoplasm and DNA, or a combination of any two or more of these components. The ROSCC detection agent may be selective for targeting ROSCC. The detection agent may detect ROS, RNS, and / or RSS in at least the cytoplasm of the sperm cell. The ROSCC detection agent may detect ROS-, RNS-, and / or RSS-induced oxidative damage in lipid components of the sperm cell. ROSCC may be a direct product of mitochondrial activity. ROSCC may be a reactive oxidant species, and the reactive oxidant species may be an ROS selected from superoxide, hydroxyl radical, and hydrogen peroxide. ROSCC may be a cellular change mediated by a reactive oxidant species, and the cellular change is lipid peroxidation.

[0014] The level of ROSCC identified in each X-CBS cell may differ from the level of ROSCC identified in each Y-CBS cell by at least 5%, at least 20%, at least 40%, or at least 60%. Alternatively, the average level of ROSCC identified for all cells in the X-CBS may differ from the average level of ROSCC identified for all cells in the Y-CBS by at least 5%, at least 20%, at least 40%, or at least 60%. The level of ROSCC identified in each X-CBS cell may be at least 5%, at least 20%, at least 40%, or at least 60% higher than the level of ROSCC identified in each Y-CBS cell. Alternatively, the average level of ROSCC identified for all cells in the X-CBS may be at least 5%, at least 20%, at least 40%, or at least 60% higher than the average level of ROSCC identified for all cells in the Y-CBS.

[0015] The plurality of sperm cells may be obtained from the proximal cauda epididymis, distal cauda epididymis, vas deferens, or ejaculated semen of the subject. The plurality of sperm cells may be obtained from a mammalian subject. The plurality of sperm cells may be obtained from a non-human mammalian subject.

[0016] The sorting step may include using a flow cytometry sorter or a microfluidic sorting device. The sorting step may be based on a visual indicator of ROS levels, such as fluorescence or absorbance. The sorting step may be based on a physiological function of the sperm, such as relative motility. The sorting step may utilize magnetic or electrostatic interactions. The sorting step may include using a flow cytometer, and the X-CBS cells and / or Y-CBS cells are optionally sorted by gating. The identifying step may be performed in situ in the flow cytometer or microfluidic device prior to the sorting step. The method may further include separately providing the sorted X-CBS cells and / or Y-CBS cells in enriched or purified form. The enriched or purified form may have a minimum purity of at least 90% X-CBS cells or a minimum purity of at least 90% Y-CBS cells. The method may further comprise the step of separately providing the sorted X-CBS cells and / or Y-CBS cells in cryopreserved form.

[0017] According to a second aspect of the present invention there is provided an enriched or purified population of X-chromosome bearing sperm (X-CBS) cells selected by the method of the first aspect above. The enriched or purified population may have a minimum purity of at least 90% X-CBS cells.

[0018] According to a third aspect of the present invention there is provided an enriched or purified population of Y chromosome-bearing sperm (Y-CBS) cells selected by the method of the first aspect above. The enriched or purified population may have a minimum purity of at least 90% Y-CBS cells.

[0019] According to a fourth aspect of the present invention, there is provided a method for identifying X-chromosome-bearing sperm (X-CBS) cells or Y-chromosome-bearing sperm (Y-CBS) cells in a sperm sample, the method comprising the steps of determining a level of reactive oxidant species and / or cellular changes mediated thereby (ROSCC) in each of a plurality of sperm cells in a starting sperm sample, and identifying at least a portion of the plurality of sperm cells in the starting sperm sample as X-CBS cells or Y-CBS cells based on the determined ROSCC level.

[0020] The starting sperm sample may contain a mixture of X-CBS cells and Y-CBS cells. The method may further comprise isolating at least a portion of the identified X-CBS cells or Y-CBS cells from the starting sperm sample to generate a concentrated sperm sample containing at least a portion of the identified and isolated X-CBS cells or Y-CBS cells. The concentrated sperm sample may contain at least 90% X-CBS cells or at least 99% X-CBS cells, or at least 90% Y-CBS cells or at least 99% Y-CBS cells. The isolating step may optionally include using a flow cytometry sorter with gating or a microfluidic sorting device. Alternatively, the method may further include inducing selective motility, sterility, or non-viability in either the identified X-CBS cells or Y-CBS cells in the starting sperm sample to generate a sperm sample with altered motility, fertility, or viability. The method may further comprise cryopreserving the concentrated sperm sample.

[0021] In one embodiment, a method for isolating X-chromosome-bearing sperm (X-CBS) cells or Y-chromosome-bearing sperm (Y-CBS) cells in a sperm sample is provided, the method comprising: determining a level of reactive oxidant species and / or cellular changes mediated thereby (ROSCC) in each of a plurality of sperm cells in a starting sperm sample; identifying at least a portion of the plurality of sperm cells in the starting sperm sample as X-CBS cells or Y-CBS cells based on the determined ROSCC level; and isolating at least a portion of the identified X-CBS cells or Y-CBS cells from the starting sperm sample to generate a concentrated sperm sample comprising at least a portion of the identified and isolated X-CBS cells or Y-CBS cells.

[0022] In another embodiment, a method for producing a sperm sample with altered motility, fertility, or viability is provided, comprising the steps of: determining a level of reactive oxidant species and / or cellular changes mediated thereby (ROSCC) in each of a plurality of sperm cells in a starting sperm sample; identifying at least a portion of the plurality of sperm cells in the starting sperm sample as X-CBS cells or Y-CBS cells based on the determined ROSCC level; and inducing selective motility, infertility, or non-viability in either the identified X-CBS cells or Y-CBS cells in the starting sperm sample to produce a sperm sample with altered motility, fertility, or viability.

[0023] According to a fifth aspect of the present invention there is provided an X-CBS cell or a Y-CBS cell identified by the method of the fourth aspect above.

[0024] According to a sixth aspect of the present invention there is provided the use of sperm selected by the method of the first aspect above, an enriched or purified population of X-chromosome-bearing sperm (X-CBS) cells or Y-chromosome-bearing sperm (Y-CBS) cells according to the second or third aspect above, or X-CBS cells or Y-CBS cells identified by the method of the fourth aspect above, in assisted reproduction techniques.

[0025] According to a seventh aspect of the present invention there is provided a non-human mammalian subject produced from sperm selected by the method of the first aspect above, an enriched or purified population of X-chromosome-bearing sperm (X-CBS) cells or Y-chromosome-bearing sperm (Y-CBS) cells according to the second or third aspect above, or X-CBS cells or Y-CBS cells identified by the method of the fourth aspect above.

[0026] According to an eighth aspect of the present invention, there is provided a sperm cell sorting system comprising: a reactive oxidant species and / or cellular change (ROSCC) detecting agent, wherein the cellular change is mediated by reactive oxidant species; and instructions for using the ROSCC detecting agent to identify a level of ROSCC in each of a plurality of sperm cells, and sorting at least a portion of the plurality of sperm cells into X chromosome-bearing sperm (X-CBS) cells and / or Y chromosome-bearing sperm (Y-CBS) cells based on the identified ROSCC level. The sperm cell sorting system may further comprise a sperm cell incubation solution for incubating the sperm cells with the ROSCC detecting agent.

[0027] According to a ninth aspect of the present invention, there is provided a method of distinguishing between X chromosome-bearing sperm (X-CBS) cells and Y chromosome-bearing sperm (Y-CBS) cells in a sperm sample, the method comprising the steps of: determining a level of reactive oxidant species and / or cellular changes mediated thereby (ROSCC) in each of a plurality of sperm cells in the sperm sample; and distinguishing between X-CBS and Y-CBS in at least a portion of the plurality of sperm cells in the sperm sample based on the determined ROSCC level.

[0028] According to a tenth aspect of the present invention, there is provided a method for distinguishing between X-chromosome-bearing sperm (X-CBS) cells and Y-chromosome-bearing sperm (Y-CBS) cells in a sperm sample, the method comprising: determining a level of reactive oxidant species and / or cellular changes mediated thereby (ROSCC) in each of a plurality of sperm cells in the sperm sample; differentiating between X-CBS and Y-CBS cells in at least a portion of the plurality of sperm cells in the sperm sample based on the determined ROSCC level; and isolating at least a portion of the differentiated X-CBS cells or Y-CBS cells from the sperm sample to produce an enriched or purified sperm sample. The enriched or purified sperm sample may be an enriched or purified population of X-CBS cells. The enriched or purified sperm sample may be an enriched or purified population of Y-CBS cells. The enriched or purified sperm sample may have a minimum purity of at least 90% X-CBS cells or a minimum purity of at least 90% Y-CBS cells. In some embodiments, the isolating step uses a flow cytometer or a microfluidic sorting device.

[0029] In an eleventh aspect of the present invention there is provided an enriched or purified population of X-CBS cells or an enriched or purified population of Y-CBS cells isolated by the method of the tenth aspect above.

[0030] In a twelfth aspect of the present invention there is provided the use of sperm differentiated by the method of the ninth or tenth aspects above, or of an enriched or purified population of X-CBS cells or Y-CBS cells according to the eleventh aspect above, in assisted reproduction techniques. [Brief explanation of the drawings]

[0031] [Figure 1]1 is a diagrammatic representation of the testis showing three locations in the reproductive tract from which sperm for experiments were obtained, as described in the Examples herein. The cauda epididymis is divided into two sections: section "1" refers to the portion of the cauda epididymis adjacent to the body of the epididymis, referred to as the "proximal tail," section "2" refers to the portion of the tail adjacent to the vas deferens, referred to as the "distal tail," and section "3" refers to the vas deferens from the distal cauda epididymis as close as possible to the ejaculatory duct, referred to as the "vas deferens." [Figure 2] Data from Hoechst 33342-sorted sperm are shown, demonstrating the level of separation of X- and Y-CBS achievable in C57BL / 6 mice: a) histogram showing significant overlap in left-peak Y-CBS and right-peak X-CBS; b) the same data presented as a contour plot. [Figure 3] Data are shown for sperm collected from the proximal tail epididymis of C57BL / 6 mice and sorted via flow cytometry into X-CBS and Y-CBS populations. a) Histogram showing high resolution of left-peak Y-CBS and right-peak X-CBS identified by differential levels of mitochondrial superoxide via MitoSOX®. b) Contour plots of mouse sperm stained and sorted with Hoechst 33342 (vertical axis) and MitoSOX® (horizontal axis). [Figure 4] Data are shown for sperm collected from the distal tail epididymis of C57BL / 6 mice and sorted via flow cytometry into X-CBS and Y-CBS populations. a) Histogram showing high resolution of left-peak Y-CBS and right-peak X-CBS identified by differential levels of mitochondrial superoxide via MitoSOX®. b) Contour plots of mouse sperm stained and sorted with Hoechst 33342 (vertical axis) and MitoSOX® (horizontal axis). [Figure 5]Data are shown for sperm collected from the vas deferens of C57BL / 6 mice and sorted via flow cytometry into X-CBS and Y-CBS populations, including a) a histogram showing high resolution of left-peak Y-CBS and right-peak X-CBS identified by differential levels of mitochondrial superoxide via MitoSOX®, and b) a contour plot of mouse sperm stained and sorted with Hoechst 33342 (vertical axis) and MitoSOX® (horizontal axis). [Figure 6] Figure 1 shows data for sperm collected from the vas deferens of C57BL / 6 mice and sorted via flow cytometry into X-CBS and Y-CBS populations, including a) a histogram of sperm stained with the broad-spectrum cytoplasmic ROS detection probe CellROX® Red, b) a histogram of sperm stained with the lipid peroxidation sensor BodipyC11®, and c) a contour plot of sperm stained with BodipyC11® (vertical axis) and CellROX® Red (horizontal axis), demonstrating the increased resolution of X-CBS and Y-CBS that can be achieved when sorting via lipid peroxidation and cytoplasmic ROS when superoxide cannot be measured directly. [Figure 7] 1 shows a bar graph illustrating the difference in mitochondrial superoxide levels in Y-CBS and X-CBS of C57BL / 6 mice, as measured by calculating the mean fluorescence intensity (MFI) of 10,000 sperm in the proximal and distal tail of the epididymis and vas deferens after staining with MitoSOX®. On average, X-CBS is 7.5 times more fluorescent than Y-CBS in mice. [Figure 8] Figure 1 shows data for cryopreserved bull sperm samples sorted via flow cytometry into X-CBS and Y-CBS populations, showing a) histograms of sperm stained with MitoSOX® Red, and b) histograms of sperm stained with Hoechst 33342. [Figure 9] 1 shows data visualizing the large difference in superoxide concentrations between equine X-CBS and Y-CBS cells by MitoSOX® detection. [Figure 10] 1 shows data visualizing the large difference in superoxide concentrations between boar X-CBS and Y-CBS cells by MitoSOX® detection. DETAILED DESCRIPTION OF THE INVENTION

