Methods for assessing sperm health and identifying sperm for use in assisted reproductive technologies

Abstract

The present invention relates to a method for assessing sperm health and distinguishing sperm for use in assisted reproductive technologies, by providing a biological sample, preferably a semen sample, from a patient, measuring the quantitative level of a biomarker in the sample, and assessing sperm health based on the quantitative level of the biomarker, wherein the biomarker is membrane-bound receptor for advanced glycation end products (mRAGE).

Description

[Technical Field]

[0001] The present invention relates to a method for assessing sperm health and distinguishing sperm for use in assisted reproductive technologies, by providing a biological sample, preferably a semen sample, from a patient, measuring the quantitative level of a biomarker in the sample, and assessing sperm health based on the quantitative level of the biomarker, wherein the biomarker is membrane-bound receptor for advanced glycation end products (mRAGE). [Background technology]

[0002] Infertility affects approximately 15% of men and women of reproductive age, and approximately 30-40% of cases can be attributed to male factors. Male infertility can arise from a variety of factors, including varicocele, genetic alterations, systemic disease, and altered semen parameters. However, the single largest contributing factor to male infertility is sperm damage due to oxidative stress and resulting DNA fragmentation, estimated to account for 30-80% of cases. DNA fragmentation not only affects sperm quality, but also has been shown to have adverse effects on embryo quality, implantation rates, and miscarriage rates.

[0003] Current investigations of male infertility include assessment of the physical characteristics of semen samples (volume, viscosity, pH, and color) and microscopic evaluation of the sperm in the sample (concentration, motility, morphology, and activity). These tests provide a functional and physical readout of the health of the sperm population in the semen sample. Assessment of double-strand breaks in sperm DNA is recommended to provide additional information regarding sperm molecular health, fertility, and DNA integrity. Tests include terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay, comet assay, or sperm chromatin dispersion (SCD) assay. Unfortunately, these tests are often not included in investigations of male infertility because they are expensive, time-consuming, invasive, and do not fully inform about sperm health.

[0004] Extracellular markers associated with the degree of sperm DNA damage and sperm quality would be extremely useful for identifying and removing damaged sperm before assisted reproductive techniques (ART). Over the past 30 years, significant efforts have been made to discover more sophisticated markers. These primarily involve intracellular markers of DNA fragmentation, oxidative DNA damage, oxidative stress, mitochondrial function, and sperm cell death. However, these require invasive internal labeling procedures, leaving behind nonviable sperm. This study evaluates extracellular markers of sperm cell health.

[0005] The receptor for advanced glycation end products (RAGE) is a central signaling molecule of the innate immune system and belongs to a class of cell surface pattern recognition receptors encoded by the MHC class III locus. This receptor exists in numerous forms as membrane-bound (full-length), soluble, or secreted proteins. Soluble RAGE (sRAGE) is produced by both proteolytic cleavage of membrane-bound / full-length RAGE (mRAGE / fl-RAGE) and alternative mRNA splicing. Soluble isoforms contain an extracellular domain but lack the transmembrane and cytoplasmic domains.

[0006] RAGE has long been recognized as a major contributor to severe chronic pathologies, including neurodegeneration, atherosclerosis, cardiovascular disease, osteoarthritis, and diabetes. Under normal conditions, RAGE expression is very low. However, during disease, RAGE production is actively induced on the cell surface by numerous different ligands, including advanced glycation end products (AGEs), high mobility group box-1 (HMGB1), S100 / calgranulins, phosphatidylserine, and C3. In addition, RAGE expression is further elevated after immune activation or infection, and many RAGE ligands are secreted by monocytes, macrophages, neutrophils, and leukocytes, which are likely crucial for the initiation and propagation of RAGE-dependent inflammatory responses. The location of membrane-bound receptors in the acrosomal cap of human sperm (the direct access point to nuclear DNA within the sperm head) has been implicated in their role in initiating and exacerbating nuclear DNA damage.

[0007] This study describes mRAGE as a diagnostic biomarker for poor sperm quality. By analyzing mRAGE expression on sperm from non-diabetic, non-obese, non-smoking men, we ruled out the role of RAGE and its ligands, which are elevated in these conditions, on sperm health. Flow cytometry was used to quantitatively assess mRAGE expression at the cellular level on sperm, and computer-assisted sperm analysis (CASA) was used to evaluate sperm motility. Cellular analyses of mRAGE and mitochondrial membrane potential (MMP), cell permeability, motility, morphology, apoptosis, and DNA fragmentation provide very strong evidence of a direct relationship.