[0032] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0033] A method for sorting sperm cells is described herein, comprising: determining a level of reactive oxidant species and / or cellular change mediated thereby (ROSCC) in each of a plurality of sperm cells; and sorting at least a portion of the plurality of sperm cells into X-chromosome-bearing sperm (X-CBS) cells and / or Y-chromosome-bearing sperm (Y-CBS) cells based on the determined ROSCC level. A method for identifying X-CBS cells or Y-CBS cells in a sperm sample is also described herein, comprising: determining a level of reactive oxidant species and / or cellular change mediated thereby (ROSCC) in each of a plurality of sperm cells in a starting sperm sample; and identifying at least a portion of the plurality of sperm cells in the starting sperm sample as X-CBS cells or Y-CBS cells based on the determined ROSCC level. The method may further comprise an isolation step to produce a sperm sample enriched in X-CBS cells or Y-CBS cells, or a step of selectively inducing motility, infertility, or non-viability in either the identified X-CBS cells or Y-CBS cells in the starting sperm sample to produce a sperm sample with altered motility, fertility, or viability. The present inventors first discovered that X-CBS and Y-CBS cells differ from each other in their levels of ROSCC and advantageously applied this finding to identify, sort, and separate X-CBS cells based on X-CBS cells having a level of ROSCC that differs from that of Y-CBS cells, such as a higher or lower level of ROSCC. Those skilled in the art will recognize that identifying X-CBS cells as distinct from Y-CBS cells is equivalent to distinguishing between two different chromosome-bearing cell types. Accordingly, in one embodiment, a method of sorting sperm cells is described herein, the method comprising: identifying a level of reactive oxidant species and / or cellular changes mediated thereby (ROSCC) in each of a plurality of sperm cells; and, based on the identified ROSCC level, sorting at least a portion of the plurality of sperm cells into X-chromosome-bearing sperm (X-CBS) having a level of ROSCC that differs from that of Y-chromosome-bearing sperm (Y-CBS) either at the cellular level or on an average across all cells.In one embodiment, the level of ROSCC identified in each X-CBS cell is different from the level of ROSCC identified in each Y-CBS cell. In another embodiment, the average level of ROSCC identified across all cells in the X-CBS is different from the average level of ROSCC identified across all cells in the Y-CBS. In one embodiment, the level of ROSCC identified in the X-CBS is higher than the level of ROSCC identified in the Y-CBS cells, either at the cellular level or average across all cells. In one embodiment, the term "higher" as used in this context refers to the level of ROSCC identified in the X-CBS being at least about 5% higher than the level of ROSCC identified in the Y-CBS cells, either at the cellular level or average across all cells. However, embodiments are also contemplated herein in which the X-CBS has a lower level of ROSCC than the Y-CBS, either at the cellular level or average across all cells. In one embodiment, the term "lower" as used in this context refers to the level of ROSCC identified in X-CBS being at least about 5% lower than the level of ROSCC identified in Y-CBS cells, either at the cellular level or on an average across all cells.

[0034] The level of ROSCC in a given sperm cell is related to the concentration of reactive oxidant species present in that cell, and is advantageously physiologically detectable in a variety of ways. This opens up the possibility of measuring the level of ROSCC in cells with various ROSCC detection agents and / or methods to optimize the identification, isolation, and / or enrichment of X-CBS and Y-CBS cells. By way of non-limiting example, the level of ROSCC detected in sperm cells herein can be the level of one or more specific reactive oxidant species and / or the level of one or more cellular changes mediated by reactive oxidant species caused by their reaction with other reactive species and / or cellular components. As will become apparent below, the ability to target a range of sperm cell components to measure ROSCC levels not only provides versatility in terms of which detection devices, detection agents, and sorting devices are used, but can also help preserve the genetic integrity of sperm DNA. For example, in certain embodiments of the methods described herein, the ROSCC detection agents do not bind to or interfere with DNA in sperm cells, thereby avoiding potential damage to DNA. In other embodiments, the fluorescence of the ROSCC detection agents used herein is activated without the use of ultraviolet light, which avoids the problems associated with ultraviolet light compromising the integrity of or permanently altering sperm DNA.

[0035] The difference in ROSCC levels between X-CBS cells and Y-CBS cells, as discovered by the present inventors, is surprisingly preserved across various sperm maturation stages, from the epididymal corpus to the vas deferens. Thus, specific identification, sorting, and enrichment of ROSCC levels in X-CBS cells and Y-CBS cells are feasible using the methods herein with sperm cell samples collected from various locations within the male reproductive system.

[0036] Furthermore, unlike previous sperm cell sorting techniques based on the DNA-binding dye Hoechst 33342, the difference in ROSCC levels between X-CBS and Y-CBS cells, as discovered by the inventors, is, in some embodiments, significantly greater than the approximately 1-7.5% difference in DNA payload size between X-CBS and Y-CBS cells. Thus, problems posed by small and variable differences in DNA size, such as the need for highly sensitive and expensive sorting equipment (including specialized lasers), can be advantageously overcome in certain embodiments of the methods described herein.

[0037] The large difference in ROSCC levels between X-CBS and Y-CBS further facilitates sperm cell identification and detection compared to using Hoechst 33342, resulting in shorter sorting times and less potential damage to sperm, potentially increasing fertility and healthier offspring. By way of explanation, sperm are time-sensitive cells that lose effectiveness the longer they remain unused. However, to ensure that small differences in DNA size are sufficiently identified for sorting, sperm must be exposed to Hoechst variants for periods of up to 1.5 hours in some species. Furthermore, the small differences in DNA content between X-CBS and Y-CBS (1-7.5% in mammals studied to date) result in overlapping fluorescence peaks, leading to slow sorting times of up to 4 hours. Therefore, the long staining and sorting times associated with this DNA-binding dye-based method can be detrimental to the recovery rate of sorted sperm. In contrast, in certain embodiments of the methods described herein, sperm staining for ROSCC can be completed in 10 minutes, and in other embodiments, in 30 minutes. The magnitude of the difference in ROSCC levels between X-CBS and Y-CBS cells is much easier to detect than differences in DNA size, and this allows for shorter identification / sorting times compared to using DNA-binding dyes such as Hoechst 33342 and other variants.

[0038] Furthermore, unlike DNA-based dye detection methods in which fluorescent events may be observed for damaged or dead sperm cells, in certain embodiments, the methods herein advantageously selectively generate signals, such as dye responses, only in living cells with functional metabolism. Such embodiments avoid the need for signal interference correction introduced by signal detection from dead sperm in the sample, particularly in the case of cryopreserved sperm samples containing dead and / or damaged sperm caused by the freezing process.

[0039] Although agents for detecting specific reactive oxygen species in cells are known, and methods using these dyes for the general assessment of sperm health have been reported, the association between ROSCC and X or Y chromosome bearing in sperm cells was previously unknown. Furthermore, although ligand activation of Toll-like receptor 7 / 8 (TLR7 / 8) was used in one study to inhibit the motility of X chromosome-bearing sperm, thereby allowing the separation of X chromosome-bearing sperm from Y chromosome-bearing sperm (Umehara et al., PLoS Biol. 2019 Aug. 13;17(8)), there is no association between TLR7 / 8 and reactive oxygen species generation in sperm. Thus, the presently described method represents a significant advance in the art.

[0040] The methods described herein are suitable for sorting a plurality of sperm cells. The term "plurality" encompasses two or more sperm cells. Thus, in some embodiments, the methods described herein are suitable for use in single cell sorting methods. In other embodiments, the methods described herein are suitable for use in multiple cell sorting methods. When employing automated cell sorting equipment, the term "multiple" refers to 10 or more sperm cells. 2 , 10 3 , 10 4 , 10 5 , 10 6 or 10 7 More than 10 sperm cells, or 10 2 , 10 3 , 10 4 , 10 5 , 10 6 or 10 7The term "sperm cells" may refer to a concentration of sperm cells exceeding 1000 / mL. The methods herein are suitable for sorting at least a portion of the plurality of sperm cells into X-chromosome-bearing sperm (X-CBS) and / or Y-chromosome-bearing sperm (Y-CBS). The plurality of sperm cells herein, prior to sorting, desirably comprises a mixture of X-CBS cells and Y-CBS cells. This advantageously allows the relative levels of ROSCC in the X-CBS cells versus the Y-CBS cells to be determined, thereby enabling the X-CBS cells (or a portion thereof) to be identified and separated from the Y-CBS cells (or a portion thereof). In some embodiments, the plurality of sperm cells, prior to sorting by the methods described herein, comprises approximately 40-60% X-CBS cells and 60-40% Y-CBS cells, such as approximately 50% X-CBS cells and 50% Y-CBS cells, or in other embodiments, any other naturally occurring ratio of X-CBS:Y-CBS cells. In some embodiments, the plurality of sperm cells may be pre-sorted or pre-enriched prior to sorting by the methods described herein so that the sperm cells have a non-naturally occurring ratio of X-CBS:Y-CBS cells. Desirably, if used, the pre-sorted or pre-enriched sperm cells comprise a mixture of X-CBS cells and Y-CBS cells. In some embodiments, pre-sorting or pre-enrichment may be performed by the methods described herein, or in other embodiments, may be performed using other methods, such as Hoechst-based methods.

[0041] The use of the term "at least a portion" is used herein to clarify that not all cells of the plurality of sperm cells need to be identified or sorted as X-CBS cells or Y-CBS cells in the described methods. As a further explanation, some sperm cells may not be identified or sorted as either X-CBS cells or Y-CBS cells because they carry variants such as chromosome diploidy, or are damaged or fragmented during sorting and are not sufficiently detected, or have insufficient ROSCC levels for determination of X-CBS cells or Y-CBS cells using a particular technique / detection agent, etc.

[0042] Sperm cells for use in the methods herein may be obtained from any suitable source. In one embodiment, sperm cells are obtained from within the subject's reproductive system, such as from the subject's corpus epididymis, e.g., from the corpus epididymis, or from the vas deferens. In one embodiment, sperm cells are obtained from the subject's proximal cauda epididymis. In another embodiment, sperm cells are obtained from the subject's distal cauda epididymis. In a further embodiment, sperm cells are obtained from the subject's vas deferens. In yet another embodiment, sperm cells are obtained from the subject's ejaculated sperm. In other embodiments, sperm cells are obtained from the proximal cauda epididymis, distal cauda epididymis, vas deferens, or from the subject's ejaculated sperm.

[0043] Those skilled in the art will be familiar with methods for isolating sperm cells from seminal plasma in semen to provide a sample of sperm cells suitable for sorting using the methods herein. Methods for isolating sperm cells from seminal plasma generally involve mixing semen containing sperm cells with a suitable aqueous solution, such as a buffer containing phosphate-buffered saline (PBS) at a pH of about 7.4, semen extender, human renal tubular fluid, cell culture medium, assisted reproduction medium, etc., centrifuging the aqueous sperm mixture, discarding the supernatant, and resuspending the sperm cells in a suitable aqueous solution. In some embodiments, the resuspended sperm cells may optionally be incubated with a detection agent, then washed and resuspended prior to detection and sorting. In some embodiments, the sperm sample used in the methods herein is one in which sperm cells have been isolated from seminal plasma prior to determining their ROSCC level. Such embodiments advantageously reduce interference from seminal plasma components during measurements, facilitate the preparation and use of clean, concentrated sperm samples, and / or prolong sperm function. In other embodiments, the sperm sample may be separated without first isolating the sperm cells from the semen. In some embodiments, the use of a ROSCC indicator or a dye selective for a particular endogenous ROSCC may avoid any influence of semen components on the measured ROSCC levels of the sperm cells in the sample.

[0044] Sperm cells for use in the methods described herein may be obtained from any suitable subject. In one embodiment, the sperm cells are obtained from a mammalian subject. In one embodiment, the mammalian subject is a domesticated animal such as a dog, cat, cow, horse, sheep, donkey, or camel. In one embodiment, the mammalian subject is a cow. In one embodiment, the mammalian subject is a livestock animal such as a horse, sheep, cow, or goat. In one embodiment, the mammalian subject is a research animal such as a mouse, rat, non-human primate, guinea pig, rabbit, or hamster. In one embodiment, the mammalian subject is a human. In one embodiment, the mammalian subject is an endangered or threatened mammalian species such as a rhinoceros, tiger, leopard, gorilla, elephant, orangutan, panda, etc. In one embodiment, the sperm cells are obtained from a non-human mammalian subject. In another embodiment, the sperm cells are obtained from a non-mammalian heterogametic male vertebrate.

[0045] As used herein, the term "reactive oxidant species and / or cellular changes mediated thereby" or "ROSCC" refers to any reactive oxidant species or cellular changes mediated by such species, including either the reactive oxidant itself or species generated by the reaction of the reactive oxidant with other chemical species (e.g., with other reactive oxidant species and / or cellular components). As used herein, the term "active" means that the ROSCC readily undergoes a chemical reaction with at least one other species, such as a species present in the cellular environment, and may refer to a radical species containing an unpaired electron, such as the hydroxyl radical (·OH), or a non-radical species, such as hydrogen peroxide (HO). As used herein, the term "oxidant" means that the ROSCC reacts with (oxidizes) another species, gaining one or more electrons in the process.

[0046] Species produced by the reaction of an active oxidant with a cellular component may also be referred to herein as a "cellular change mediated by an active oxidant species." For the avoidance of doubt, a cellular change may be the product formed in a single reaction between a cellular component and an active oxidant, or may form after a chain of reactions in one or more cellular components triggered by the active oxidant. Cellular components susceptible to reaction with active oxidant species include lipids, proteins, carbohydrates, and nucleic acids. It should be appreciated that any cellular change mediated by active oxidant species may be the product of oxidative attack, and that some cellular components may be converted to active oxidant species during this process. Thus, in some embodiments, there is an overlap between what is considered an active oxidant species and what is considered a cellular change mediated by an active oxidant species. Thus, the term "ROSCC" is used herein for convenience to emphasize that the present invention is not limited to screening based on the detection of reactive oxidant species, primarily ·OH, ·O2, and NO·, that are produced directly by metabolic processes, but also encompasses screening based on the detection of reactive oxidant species that are formed when ·OH, ·O2, and / or NO· react with other radicals, ions, and / or molecules within the cell to cause cellular changes.

[0047] The nature of the level of ROSCC identified in the methods herein is not particularly limited, provided that it is a level of ROSCC that can be identified in sperm cells. In one embodiment, the level of ROSCC identified in the methods herein is advantageously at a level that is significantly different between X-CBS cells and Y-CBS cells. In another embodiment, the level of ROSCC identified in the methods herein is advantageously at a level that is sufficiently different to distinguish a subpopulation of X-CBS cells in a sperm cell sample, to distinguish a subpopulation of Y-CBS cells in a sperm cell sample, or to distinguish a subpopulation of X-CBS cells in a sperm cell sample from a subpopulation of Y-CBS cells in a sperm cell sample, optionally wherein the subpopulation of X-CBS cells and / or the subpopulation of Y-CBS cells form only a portion of the plurality of sperm cells initially designated for sorting.

[0048] As used herein, the "level" of ROSCC encompasses any means of quantifying the amount or concentration of ROSCC. The level of ROSCC identified in each sperm cell generally does not represent the total concentration of ROSCC (whether of any single species or combination thereof) in the cell. Instead, the level of ROSCC identified in each sperm cell, in some embodiments, represents the total ROSCC level (whether of any single species or combination thereof) in the cell, and in some embodiments, represents proportionally the total ROSCC level (whether of any single species or combination thereof) in the cell. In embodiments in which a detection agent is used, this refers to the use of a detection agent at a concentration significantly lower than the concentration of ROSCC (whether of any single species or combination thereof) in the cell, such as a detection agent concentration of less than 1 μM, less than 1.5 μM, less than 2.0 μM, less than 5 μM, less than 10 μM, less than 50 μM, 0.01-50 μM, 0.1-25 μM, or 0.5-5 μM. Advantageously, such embodiments avoid altering or substantially altering the total level of ROSCC in cells when identifying the level of ROSCC for purposes of sorting cells into X-CBS cells and / or Y-CBS cells. For completeness, there are detection agents known to have catalytic neutralizing activity against one or more ROSCCs, and thus may be able to alter the level of ROSCC within cells, which may be suitable for use in certain embodiments of the present invention.

[0049] It should be understood that each sperm cell in a plurality of sperm cells in a given sample desirably has its ROSCC level determined using the same methodology to allow for sorting / separation into X-CBS cells and / or Y-CBS cells by comparing ROSCC levels between X-CBS cells and Y-CBS cells in a given sample. In some embodiments, the level of ROSCC is a concentration. In some embodiments, the level of ROSCC is a detectable level of ROSCC, such as a level detectable by a particular technique and, if applicable, a detection agent. In one embodiment, the level of ROSCC is a level of fluorescence intensity, such as a detectable fluorescence intensity. In other embodiments, the level of ROSCC is a level of optical density or absorbance, such as a detectable level of optical density or absorbance. In some embodiments, the level of ROSCC is a relative level of ROSCC.

[0050] In one embodiment, ROSCC is reactive oxidant species. In one embodiment, ROSCC is cellular changes mediated by reactive oxidant species. In one embodiment, ROSCC is endogenous reactive oxidant species and / or cellular changes mediated by endogenous reactive oxidant species. In other embodiments, ROSCC encompasses reactive oxygen species (ROS), reactive nitrogen species (RNS), reactive sulfur species (RSS), ROS-mediated cellular changes, RNS-mediated cellular changes, and / or RSS-mediated cellular changes. In one embodiment, the level of ROSCC is the level of reactive oxygen species (ROS), reactive nitrogen species (RNS), or reactive sulfur species (RSS), or a combination of any two or more thereof. Reactive oxygen species (ROS) include superoxide anion radical (·O2 - ), hydroxyl radical (·OH), hydroperoxyl radical (HO2·), singlet oxygen ( 1 Reactive nitrogen species (RNS) include nitric oxide radicals (NO·), peroxynitrite (ONOO), ozone (O₃), and hydrogen peroxide (H₂O₂). - ) and nitrogen dioxide (NO2). Reactive sulfur species (RSS) include hydrogen sulfide (H2S), low molecular weight thiols (RSH), hydrogen persulfides / polysulfides (H2S n;n≧2), low molecular weight thiol persulfides (RSSH), protein persulfides (PS-SH), various polysulfides (RSS (n) H, RSS (n) R and H2S n ;n>1), sulfenic acid (RSOH), nitrosothiol (RSNO), various sulfide crosslinked compounds (PS-SR, RS-S-SR and RS-S n -SH). In other embodiments, the level of ROSCC is the level of ROS-mediated cellular changes, RNS-mediated cellular changes, and RSS-mediated cellular changes, or a combination of any two or more thereof. In one embodiment, the cellular changes mediated by reactive oxidant species are selected from ROS-induced oxidative changes, RNS-induced oxidative changes, and RSS-induced oxidative changes, or a combination of two or more thereof.

[0051] In one embodiment, the level of reactive oxidant species determined in the methods herein is any suitable reactive oxidant species. In one embodiment, the level of ROSCC is the level of a single reactive oxidant species. In other embodiments, the level of ROSCC is the level of a mixture of two or more reactive oxidant species. In one embodiment, the reactive oxidant species is a radical species. In such embodiments, the radical species is a superoxide anion radical (·O2 - ), hydroxyl radical (·OH), nitric oxide radical (NO·), or hydroperoxyl radical (HO2·). The levels of radical species in sperm cells can be determined using the techniques and, if applicable, detection agents summarized in Tables 1 and 2 below. The radicals can be detected in any suitable component of the sperm cell, such as in the lipid component, protein component, organelle, extracellular component, cytoplasm, or DNA. In one embodiment, the radical species is a primary radical species and is a direct product of cellular metabolic or respiratory activity. In such an embodiment, the primary radical is a superoxide radical ion (·O 2- ), hydroxyl radical (·OH), or nitric oxide radical (NO·). In other embodiments, the active oxidant species is a non-radical species. In such embodiments, the non-radical species is singlet oxygen ( 1O2), ozone (O3), hydrogen peroxide (H2O2), hypochlorous acid (HOCl) or peroxynitrite (ONOO - ). The levels of non-radical species in sperm cells can be determined using the techniques and, if applicable, detection agents summarized in Tables 1 and 2 below. Radicals can be detected in any suitable component of the sperm cell, such as in the lipid component, protein component, organelle, extracellular component, cytoplasm, or DNA. In one embodiment, the reactive oxidant species is a reaction product of the primary radical with another radical, ion, or molecule. In such an embodiment, the reaction product can be hydroperoxyl radical (HO2·), peroxynitrite (ONOO), or hydroxybenzoate (H2O). - ), hypochlorous acid (HOCl), or hydrogen peroxide (HO). In one embodiment, ROSCC is a reaction product of ROS·O 2- and peroxynitrite (ONOO), a reaction product of RNS with NO. - In one embodiment, the ROSCC is a reaction product of two ROS, RNS, or RSS, such as superoxide, hydroxyl radical, and / or hydrogen peroxide. Tables 1 and 2: Tables of selected techniques and detection agents suitable for use in identifying levels of ROSCC by the methods described herein. [Table 1] [Table 2]

[0052] In another embodiment, ROSCC is a cellular change mediated by reactive oxidant species. The cellular change mediated by reactive oxidant species can be any suitable cellular change. "Cellular change" refers to any change, but generally refers to a chemical change that occurs within or on a cell or any cellular component thereof that is mediated by reactive oxidant species. "Mediated" means that the reactive oxidant species participates in a cellular change pathway in a manner that contributes to initiating or bringing about the cellular change, optionally through the formation of secondary or higher order reactive by-products.

[0053] In one embodiment, the cellular changes are oxidative and / or nitrosative damage. In one embodiment, ROSCC is a cellular change mediated by reactive oxidant species, and the cellular changes are lipid peroxidation, protein (amino acid) oxidation, and DNA oxidation. In one embodiment, ROSCC is a cellular change mediated by reactive oxidant species, and the cellular change is lipid peroxidation. Lipid peroxidation refers to a process in which reactive species, particularly ·OH and HO· radicals, attack carbon-carbon double bond-containing lipids (L), thereby inserting oxygen into the lipid chain and resulting in the formation of lipid peroxyl radicals (LOO·) and hydroperoxides (L-OOH). Lipid peroxyl radicals and hydroperoxides can be identified in sperm using any suitable method known to those skilled in the art. One or more suitable techniques or agents are listed in Tables 1 and 2 above.

[0054] In another embodiment, ROSCC is a cellular change mediated by reactive oxidant species, and the cellular change is DNA oxidation. DNA oxidation can take several forms, but can occur as hydrogen abstraction from the deoxyribose sugar backbone and / or bases, base oxidation, and strand scission, often involving ·OH and O2. The level of DNA oxidation can be determined in sperm using any suitable method known to those of skill in the art. One or more suitable techniques or agents are listed in Tables 1 and 2 above.

[0055] In a further embodiment, ROSCC is a cellular change mediated by reactive oxidant species, and the cellular change is protein oxidation. In one embodiment, protein oxidation consists of oxidation of one or more amino acids comprising the protein, either in their side chains or backbone. In one embodiment, protein oxidation is amino acid carbonylation. Protein carbonylation occurs when reactive oxidant species, in the presence of transition metals, attack the amino acid side chains of proline, arginine, lysine, and threonine to form reactive ketones or aldehydes that can then further react with other amino acid residues. In another embodiment, protein oxidation is thiol oxidation. Thiol oxidation occurs when thiol (-SH) side chains in amino acid residues such as cysteine ​​and methionine are oxidized to form disulfide bridges (-SS-). Proteins in sperm that contain thiol groups and are therefore susceptible to thiol oxidation include enzymes, antioxidant molecules, and structural proteins in sperm axons. In one embodiment, protein oxidation is amino acid nitration, such as tyrosine nitration. Other forms of protein oxidation suitable for use in the methods of the present invention are known to those of skill in the art, such as tyrosine nitration, S-nitrosylation, S-glutathionylation, 4-HNE protein adducts, etc. The level of protein oxidation can be determined in sperm using any suitable method known to those of skill in the art. One or more suitable techniques or agents are set forth in Tables 1 and 2 above.

[0056] In one embodiment, ROSCC is a combination of two or more specific reactive oxidant species, as described above, a combination of two or more cellular changes mediated by such reactive oxidant species, or a combination of one or more specific reactive oxidant species and one or more cellular changes. In embodiments in which ROSCC consists of a combination of species and / or cellular changes, the level of ROSCC can be the sum of all species / cellular changes measured. In some embodiments, the specific ROSCC identified in the methods herein will be determined by the choice of instrument and, if applicable, detection agent. By way of example only, when a fluorescent dye such as MitoSOX® (Invitrogen) is used in a flow cytometry system, the level of ROSCC can be the level of mitochondrial superoxide, quantified by fluorescence intensity.

[0057] The source of ROSCC whose levels are identified in sperm cells by the methods described herein is not particularly limited, as described above. ROSCC is endogenous ROSCC generated by cellular processes such as respiration and metabolism, and can arise from the activity of mitochondria in sperm cells, enzymes such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), xanthine oxidase (XO), nitric oxide synthase (NOS), or cytochrome P450, or the mitochondrial electron transport chain (ETC).

[0058] In one embodiment, ROSCC is detectable in a sperm cell component selected from lipid components, protein components, organelles, extracellular components, mitochondria, cytoplasm, and DNA, or a combination of any two or more of these components. In one embodiment, ROSCC is selectively detectable in a sperm cell component selected from lipid components, protein components, organelles, extracellular components, mitochondria, cytoplasm, and DNA. In one embodiment, ROSCC is detectable in two or more sperm cell components selected from lipid components, protein components, organelles, extracellular components, mitochondria, cytoplasm, and DNA.

[0059] In one embodiment, ROSCC is a direct or indirect product of mitochondrial activity in sperm cells. In some embodiments, a detection agent selective for ROSCC generated by mitochondria in sperm cells is used. Non-limiting examples of suitable measurement techniques and, if appropriate, detection agents that can identify ROSCC generated by mitochondria are summarized in Tables 1 and 2.

[0060] In one embodiment, the level of ROSCC corresponds to the level of ROSCC detectable within a sperm cell organelle. In one embodiment, the level of ROSCC corresponds to the level of ROSCC detectable within the mitochondria of the sperm cell. Because mitochondria are located in the sperm midpiece, embodiments that determine the level of reactive oxidant species within or produced by mitochondria may be less susceptible to sperm orientation effects caused by mutual interference between sperm heads and, therefore, may advantageously allow easier and / or more accurate selection than the level of ROSCC detectable in the sperm head. In some embodiments, a detection agent selective for ROSCC within the mitochondria of sperm cells is used. Non-limiting examples of suitable measurement techniques and, if appropriate, detection agents that can determine the level of ROSCC within organelles such as mitochondria are summarized in Tables 1 and 2, although other suitable techniques and / or agents may be known to those of skill in the art. In one embodiment, the level of ROSCC corresponds to the level of ROSCC detectable within the sperm midpiece.

[0061] In other embodiments, the level of ROSCC corresponds to the level of ROSCC detectable in the cytoplasm of sperm cells. In some embodiments, a detection agent selective for ROSCC in the cytoplasm of sperm cells is used. Non-limiting examples of suitable measurement techniques and, if appropriate, detection agents that can identify ROSCC levels in the cytoplasm of sperm cells are summarized in Tables 1 and 2, although other suitable techniques and / or agents may be known to those of skill in the art.

[0062] In other embodiments, the level of ROSCC corresponds to the level of ROSCC detectable in the lipid component of sperm cells. In some embodiments, the lipid component includes the cellular membrane and the mitochondrial membrane. In some embodiments, a detection agent selective for ROSCC in the lipid component of sperm cells is used, such that it is selective for oxidative changes in the cellular membrane and / or the mitochondrial membrane. Non-limiting examples of suitable measurement techniques and, if appropriate, detection agents that can identify ROSCC levels in the lipid component of sperm cells are summarized in Tables 1 and 2, although other suitable techniques and / or agents may be known to those of skill in the art.

[0063] In further embodiments, the level of ROSCC corresponds to the level of ROSCC detectable in the protein component of the sperm cell. In some embodiments, the protein component comprises an enzyme. In some embodiments, the protein component may be a protein component of chromatin. In some embodiments, a detection agent is used that is selective for ROSCC in the protein component of the sperm cell, such as being selective for protein oxidation, such as protein carbonylation. Non-limiting examples of suitable measurement techniques and, if appropriate, detection agents that can identify ROSCC levels in the protein component of the sperm cell are summarized in Tables 1 and 2, although other suitable techniques and / or agents may be known to those of skill in the art.

[0064] In yet other embodiments, the level of ROSCC corresponds to the level of ROSCC detectable in the extracellular compartment of sperm cells. The extracellular compartment may refer to the extracellular space immediately surrounding the sperm cells. In some embodiments, extracellular ROSCC results from the activity of a protein component of the sperm cell that is embedded within the cell membrane and faces outward from the cell membrane. In one embodiment, the protein component of the sperm cell embedded in the cell membrane is a cell surface enzyme, such as NOX. Thus, in such embodiments, ROSCC is detected extracellularly rather than in the intracellular space. In some embodiments, a detection agent selective for ROSCC in the extracellular compartment of sperm cells is used. Non-limiting examples of suitable measurement techniques and, if appropriate, detection agents that can identify ROSCC levels in the extracellular compartment of sperm cells are summarized in Tables 1 and 2 and include WST-1 dye, which contains a lipid linker that anchors it in the cell membrane, although other suitable techniques and / or agents may be known to those of skill in the art.

[0065] In further embodiments, the level of ROSCC corresponds to the level of ROSCC detectable in the DNA of sperm cells. In some embodiments, a detection agent selective for ROSCC in the DNA of sperm cells is used, such as one that is selective for hydrogen abstraction or oxidation of bases and / or deoxyribose sugar moieties. Non-limiting examples of suitable measurement techniques and, if appropriate, detection agents that can identify ROSCC levels in the DNA of sperm cells are summarized in Tables 1 and 2, although other suitable techniques and / or agents may be known to those of skill in the art.

[0066] In one embodiment, determining the level of ROSCC comprises treating a plurality of sperm cells with a detection agent that detects ROS, RNS, and / or RSS in at least the cytoplasm of the sperm cells. In another embodiment, determining the level of ROSCC by the methods herein comprises treating a plurality of sperm cells with a ROSCC detection agent that detects ROS-, RNS-, and / or RSS-mediated cellular changes in lipid components of the sperm cells.

[0067] Those skilled in the art will readily appreciate that ROSCC and corresponding measurement techniques, including applicable detection agents, can be identified as suitable for use in identifying sperm cells as X-CBS cells and Y-CBS cells and / or sorting sperm cells into X-CBS cells and Y-CBS cells, or in generating samples enriched in X-CBS cells or Y-CBS cells, by confirming that the determined levels of ROSCC are detectably different between, or at least a portion of, X-CBS cells and Y-CBS cells. In some embodiments, ROSCC and corresponding measurement techniques, including applicable detection agents, can be selected such that the determined levels of ROSCC in X-CBS cells and Y-CBS cells in a sample do not overlap. In other embodiments, ROSCC and corresponding measurement techniques, including applicable detection agents, can be identified as suitable for use in identifying sperm cells as X-CBS cells and Y-CBS cells and / or sorting sperm cells into X-CBS cells and Y-CBS cells, or in generating samples enriched in X-CBS cells or Y-CBS cells, by confirming that the specified level of ROSCC is sufficiently different to distinguish the subpopulation of X-CBS cells from the subpopulation of Y-CBS cells. Generally, the subpopulations of X-CBS cells and Y-CBS cells form only a portion of the plurality of sperm cells originally designated for identification, sorting, or enrichment. In such embodiments, ROSCC and corresponding measurement techniques, including applicable detection agents, can be selected in the following manner. In this embodiment, there is some overlap in the levels of ROSCC between some X-CBS cells and some Y-CBS cells in a sample, but the overlap only affects a sufficiently small portion of the sample to allow for the separate identification, separation and / or collection of X-CBS cells and / or Y-CBS cells in non-overlapping regions through processing such as gating, or to allow for the selective induction of motility, infertility or non-viability in either the X-CBS cells or the Y-CBS cells identified in the sperm sample.

[0068] In this regard, it should be understood that a combination of measurement techniques and, if appropriate, detection agents can be used in the methods described herein to identify and / or sort sperm. In one embodiment, two or more detection agents are used simultaneously or sequentially to identify ROSCC levels and thus identify and / or sort sperm in the methods herein. In other embodiments, two or more techniques and, optionally, one or more detection agents are used sequentially to identify ROSCC levels and thus identify and / or sort sperm in the methods herein.

[0069] As is apparent from the range of techniques listed in Tables 1 and 2, the means for determining the level of ROSCC in sperm cells are not particularly limited in the methods herein. In some embodiments, the level of ROSCC is measured by spectroscopic means. Spectroscopic means may include fluorescence spectroscopy and photometry and related techniques, optionally combined with other imaging techniques such as microscopy or tomography. In some embodiments, the spectroscopic means is combined with techniques that aid in visualizing, processing, and / or aligning the cells, such as image cytometry, flow cytometry, or microfluidics. Other means for measuring ROSCC levels will be known to those skilled in the art, including, but not limited to, measuring physiological indicators of ROSCC levels, such as sperm motility.

[0070] In some embodiments, the methods herein comprise identifying a level of ROSCC in each of a plurality of sperm cells, where the identifying step comprises treating the plurality of sperm cells with a ROSCC detection agent. The ROSCC detection agent may detect reactive oxidant species, cellular changes mediated by reactive oxidant species, or a combination of the two. In one embodiment, the detection agent comprises a dye. In some embodiments, the detection agent, optionally a dye, directly detects ROSCC, e.g., interacts with ROSCC, to produce a detectable response (e.g., a visible response) indicative of the presence and quantity of one or more ROSCC. In some embodiments, light, illumination, or other stimulus may be required to activate the detectable response. In one embodiment, the detection agent is photoactivated, e.g., activated to produce a detectable signal. In one embodiment, the detection agent used herein is activated by visible light, e.g., having a wavelength in the range of 400 nm to 700 nm, to produce a detectable signal. Examples of suitable detection agents activated by visible light include the fluorescent dyes MitoSOX® (Invitrogen®) and CellROX® deep red (Invitrogen®). In some embodiments, the detection agent used herein is activated by ultraviolet light to produce a detectable signal, such as one having a wavelength of 100 nm to 400 nm, 200 nm to 400 nm, or 300 nm to 400 nm. An example of a suitable detection agent activated by ultraviolet light includes luminol, which has an excitation wavelength of 355 nm and an emission wavelength of 411 nm. In some embodiments, the detection agent used herein is not activated by ultraviolet light to produce a detectable signal. In one embodiment, the detection agent is a naturally occurring antioxidant compound.

[0071] In some embodiments, the detecting agent is a positive detecting agent, i.e., the agent gives a positive signal, such as fluorescence, in the presence of ROSCC. Examples of such detecting agents include MitoSOX® and CellROX® deep red (commercially available from Invitrogen), both of which fluoresce in the presence of specific reactive oxidant species in response to excitation with visible light. Such detecting agents are sometimes referred to as "activated" detecting agents because they are activated and fluoresce in the presence of ROSCC. In other embodiments, the detecting agent is a negative detecting agent, i.e., its signal remains detectable until it is abolished in the presence of ROSCC. Examples of such detecting agents include nitroxide dye conjugates (see Morrow et al., Free Radic Biol Med. 2010 Jul. 1;49(1):67-76) and beta-carotene. Such detecting agents are sometimes referred to as "inactivated" detecting agents because their fluorescence is inactivated in the presence of ROSCC. In some embodiments, the negative detecting agent is a naturally occurring antioxidant present in sperm. In such embodiments, the negative detector agent becomes non-fluorescent upon oxidation, such as in the presence of ROSCC. It should be understood that the negative detector agent must be excited by light, such as visible or ultraviolet light, to observe fluorescence and thus loss of fluorescence in response to oxidation, such as in the presence of ROSCC.

[0072] Because sperm cells with levels of reactive oxidant species outside the biologically ideal range are infertile and because extremely high levels of reactive oxidant species contribute to sperm cell death, in certain embodiments, the methods herein employ detection agents that neutralize reactive oxidant species, i.e., chemically remove the reactive radical and / or oxidant properties of the active oxidant species. Illustratively, detection agents may be selected that generate a detectable signal, in one embodiment, fluorescence, activated upon neutralization of the active oxidant species. Examples of such detection agents include MitoSOX® and CellROX® deep red. In such embodiments, the methods herein may advantageously, in some embodiments, reduce the concentration of reactive oxidant species in sorted sperm, thereby improving sperm health.

[0073] In one embodiment, the detection agent comprises a fluorescent dye. In some embodiments, the fluorescence is activated by reaction with, such as neutralization of, one or more ROSCC in the cell. In other embodiments, the fluorescence is quenched by reaction with, such as neutralization of, one or more ROSCC in the cell. Examples of fluorescent dyes suitable for use as detection agents herein include MitoSOX® and CellROX® deep red.

[0074] In one embodiment, the detection agent produces a detectable signal sufficient to identify or sort sperm as X-CBS cells or Y-CBS cells after incubation with the sperm for a period of less than 90 minutes, such as less than 75 minutes, less than 60 minutes, less than 45 minutes, less than 30 minutes, or less than 15 minutes, or for a period of 5 to 90 minutes, 10 to 75 minutes, 10 to 30 minutes, 15 to 30 minutes, or 30 to 60 minutes.

[0075] In one embodiment, the detection agent is selective for the target ROSCC. In some embodiments, the target ROSCC is predetermined for sperm of a particular species or sperm sampled from a particular region of the male reproductive tract, as a ROSCC for which sperm cell identification or sorting is optimized, such as by maximizing a predetermined difference in target ROSCC levels between X-CBS and Y-CBS cells and / or minimizing overlap between ROSCC levels in X-CBS and Y-CBS cell subpopulations. Examples of such detection agents include MitoSOX®, which is highly selective for mitochondrial superoxide.

[0076] The identifying or sorting step in the methods described herein is by the level of ROSCC in the sperm cells. In one embodiment, the X-CBS cells have a specified level of ROSCC that is detectably different from the level of ROSCC in the Y-CBS cells, and therefore the identifying or sorting step in the methods described herein can comprise identifying or sorting to X-CBS cells that have a specified level of ROSCC that is detectably different compared to the Y-CBS cells. In one embodiment, the specified level of ROSCC in each X-CBS cell differs from the specified level of ROSCC in each Y-CBS cell by at least 5%, at least 10%, at least 20%, at least 40%, at least 50%, at least 60%, at least 75%, at least 100%, at least 200%, or at least 300% or more, or by between 5% and 500%, between 100% and 300%, between 25% and 100%, or between 250% and 400%. In other embodiments, the average level of ROSCC identified for all cells in the X-CBS subpopulation differs from the average level of ROSCC identified for all cells in the Y-CBS subpopulation by at least 5%, at least 10%, at least 20%, at least 40%, at least 50%, at least 60%, at least 75%, at least 100%, at least 200%, or at least 300% or more, or by between 5% and 500%, between 100% and 300%, between 25% and 100%, or between 250% and 400%.

[0077] In one embodiment, the detectably different specified level is a detectably higher level of ROSCC in the X-CBS cells compared to the Y-CBS cells. In embodiments in which the X-CBS cells have a higher level of ROSCC than the Y-CBS cells, the methods herein can include identifying at least a portion of the plurality of sperm cells as X-CBS cells, each having a specified level of ROSCC that is higher than the level of ROSCC in each of the Y-CBS cells, or sorting at least a portion of the plurality of sperm cells into X-CBS cells, each having a specified level of ROSCC that is higher than the level of ROSCC in each of the Y-CBS cells. In one embodiment, each X-CBS cell has a specified level of ROSCC that is at least 5% higher, at least 10% higher, at least 20% higher, at least 40% higher, at least 50% higher, at least 75% higher, at least 100% higher, at least 200% higher, at least 300% higher, at least 400% higher, at least 500% higher, at least 600% higher, at least 700% higher, or at least 800% or more higher than each Y-CBS cell, or that is 5% to 800% higher, 100% to 300% higher, 25% to 100% higher, 250% to 400% higher, 100% to 800% higher, or 500% to 800% higher than each Y-CBS cell. % higher" is [(ROSCC level X-CBS -ROSCC level Y-CBS ) / ROSCC level Y-CBS ] x 100 It is measured as:

[0078] In other embodiments, the average level of ROSCC determined for all cells in the X-CBS is at least 5%, at least 10%, at least 20%, at least 40%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, or at least 800% or more higher than the average level of ROSCC determined for all cells in the Y-CBS, or is 5% to 500%, 100% to 300%, 25% to 100%, 250% to 400%, 100% to 800%, or 500% to 800% higher than the average level of ROSCC determined for all cells in the Y-CBS.

[0079] As used herein, the term "identifying" refers to a process in which specific cells are distinguished from others in a sample. As used herein, the term "sorting" refers to a process in which specific cells are identified or selected and then separated from others in a sample. In the present disclosure, the basis for identification or selection is a specified level of ROSCC. In one embodiment, the methods herein comprise identifying a portion of the plurality of cells as X-CBS cells, such as an X-CBS cell subpopulation, or sorting a portion of the plurality of cells into X-CBS cells, such as an X-CBS cell subpopulation. In such embodiments, the plurality of cells that are not identified or sorted into an X-CBS cell subpopulation are further identified, sorted, or discarded. In other embodiments, the methods herein comprise identifying a portion of the plurality of cells as Y-CBS cells, such as a Y-CBS cell subpopulation, or sorting a portion of the plurality of cells into Y-CBS cells, such as a Y-CBS cell subpopulation. In such embodiments, cells of the plurality that are not identified or sorted into a Y-CBS cell subpopulation are to be further identified, sorted, or discarded. In one embodiment, a portion of the cells of the plurality are identified or sorted into X-CBS cells, and a distinct portion of the same plurality are identified or sorted into Y-CBS cells, sorted into distinct X-CBS subpopulations and distinct Y-CBS subpopulations, etc. In such embodiments, cells of the plurality that are not identified or sorted into X-CBS or Y-CBS cell subpopulations are to be further identified, sorted, or discarded. In one embodiment, all or substantially all cells of the plurality are identified or sorted into X-CBS or Y-CBS cells, e.g., up to 99%, up to 98%, or up to 95% of the cells are sorted into X-CBS or Y-CBS cells.

[0080] In one embodiment, the level of ROSCC is determined in the methods herein using a flow cytometer. In one embodiment, the level of ROSCC is determined in a microfluidic device. In one embodiment, the level of ROSCC is determined based on a physiological response of sperm cells, such as sperm motility. In one embodiment, the level of ROSCC is determined in situ in a flow cytometer or microfluidic device prior to sorting, such as immediately prior to sorting, or to activate sorting. In one embodiment, the sorting step is performed in a flow cytometric sorter. In such embodiments, X-CBS cells and Y-CBS cells can be optionally sorted by gating, such as gating on X-CBS cell subpopulations and Y-CBS cell subpopulations, each having different levels of ROSCC. In one embodiment, sperm are sorted based on the average level of ROSCC of a group of cells, such as the average level of ROSCC for a cell subpopulation. In another embodiment, the sorting step is performed in a microfluidic sorting device. In other embodiments, the step of enriching or isolating at least a portion of the identified X-CBS cells or Y-CBS cells from the starting sperm sample to produce a concentrated sperm sample is performed using a microfluidic sorting device.

[0081] Those skilled in the art will be familiar with the operation and function of flow cytometers and methods that can be employed in the methods of the present disclosure. Briefly, a flow cytometer has a tube through which a stream of aqueous solution in which cells are suspended flows. The cells flow through the aqueous stream until they reach a laser and detector. At that point, the instrument delivers and presents to the detector either a slow continuous stream or drops of solution containing isolated cells whose scatter or fluorescence signal can be measured. The signal can then be used to activate a sorting mechanism, such as the application of an electric charge or a magnet, to separate the cells into different collection vessels.

[0082] Those skilled in the art will be familiar with the operation and function of microfluidic devices and methods that can be employed in the methods of the present disclosure. Briefly, microfluidic devices utilize patterns of microchannels molded or imprinted into a chip. The microchannels are connected to larger cavities through which fluids can be injected and / or ejected from the chip. By adjusting the microchannel pattern and / or the characteristics of the flow into or out of the cavities, cells can be directed into specific paths for sorting and collection or disposal. Examples of microfluidic devices suitable for use in the methods described herein can include the Wolf® and Wolf G2® cell sorters manufactured by NanoCellect Biological, Inc.

[0083] The methods described herein may further comprise separately providing the identified or selected X-CBS cells and / or Y-CBS cells in enriched or purified form. Naturally occurring sperm samples prior to sorting typically consist of, but are not limited to, approximately 50% X-CBS cells and 50% Y-CBS cells. Other variations in the ratio of X-CBS:Y-CBS sperm cells prior to sorting by the methods herein are described above.

[0084] In one embodiment, enriched sperm populations produced by certain methods described herein contain a relatively large population of either X-CBS cells or Y-CBS cells in the sorted sperm relative to the sperm population before sorting (also referred to herein as the starting sperm sample or simply the sperm sample). In other embodiments, enriched sperm populations produced by certain methods described herein may contain 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more X-CBS cells or Y-CBS cells by number as a percentage of the total cell content of the sample. In one embodiment, a purified sperm population consists of either only X-CBS cells or only Y-CBS cells.

[0085] In one embodiment, the methods described herein may further comprise separately providing, after either identification or sorting, the X-CBS cells and / or Y-CBS cells in enriched or purified form, having a minimum purity of at least 90% X-CBS cells or at least 90% Y-CBS cells, such as a minimum purity of at least 95% X-CBS cells or at least 95% Y-CBS cells, or at least 99% X-CBS cells or at least 95% Y-CBS cells. In some embodiments, these levels of enrichment or purity are achieved using a detection agent activated by visible light, a detection agent that neutralizes reactive oxidant species, and / or under conditions in which the detection agent is incubated with the sperm for a period of less than 90 minutes, such as less than 75 minutes, less than 60 minutes, less than 45 minutes, less than 30 minutes, or less than 15 minutes, or for a period of 5-90 minutes, 10-75 minutes, 10-30 minutes, 15-30 minutes, or 30-60 minutes. In one embodiment, the method herein further comprises separately providing the X-CBS cells and / or Y-CBS cells in a cryopreserved form after identification or selection. Methods for cryopreserving sperm will be well known to those skilled in the art.

[0086] In one embodiment, the method for sorting sperm cells described herein is a method for sex-sorting sperm cells. In another embodiment, the method for sorting sperm cells described herein is a sperm sorting method. In one embodiment, the method for sorting sperm cells described herein is a method for separating sperm cells into X-CBS cells and / or Y-CBS cells. In another embodiment, the method for sorting sperm cells described herein is a sperm sorting method for separating sperm cells into X-CBS cells and / or Y-CBS cells. In one embodiment, the method for sorting sperm cells described herein is a method for enriching the concentration of X-CBS cells or Y-CBS cells in a sperm cell sample. In another embodiment, the method for sorting sperm cells described herein is a method for purifying X-CBS cells or Y-CBS cells in a sperm sample.

[0087] As is apparent from the above, the present disclosure also relates to enriched or purified populations of X-CBS cells produced by identifying or sorting sperm cells by the methods described herein. In some embodiments, the enriched or purified populations have a minimum purity of at least 75% X-CBS cells as a percentage of the total cell population of the enriched sample, such as a minimum purity of at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%. The present disclosure also relates to enriched or purified populations of Y-CBS cells produced by identifying or sorting sperm cells by the methods described herein. In some embodiments, the enriched or purified populations have a minimum purity of at least 75% Y-CBS cells as a percentage of the total cell population of the enriched sample, such as a minimum purity of at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%.

[0088] In another aspect, described herein is a method for identifying X-chromosome-bearing sperm (X-CBS) cells or Y-chromosome-bearing sperm (Y-CBS) cells in a sperm sample, the method comprising: determining a level of reactive oxidant species and / or cellular changes mediated thereby (ROSCC) in each of a plurality of sperm cells in a starting sperm sample; and identifying at least a portion of the plurality of sperm cells in the starting sperm sample as X-CBS cells or Y-CBS cells based on the determined ROSCC level.

[0089] In one embodiment, the starting sperm sample includes a mixture of X-CBS cells and Y-CBS cells, such as a mixture as described above for "plurality of sperm." Other terms, such as "identify," "level," "ROSCC," and "at least a portion," are as described above. The method may include isolating at least a portion of the identified X-CBS cells or Y-CBS cells from the starting sperm sample to generate a concentrated sperm sample containing at least a portion of the identified and isolated X-CBS cells or Y-CBS cells. The concentrated sperm sample may contain at least 90% X-CBS cells or at least 99% X-CBS cells, or at least 90% Y-CBS cells or at least 99% Y-CBS cells. The isolating step may include using a flow cytometry sorter, optionally with gating, or a microfluidic device. Alternatively, the method may further comprise inducing selective motility, infertility, or non-viability in either the identified X-CBS cells or Y-CBS cells in the starting sperm sample to produce a sperm sample with altered motility, fertility, or viability. Inducing selective motility or infertility can take the form of inducing non-motility or inducing incapacitation in either the identified X-CBS cells or Y-CBS cells, effectively rendering that portion of the sperm sample unable to produce progeny without requiring physical separation / removal of unwanted CBS cells. Inducing selective infertility in the form of non-viability can take the form of inducing cell death in the identified X-CBS cells or Y-CBS cells. In one embodiment, inducing selective infertility or non-viability utilizes a difference in the identified level of one or more ROSCCs between the identified X-CBS cells and Y-CBS cells.

[0090] The present disclosure also provides for the use of X-CBS cells or Y-CBS cells identified or selected by the methods described herein, or the use of samples enriched in X-CBS cells or Y-CBS cells produced by the methods herein, in assisted reproductive technologies. Assisted reproductive technologies can include any experimental or clinical technique applied to isolated gametes (eggs or sperm) for the purpose of reproduction. Such techniques include in vitro insemination (IVF: aspiration of eggs, insemination in a laboratory, and transfer of the embryo into a recipient), artificial insemination (AI: placement of sperm into the pelvic cavity, such as the cervix or uterine cavity), gamete intrafallopian transfer (GIFT: placement of eggs and sperm into the fallopian tubes), zygote intrafallopian transfer (ZIFT: placement of fertilized eggs into the fallopian tubes), tubal embryo transfer (TET: placement of cleaved embryos into the fallopian tubes), peritoneal egg and sperm transfer (POST: placement of eggs and sperm into the pelvic cavity), intracytoplasmic sperm injection (ICSI), intratesticular sperm extraction (TESE), and microsurgical epididymal sperm aspiration (MESA), or any other in vitro technique for generating embryos in humans and / or animals, such as nuclear transfer, parthenogenetic activation, embryonic stem cell production, and the use of totipotent cells. In one embodiment, the assisted reproductive technique is in vitro insemination. In another embodiment, the assisted reproductive technique is artificial insemination. In some embodiments, sperm provided for use in assisted reproductive technologies may have a minimum concentration or purity that may be at least 90% X-CBS (or Y-CBS), at least 95% X-CBS (or Y-CBS), or at least 99% X-CBS (or Y-CBS), etc. Those skilled in the art will be familiar with the preparations necessary to provide sperm selected by the methods described herein in a form suitable for use in assisted reproductive technologies.

[0091] In one embodiment, the present disclosure relates to a non-human mammalian subject produced from sperm identified, selected, or enriched by the methods described herein, such as from a selected or enriched population of X-CBS cells or Y-CBS cells.

[0092] Also disclosed herein is a sperm cell sorting system, comprising: a reactive oxidant species and / or cellular transformation (ROSCC) detecting agent, wherein the cellular transformation is mediated by reactive oxidant species; and instructions for using the ROSCC detecting agent to identify a level of ROSCC in each of a plurality of sperm cells and sorting at least a portion of the plurality of sperm cells into X-chromosome-bearing sperm (X-CBS) cells and / or Y-chromosome-bearing sperm (Y-CBS) cells based on the identified ROSCC level. In one embodiment, the system further comprises a sperm cell incubation solution for incubating the sperm cells with the ROSCC detecting agent. The sperm cell sorting system may be in the form of a kit, such as a box, container, bag, etc., in which the ROSCC detecting agent and, optionally, the sperm cell incubation solution are provided in separate, sealed containers. In some embodiments, the system further comprises one or more components necessary for sorting the sperm after incubation with the ROSCC detecting agent. In one embodiment, the system includes a microfluidic sorting device for sorting sperm adapted for use with the detection agent included in the system. In one embodiment, the microfluidic sorting device is reusable. The detection agent and ROSCC in the system are as described above.

[0093] In one embodiment, the system comprises two or more separately packaged detection agents, such as two, three, four, five, ten, or twenty. In one embodiment, the system comprises two or more separately packaged incubation solutions, such as two, three, four, five, ten, or twenty. In one embodiment, the detection agents and incubation solutions are the same. In one embodiment, the detection agents and / or incubation solutions are included in the system in a ready-to-use form. In other embodiments, the detection agents and / or incubation solutions are provided in the system in a form that requires reconstitution or dilution before use. A system comprising two or more detection agents and optionally two or more incubation solutions may advantageously be packaged with a reusable microfluidic device adapted for use with the detection agents and incubation solutions included in the system, such that a single system may analyze two or more different sperm samples at different times. In one embodiment, the detection agents and incubation solutions are different and complementary. In such embodiments, two or more different microfluidic sorting devices may be included in the system. Other components of the kit may include storage solutions for the sorted or concentrated sperm, and devices to assist in the administration of the sorted or concentrated sperm in assisted reproductive techniques.

[0094] Any numerical range recited herein should be understood to include all subranges subsumed therein, for example, a range "from x to y" or "between x and y" includes all subranges between x and y, as well as ranges having endpoints x and y.

[0095] As used herein, the singular forms "a," "an," and "the" may refer to plural items unless the context clearly indicates otherwise.

[0096] Embodiment The following embodiments constitute a part of the present disclosure.

[0097] Embodiment 1 1. A method for sorting sperm cells, comprising: identifying a level of reactive oxidant species and / or cellular changes mediated thereby (ROSCC) in each of the plurality of sperm cells; sorting at least a portion of the plurality of sperm cells into X-chromosome-bearing sperm (X-CBS) cells and / or Y-chromosome-bearing sperm (Y-CBS) cells based on the determined ROSCC level; A method for providing the above.

[0098] Embodiment 2 2. The method of embodiment 1, comprising sorting at least a portion of the plurality of sperm cells into X-CBS cells and Y-CBS cells.

[0099] Embodiment 3 ROSCC is reactive oxidant species selected from reactive oxygen species (ROS), reactive nitrogen species (RNS) and reactive sulfur species (RSS), or a combination of two or more thereof; and / or Cellular changes mediated by reactive oxidant species selected from ROS-induced oxidative changes, RNS-induced oxidative changes, and RSS-induced oxidative changes, or a combination of two or more thereof. 3. The method of embodiment 1 or embodiment 2, wherein the

[0100] Embodiment 4 4. The method of any one of embodiments 1 to 3, wherein identifying the level of ROSCC comprises treating the plurality of sperm cells with a ROSCC detection agent.

[0101] Embodiment 5 5. The method of any one of embodiments 1 to 4, wherein identifying the level of ROSCC comprises treating the plurality of sperm cells with a ROSCC detection agent comprising a dye.

[0102] Embodiment 6 6. The method of any one of embodiments 1 to 5, wherein identifying the level of ROSCC comprises treating the plurality of sperm cells with a ROSCC detection agent comprising a fluorescent dye, optionally wherein the fluorescence is activated by reaction with one or more ROSCC in the cells.

[0103] Embodiment 7 7. The method of any one of embodiments 1 to 6, wherein the ROSCC is detectable in a component of the sperm cell selected from a lipid component such as the cell membrane or mitochondrial membrane, a protein component such as chromatin or enzymes, an organelle such as mitochondria, an extracellular component such as a cell surface enzyme, cytoplasm and DNA, or a combination of any two or more of these components.

[0104] Embodiment 8 8. The method of any one of embodiments 1 to 7, wherein identifying the level of ROSCC comprises treating the plurality of sperm cells with a ROSCC detection agent selective for the target ROSCC.

[0105] Embodiment 9 9. The method of any one of embodiments 1 to 8, wherein the ROSCC is a reactive oxidant species, and the reactive oxidant species is a ROS selected from superoxide, hydroxyl radical, and hydrogen peroxide.

[0106] Embodiment 10 10. The method of any one of embodiments 1 to 9, wherein the step of identifying the level of ROSCC comprises treating the plurality of sperm cells with a detection agent that detects ROS, RNS and / or RSS in at least the cytoplasm of the sperm cells.

[0107] Embodiment 11 9. The method of any one of embodiments 1 to 8, wherein ROSCC is a cellular change mediated by reactive oxidant species, and the cellular change is lipid peroxidation.

[0108] Embodiment 12 12. The method of any one of embodiments 1 to 8 or 11, wherein identifying the level of ROSCC comprises treating the plurality of sperm cells with a ROSCC detection agent that detects cellular changes mediated by reactive oxidant species, wherein the cellular changes are in lipid components of the sperm cells.

[0109] Embodiment 13 13. The method of any one of embodiments 1 to 12, wherein the level of ROSCC identified in each X-CBS cell differs from the level of ROSCC identified in each Y-CBS cell by at least 5%, at least 20%, at least 40%, or at least 60%.

[0110] Embodiment 14 14. The method of any one of embodiments 1 to 13, wherein the level of ROSCC identified in each X-CBS cell is at least 5%, at least 20%, at least 40%, or at least 60% higher than the level of ROSCC identified in each Y-CBS cell.

[0111] Embodiment 15 13. The method of any one of embodiments 1 to 12, wherein the average level of ROSCC identified for all cells in the X-CBS is at least 5%, at least 20%, at least 40%, or at least 60% different from the average level of ROSCC identified for all cells in the Y-CBS.

[0112] Embodiment 16 16. The method of any one of embodiments 1 to 12 or 15, wherein the average level of ROSCC identified for all cells in the X-CBS is at least 5%, at least 20%, at least 40%, or at least 60% higher than the average level of ROSCC identified for all cells in the Y-CBS.

[0113] Embodiment 17 17. The method of any one of embodiments 1 to 16, wherein the plurality of sperm cells is obtained from the proximal cauda epididymis, distal cauda epididymis, vas deferens or ejaculate of the subject.

[0114] Embodiment 18 18. The method of any one of embodiments 1 to 17, wherein the plurality of sperm cells is obtained from a mammalian subject.

[0115] Embodiment 19 19. The method of any one of embodiments 1 to 18, wherein the plurality of sperm cells is obtained from a non-human mammalian subject.

[0116] Embodiment 20 20. The method of any one of embodiments 1 to 19, wherein the sorting step comprises using a flow cytometry sorter or a microfluidic device.

[0117] Embodiment 21 21. The method of any one of embodiments 1 to 20, wherein the sorting step comprises using a flow cytometry sorter, and the X-CBS and / or Y-CBS are sorted by gating.

[0118] Embodiment 22 22. The method of any one of embodiments 1 to 21, wherein the identifying step is carried out in situ in a flow cytometer or a microfluidic device prior to the sorting step.

[0119] Embodiment 23 23. The method of any one of embodiments 1 to 22, further comprising separately providing the selected X-CBS and / or Y-CBS in concentrated or purified form.

[0120] Embodiment 24 24. The method of any one of embodiments 1 to 23, further comprising separately providing the sorted X-CBS cells and / or Y-CBS cells in an enriched or purified form having a minimum purity of at least 90% X-CBS cells or a minimum purity of at least 90% Y-CBS cells.

[0121] Embodiment 25 25. The method of any one of embodiments 1 to 24, further comprising the step of separately providing the sorted X-CBS cells and / or Y-CBS cells in cryopreserved form.

[0122] Embodiment 26 26. The method of any one of embodiments 1 to 25, wherein the plurality of sperm cells comprises a mixture of X-CBS cells and Y-CBS cells.

[0123] Embodiment 27 27. An enriched or purified population of X chromosome-bearing sperm (X-CBS) cells sorted by the method of any one of embodiments 1 to 26.

[0124] Embodiment 28 28. The enriched or purified population of X-CBS cells according to embodiment 27, having a minimum purity of at least 90% X-CBS cells.

[0125] Embodiment 29 27. An enriched or purified population of Y chromosome-bearing sperm (Y-CBS) cells sorted by the method of any one of embodiments 1 to 26.

[0126] Embodiment 30 30. The enriched or purified population of Y-CBS cells according to embodiment 29, having a purity of at least 90% Y-CBS cells.

[0127] Embodiment 31 1. A method for identifying X-chromosome-bearing sperm (X-CBS) cells or Y-chromosome-bearing sperm (Y-CBS) cells in a sperm sample, the method comprising: determining a level of reactive oxidant species and / or cellular change mediated thereby (ROSCC) in each of a plurality of sperm cells in a starting sperm sample; and identifying at least a portion of the plurality of sperm cells in the starting sperm sample as X-CBS cells or Y-CBS cells based on the determined ROSCC level.

[0128] Embodiment 32 32. The method of embodiment 31, wherein the starting sperm sample comprises a mixture of X-CBS cells and Y-CBS cells.

[0129] Embodiment 33 33. The method of embodiment 31 or 32, wherein the ROSCC is selected from reactive oxidant species selected from reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS), or a combination of two or more thereof, and / or cellular changes mediated by reactive oxidant species selected from ROS-induced oxidative changes, RNS-induced oxidative changes, and RSS-induced oxidative changes, or a combination of two or more thereof.

[0130] Embodiment 34 34. The method of any one of embodiments 31 to 33, wherein identifying the level of ROSCC comprises treating a plurality of sperm cells with a ROSCC detection agent.

[0131] Embodiment 35 35. The method of any one of embodiments 31 to 34, wherein identifying the level of ROSCC comprises treating the plurality of sperm cells with a ROSCC detection agent comprising a dye.

[0132] Embodiment 36 36. The method of any one of embodiments 31 to 35, wherein the step of identifying the level of ROSCC comprises treating the plurality of sperm cells with a ROSCC detection agent comprising a fluorescent dye, optionally wherein the fluorescence is activated by reaction with one or more ROSCC in the cells.

[0133] Embodiment 37 37. The method of any one of embodiments 31 to 36, wherein ROSCC is detectable in a component of the sperm cell selected from lipid components such as the cell membrane or mitochondrial membrane, protein components such as chromatin or enzymes, organelles such as mitochondria, extracellular components such as cell surface enzymes, cytoplasm and DNA, or a combination of any two or more of these components.

[0134] Embodiment 38 38. The method of any one of embodiments 31 to 37, wherein identifying the level of ROSCC comprises treating the plurality of sperm cells with a ROSCC detection agent selective for the target ROSCC.

[0135] Embodiment 39 39. The method of any one of embodiments 31 to 38, wherein the ROSCC is a reactive oxidant species, and the reactive oxidant species is a ROS selected from superoxide, hydroxyl radical, and hydrogen peroxide.

[0136] Embodiment 40 40. The method of any one of embodiments 31 to 39, wherein the step of identifying the level of ROSCC comprises treating the plurality of sperm cells with a detection agent that detects ROS, RNS and / or RSS in at least the cytoplasm of the sperm cells.

[0137] Embodiment 41 40. The method of any one of embodiments 31 to 39, wherein ROSCC is a cellular change mediated by reactive oxidant species, and the cellular change is lipid peroxidation.

[0138] Embodiment 42 42. The method of any one of embodiments 31 to 39 or 41, wherein the step of identifying the level of ROSCC comprises treating the plurality of sperm cells with a ROSCC detection agent that detects cellular changes mediated by reactive oxidant species, wherein the cellular changes are in lipid components of the sperm cells.

[0139] Embodiment 43 43. The method of any one of embodiments 31 to 42, wherein the level of ROSCC in each cell identified as an X-CBS cell differs from the level of ROSCC in each cell identified as a Y-CBS cell by at least 5%, at least 20%, at least 40%, or at least 60%.

[0140] EMBODIMENT 44 44. The method of any one of embodiments 31 to 43, wherein the level of ROSCC in each cell identified as an X-CBS cell is at least 5%, at least 20%, at least 40%, or at least 60% higher than the level of ROSCC in each cell identified as a Y-CBS cell.

[0141] Embodiment 45 44. The method of any one of embodiments 31 to 43, wherein the average level of ROSCC identified for the portion of cells identified as X-CBS cells differs from the average level of ROSCC identified for the portion of cells identified as Y-CBS by at least 5%, at least 20%, at least 40%, or at least 60%.

[0142] Embodiment 46 46. ​​The method of any one of embodiments 31 to 43 or 45, wherein the average level of ROSCC identified for the portion of cells identified as X-CBS cells is at least 5%, at least 20%, at least 40%, or at least 60% higher than the average level of ROSCC identified for the portion of cells identified as Y-CBS.

[0143] Embodiment 47 47. The method of any one of embodiments 31 to 46, wherein the sperm cells are obtained from the proximal cauda epididymis, distal cauda epididymis, vas deferens or ejaculate of the subject.

[0144] Embodiment 48 48. The method of any one of embodiments 31 to 47, wherein the sperm cells are obtained from a mammalian subject.

[0145] Embodiment 49 49. The method of any one of embodiments 31 to 48, wherein the sperm cells are obtained from a non-human mammalian subject.

[0146] Embodiment 50 50. The method of any one of embodiments 31 to 49, further comprising isolating at least a portion of the identified X-CBS cells or Y-CBS cells from the starting sperm sample to produce a concentrated sperm sample comprising at least a portion of the identified and isolated X-CBS cells or Y-CBS cells.

[0147] Embodiment 51 51. The method of embodiment 50, wherein the concentrated sperm sample comprises at least 90% X-CBS cells, optionally at least 99% X-CBS cells, or at least 90% Y-CBS cells, optionally at least 99% Y-CBS cells.

[0148] Embodiment 52 52. The method of embodiment 50 or embodiment 51, wherein the isolating step comprises using a flow cytometry sorter or a microfluidic device.

[0149] Embodiment 53 53. The method of any one of embodiments 50 to 52, wherein the isolating step comprises using a flow cytometry sorter and gating on either the identified X-CBS cells or the Y-CBS cells.

[0150] EMBODIMENT 54 54. The method of any one of embodiments 50 to 53, wherein the identifying step is performed in situ in a flow cytometer or microfluidic device prior to the step of isolating at least a portion of the identified X-CBS cells or Y-CBS cells.

[0151] Embodiment 55 50. The method of any one of embodiments 31 to 49, further comprising inducing selective motility, fertility, or non-viability in any of the identified X-CBS cells or Y-CBS cells in the starting sperm sample to produce a sperm sample with altered motility, fertility, or viability.

[0152] Embodiment 56 56. The method of any one of embodiments 31 to 55, further comprising the step of cryopreserving the concentrated sperm sample.

[0153] Embodiment 57 Use of sperm sorted by the method of any one of embodiments 1 to 26, an enriched or purified population of X chromosome-bearing sperm (X-CBS) cells or Y chromosome-bearing sperm (Y-CBS) cells according to any one of embodiments 27 to 30, or sperm identified as X-CBS or Y-CBS according to the method of any one of embodiments 31 to 56, in assisted reproductive technologies.

[0154] Embodiment 58 A non-human mammalian subject produced from sperm sorted by the method of any one of embodiments 1 to 26, an enriched or purified population of X chromosome-bearing sperm (X-CBS) cells or Y chromosome-bearing sperm (Y-CBS) cells according to any one of embodiments 27 to 30, or sperm identified as X-CBS or Y-CBS according to the method of any one of embodiments 31 to 56.

[0155] Embodiment 59 1. A sperm cell sorting system comprising: a reactive oxidant species and / or cellular change (ROSCC) detection agent, wherein the cellular change is mediated by reactive oxidant species; and instructions for using the ROSCC detection agent to identify a level of ROSCC in each of a plurality of sperm cells, and sorting at least a portion of the plurality of sperm cells into X chromosome-bearing sperm (X-CBS) cells and / or Y chromosome-bearing sperm (Y-CBS) cells based on the identified ROSCC levels.

[0156] Embodiment 60 60. The sperm cell sorting system of embodiment 59, further comprising a sperm cell incubation solution for incubating the sperm cells with the ROSCC detection agent. [Example]

[0157] Example 1 1.1. Methods and Materials Mice of the C57BL / 6 strain were obtained and euthanized by cervical dislocation before removal of the epididymis body and vas deferens. The tail was divided into two main regions: the "proximal tail," which was the region of the cauda epididymis adjacent to the body of the epididymis, and the "distal tail," which was the section adjacent to the vas deferens (see Figure 1). The vas deferens was divided at the tail and as close as possible to the ejaculatory duct. The tissue from the epididymis body and vas deferens was covered with prewarmed phosphate-buffered saline (PBS) at pH 7.4. The tissue was carefully dissected open, and the semen was washed from the tissue. The semen-PBS mixture was carefully mixed and centrifuged at 400 g. The supernatant was discarded, and the sperm were resuspended and subjected to one of the following protocols.

[0158] 1.1a. Mitochondrial superoxide Mitochondrial superoxide was measured by incubating sperm with 1.25 μM MitoSOX® red mitochondrial superoxide indicator (excitation 488 nm, emission 585 nm, ThermoFisher Scientific Australia, M36008) for 10 minutes in the dark at 37°C, and sperm were washed by centrifugation at 400 g, resuspension in 200 μL of prewarmed PBS, and dilution 1:5 in prewarmed PBS before immediate analysis via flow cytometry.

[0159] 1.1b. Cytosolic reactive oxygen species Cytosolic ROS was measured by incubating sperm with 2.5 μM of the broad-spectrum ROS-detecting molecular probe CellROX® deep red (excitation 644 nm, emission 665 nm, ThermoFisher Scientific Australia Pty Ltd, C10491) in the dark for 30 min at 37°C. Sperm were then washed by centrifugation at 400 g, resuspension in 200 μL of prewarmed PBS, and dilution 1:5 in prewarmed PBS before immediate analysis via flow cytometry.

[0160] 1.1c. Lipid peroxidation Lipid peroxidation was measured by incubating sperm with 1.25 μM of the lipid peroxidation sensor Bodipy581 / 591C11 (excitation 488 nm, emission 585 nm, ThermoFisher Scientific Australia, D3861) in the dark for 30 min at 37°C, and sperm were washed by centrifugation at 400g, resuspension in 200 μL of prewarmed PBS, and dilution 1:5 in prewarmed PBS before immediate analysis via flow cytometry.

[0161] 1.2.Flow cytometry Samples were analyzed on a BD FACS Canto II. Forward scatter, side scatter, and PMT voltage were optimized so that the negative control (unstained sperm) was located approximately in the second decade of the logarithmic scale. The flow rate was set low, and 10,000 events were recorded. Data were analyzed with FCS Express 6plus software.

[0162] 1.3. Results and Discussion At all locations in the reproductive tract, X-CBS has more mitochondrial superoxide, more cytoplasmic ROS, and a higher degree of lipid peroxidation than Y-CBS. Mitochondrial superoxide is superior to Hoechst 33342 as a dye to aid in the differentiation of X-CBS from Y-CBS. The degree of lipid peroxidation and cytoplasmic ROS are comparable to the current Hoechst method when used simultaneously (see Figures 2-7).

[0163] Referring to Figure 1, a diagrammatic representation of the testis is shown showing three locations in the reproductive tract from which sperm were obtained for experiments, as described in the Examples above. The cauda epididymis is divided into two parts: the portion of the cauda epididymis adjacent to the body of the epididymis 1 is referred to as the "proximal tail," the portion of the tail adjacent to the vas deferens 2 is referred to as the "distal tail," and the vas deferens 3 from the tail as close as possible to the ejaculatory duct is referred to as the "vas deferens." Also shown are the head of the epididymis 13, seminiferous tubules 10, tunica albuginea 11, body of the epididymis 12, tail of the epididymis 15, and vas deferens 14.

[0164] Referring to Figure 2, data show the degree of separation between X- and Y-CBS using current sperm sorting techniques. Sperm were stained with 111 μM Hoechst 33342 for 30 minutes. The X-CBS population is 1.9 times brighter in fluorescence intensity than the Y-CBS. The histogram in Figure 2a) shows a moderate overlap in fluorescence intensity, while the contour plot in Figure 2b) reveals that there is a significant overlap between the two chromosome-carrying sperm types.

[0165] Figure 3 shows sorting data from sperm cells isolated from the proximal tail of the epididymis, where ROSCC levels were detected by MitoSOX® using the method described here. These data demonstrate the superiority of superoxide as a detection property for separating X-CBS and Y-CBS compared to available techniques. As shown in Figure 3, the fluorescence intensity of X-CBS is 8.5-fold higher than that of Y-CBS in sperm in the proximal tail of the epididymis. The histogram in Figure 3a shows a mild overlap in fluorescence intensity between X-CBS and Y-CBS. The contour plot in Figure 3b reveals that the two peaks in Figure 3a only overlap by a few orders of magnitude.

[0166] Figure 4 shows sorting data from sperm cells isolated from the distal tail of the epididymis, where ROS levels were detected by MitoSOX® using the method described here. These data reinforce the superiority of superoxide as a detection property for separating X-CBS and Y-CBS compared to available techniques. In sperm in the distal tail of the epididymis, the fluorescence intensity of X-CBS in Figure 4 is 6.5-fold higher than that of Y-CBS. The histogram in Figure 4a shows a slight overlap in fluorescence intensity between X-CBS and Y-CBS. The contour plot in Figure 4b reveals that only a few orders of magnitude of cells overlap in the two peaks in Figure 4a, representing only a very small number of cells.

[0167] Figure 5 shows sorting data from sperm cells isolated from the vas deferens, where ROS levels were detected by MitoSOX® using the method described herein. These data reinforce the superiority of superoxide as a detection property for separating X-CBS and Y-CBS compared to available techniques. In spermatozoa in the vas deferens, the fluorescence intensity of X-CBS in Figure 5 is 7.6-fold higher than that of Y-CBS. The histogram in Figure 5a shows a slight overlap in fluorescence intensity between X-CBS and Y-CBS. The contour plot in Figure 5b reveals that the numerical scale of overlap between the two peaks in Figure 5a is small, with only a small number of cells.

[0168] The data show that when sorted via cytoplasmic ROS detected with CellROX deep Red® (Figure 6a), the resolution of X-CBS and Y-CBS is low. When sorted via membrane lipid peroxidation detected with Bodipy C11® (Figure 6b), the resolution of X-CBS and Y-CBS is also low. On the other hand, when sperm are sorted via cytoplasmic ROS and lipid peroxidation (Figure 6c), high resolution can be achieved. Combining cytoplasmic ROS and lipid peroxidation in two-dimensional sorting achieves higher resolution than Hoechst.

[0169] Figure 7 visualizes the significant difference in superoxide concentrations between X-CBS and Y-CBS detected by MitoSOX®, as shown by the histograms and contour plots in Figures 3-6. The significant difference in superoxide observed between X-CBS and Y-CBS in pre-ejaculated sperm using the method described here and shown in Figure 7 may enable faster sorting times, higher sperm viability after sorting, and reduced potential for oxidative stress, a common inhibitor of fertility in mammals. Current technology utilizing a variant of the nucleotide-binding Hoechst stain results in X-CBS emitting 1.9 times more light than Y-CBS. The method described here surpasses current technology by utilizing superoxide, a potential inhibitor of sperm function, as a detection feature for sperm sorting.

[0170] Example 2 2.1. Methods and Materials 2.1.1. Cryopreserved bull semen was thawed at 37°C for 60 seconds, and samples were diluted 1:3 in PBS, washed, and incubated with 2.5 μM MitoSOX Red in PBS for 10 minutes in the dark. Samples were washed by diluting 1:5 in PBS before centrifugation at 600g for 2 minutes. Samples were resuspended in 200 μL of PBS before analysis via flow cytometry.

[0171] 2.1.2. Horse semen was purchased from Elderslie Horse Care and Spelling Tasmania. Semen was provided in a 1:1 ratio in Equiplus equine semen extender (Minitube, Australia). Cells were concentrated by first diluting 1000 μL of semen diluent into 4000 μL of PBS, pH 7.4, and centrifuging at 6000 g for 20 minutes. The pellet was washed again in 1000 μL of PBS. The pellet was then resuspended in 2.5 μM MitoSOX Red in PBS and incubated in the dark for 10 minutes. Samples were washed by diluting 1:5 in PBS before centrifugation at 600 g for 2 minutes. Samples were resuspended in 200 μL of PBS before analysis via flow cytometry.

[0172] 2.1.3. Boar semen was purchased from Sabor Limited. 200 μL of semen diluent was incubated with 5 μM MitoSOX Red in PBS and incubated for 10 minutes in the dark. Samples were washed by diluting 1:5 in PBS before analysis via flow cytometry. Samples were washed by diluting 1:5 in PBS before centrifugation at 600 g for 2 minutes. Samples were resuspended in 200 μL of PBS before analysis via flow cytometry.

[0173] Flow cytometry Cells were analyzed on a Beckman Dickinson BD FACS Canto II. Forward scatter, side scatter, and PMT voltage were optimized so that the negative control (unstained spermatozoa) was located approximately in the second decade of the logarithmic scale. The flow rate was set low, and 50,000 events were recorded. Data were analyzed with FCS Express 6plus software.

[0174] 2.3. Results and Discussion Figure 8 shows data visualizing the large difference in superoxide concentrations between (a) X-CBS and Y-CBS of bulls detected by MitoSOX® compared to (b) sperm stained with Hoechst 33342, as shown by histograms. Cryopreserved semen is difficult to separate, and there are usually many steps involved in increasing the permeability of Hoechst and reducing signal interference from dead sperm (caused by the freezing process). The method of the present invention provides better resolution of the different subpopulations of X-CBS and Y-CBS compared to the current Hoechst method.

[0175] FIG. 9 shows data visualizing the large difference in superoxide concentrations between equine X-CBS and Y-CBS by MitoSOX® detection, as shown by histograms.

[0176] FIG. 10 shows data visualizing the large difference in superoxide concentrations between X-CBS and Y-CBS of wild boar by MitoSOX® detection, as shown by histograms.

[0177] Although the invention has been described with reference to the above examples, it should be understood that the examples are illustrative of the invention described herein and are not intended to limit the invention.

[0178] Although the present invention has been described in some detail for purposes of clarity and understanding, it should be apparent to those skilled in the art that various changes and modifications to the embodiments and methods described herein can be made without departing from the scope of the inventive concepts disclosed herein.

Claims

1. 1. A method for distinguishing between X chromosome-bearing sperm (X-CBS) cells and Y chromosome-bearing sperm (Y-CBS) cells in a sperm sample, comprising: identifying a level of reactive oxidant species and / or cellular changes mediated thereby (ROSCC) in each of a plurality of sperm cells in the sperm sample; distinguishing between X-CBS and Y-CBS cells in at least a portion of said plurality of sperm cells based on the determined ROSCC level; A method for providing the above.

2. The ROSCC is reactive oxidant species selected from reactive oxygen species (ROS), reactive nitrogen species (RNS) and reactive sulfur species (RSS), or a combination of two or more thereof; and / or Cellular changes mediated by reactive oxidant species selected from ROS-induced oxidative changes, RNS-induced oxidative changes, and RSS-induced oxidative changes, or a combination of two or more thereof. The method of claim 1 , wherein the compound is selected from the group consisting of:

3. 3. The method of claim 1, wherein the step of identifying the level of ROSCC comprises treating the plurality of sperm cells with a ROSCC detecting agent.

4. 4. The method of claim 1, wherein identifying the level of ROSCC comprises treating the plurality of sperm cells with a ROSCC detection agent comprising a dye.

5. 5. The method of claim 1, wherein identifying the level of ROSCC comprises treating the plurality of sperm cells with a ROSCC detection agent comprising a fluorescent dye, optionally wherein the fluorescence is activated by reaction with one or more ROSCCs in the cells.

6. 6. The method of any one of claims 1 to 5, wherein the ROSCC is detectable in a component of the sperm cell selected from lipid components such as the cellular membrane or mitochondrial membrane, protein components such as chromatin or enzymes, organelles such as mitochondria, extracellular components such as cell surface enzymes, cytoplasm and DNA, or a combination of any two or more of these components.

7. 7. The method of claim 1, wherein identifying the level of ROSCC comprises treating the plurality of sperm cells with a ROSCC detection agent selective for target ROSCC.

8. 8. The method of any one of claims 1 to 7, wherein the ROSCC is a reactive oxidant species, the reactive oxidant species being a ROS selected from superoxide, hydroxyl radical, and hydrogen peroxide.

9. 9. The method of claim 1, wherein identifying the level of ROSCC comprises treating the plurality of sperm cells with a detection agent that detects ROS, RNS, and / or RSS in at least the cytoplasm of the sperm cells.

10. 9. The method of any one of claims 1 to 8, wherein the ROSCC is a cellular change mediated by reactive oxidant species, the cellular change being lipid peroxidation.

11. 11. The method of any one of claims 1 to 8, or 10, wherein identifying the level of ROSCC comprises treating the plurality of sperm cells with a ROSCC detection agent that detects cellular changes mediated by reactive oxidant species, wherein the cellular changes are in lipid components of the sperm cells.

12. 9. The method of claim 1, wherein identifying the level of ROSCC comprises treating the plurality of sperm cells with a ROSCC detection agent that detects cellular changes mediated by reactive oxidant species, wherein the cellular changes are in protein components of sperm cells, and optionally, the cellular changes are carbonylation or nitration of amino acids.

13. 13. The method of any one of claims 1 to 12, wherein the mean level of ROSCC for X-CBS cells differs from the mean level of ROSCC for Y-CBS cells by at least 5%, at least 20%, at least 40%, or at least 60%.

14. 14. The method of any one of claims 1 to 13, wherein the average level of ROSCC for X-CBS cells is at least 5%, at least 20%, at least 40%, or at least 60% higher than the average level of ROSCC for Y-CBS cells.

15. 15. The method of any one of claims 1 to 14, wherein the sperm cells are obtained from the proximal cauda epididymis, distal cauda epididymis, vas deferens or ejaculated semen of the subject.

16. 16. The method of any one of claims 1 to 15, wherein the sperm cells are obtained from a mammalian subject.

17. 17. The method of any one of claims 1 to 16, wherein the sperm cells are obtained from a non-human mammalian subject.

18. 18. The method of any one of claims 1 to 17, wherein identifying the level of ROSCC comprises using a flow cytometer or a microfluidic device.

19. 19. The method of any one of claims 1 to 18, further comprising isolating at least a portion of the differentiated X-CBS cells or Y-CBS cells from the sperm sample to produce an enriched or purified sperm sample, optionally using a flow cytometer or a microfluidic device.

20. 20. The method of any one of claims 1 to 19, wherein the identifying step is optionally performed in situ in a flow cytometer or microfluidic device prior to isolating the differentiated X-CBS or Y-CBS cells.

21. 21. An enriched or purified population of X chromosome-bearing sperm (X-CBS) cells isolated by the method of claim 19 or 20, or an enriched or purified population of Y chromosome-bearing sperm (Y-CBS) cells isolated by the method of claim 19 or 20.

22. 22. Use of sperm distinguished by the method of any one of claims 1 to 20, or of an enriched or purified population of X-chromosome-bearing sperm (X-CBS) cells or Y-chromosome-bearing sperm (Y-CBS) cells according to claim 21, in assisted reproductive technology.

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