[0008] We observed that expression of mRAGE on sperm is associated with mitochondrial dysfunction, cell death, DNA fragmentation, and immotile sperm. Furthermore, sperm purification using conventional methods (density gradient centrifugation and direct swim-up) successfully removed mRAGE-expressing sperm. Therefore, a reliable, minimally invasive extracellular marker for assessing sperm health is needed. Because RAGE is membrane-bound, it can also be used as a means to remove damaged sperm. Summary of the Invention [Means for solving the problem]

[0009] According to a first aspect of the present invention there is provided a method of assessing sperm health, the method comprising: (a) a procedure for providing a biological sample from a patient; (b) a procedure for measuring the quantitative or qualitative level of a biomarker in the sample; and (c) assessing sperm health based on the quantitative or qualitative levels of said biomarkers; wherein the biomarker is membrane-associated receptor for advanced glycation end products (mRAGE).

[0010] The method for assessing sperm health may be a method for in vitro fertilization.

[0011] mRAGE may be membrane-bound to cells.

[0012] mRAGE may be bound to the sperm membrane.

[0013] mRAGE may be bound to the membrane of the sperm head.

[0014] mRAGE may be bound to the membrane in the equatorial region of the sperm head.

[0015] mRAGE may be a transmembrane receptor.

[0016] mRAGE may comprise three extracellular domains.

[0017] mRAGE may comprise three extracellular domains: one V-type Ig domain and two C-type Ig domains (C1 and C2).

[0018] mRAGE may comprise three extracellular domains: one V-type Ig domain and two C-type Ig domains (C1 and C2), and one transmembrane domain.

[0019] mRAGE may comprise three extracellular domains: one V-type Ig domain and two C-type Ig domains (C1 and C2), one transmembrane domain, and a cytoplasmic tail.

[0020] mRAGE may be full-length RAGE (fl-RAGE).

[0021] Preferably, the biological sample is a semen sample.

[0022] The biomarker may be a gene. The biomarker may be a nucleic acid. The biomarker may be a deoxyribonucleic acid. The biomarker may be a ribonucleic acid. The biomarker may be a protein. The biomarker may be a peptide.

[0023] The biomarker for assessing sperm health may be the gene with GenBank Accession Version Number AQY76652.

[0024] Preferably, said biomarker for assessing sperm health is the protein with UniProt ID Q15109.

[0025] Preferably, said measuring step (b) comprises measuring the quantitative level of mRAGE on sperm cells from the semen sample.

[0026] The measuring step (b) may comprise measuring the quantitative level of mRAGE on sperm cells from the semen sample by any quantitative analytical technique.

[0027] The measuring step (b) may comprise measuring quantitative or qualitative levels of mRAGE on sperm cells from the semen sample by cell-based analysis.

[0028] The measuring step (b) may comprise measuring quantitative or qualitative levels of mRAGE on sperm cells from the semen sample by flow cytometry.

[0029] The measuring step (b) may comprise measuring the quantitative or qualitative level of mRAGE on sperm cells from the semen sample, as determined using a RAGE antibody.

[0030] The measuring step (b) may involve measuring the quantitative or qualitative level of mRAGE on sperm cells from the semen sample, as determined using an sRAGE antibody.

[0031] The measuring step (b) may involve measuring the quantitative or qualitative level of mRAGE on sperm cells from the semen sample, as determined using an mRAGE antibody.

[0032] The measuring step (b) may comprise measuring the quantitative or qualitative level of mRAGE on sperm cells from the semen sample, as determined using a mouse anti-human mRAGE antibody.

[0033] The measuring step (b) may comprise measuring the quantitative or qualitative level of mRAGE on sperm cells from the semen sample, as determined using a rabbit anti-human mRAGE antibody.

[0034] The measuring step (b) may comprise measuring the quantitative or qualitative level of mRAGE on sperm cells from the semen sample, as determined using a murine anti-human mRAGE antibody.

[0035] The measuring step (b) may comprise measuring the quantitative or qualitative level of mRAGE on sperm cells from the semen sample, as determined using a sheep anti-human mRAGE antibody.

[0036] The measuring step (b) may comprise measuring the quantitative or qualitative level of mRAGE on sperm cells from the semen sample, as determined using a monoclonal rabbit anti-human mRAGE antibody.

[0037] The measuring step (b) may comprise measuring the quantitative or qualitative level of mRAGE on sperm cells from the semen sample, as determined using a monoclonal murine anti-human mRAGE antibody.

[0038] The measuring step (b) may comprise measuring the quantitative or qualitative level of mRAGE on sperm cells from the semen sample, as determined using a monoclonal sheep anti-human mRAGE antibody.

[0039] The measuring step (b) may comprise measuring the quantitative or qualitative level of mRAGE on sperm cells from the semen sample, as determined using a monoclonal mouse anti-human mRAGE antibody.

[0040] The measuring step (b) may comprise measuring the quantitative or qualitative level of mRAGE on sperm cells from the semen sample, as determined using the monoclonal mouse anti-human mRAGE antibody AF647.

[0041] The measuring step (b) may comprise measuring the quantitative or qualitative level of mRAGE on sperm cells from the semen sample, which may be determined using a DNA aptamer.

[0042] The measuring step (b) may comprise measuring the quantitative or qualitative level of mRAGE on sperm cells from the semen sample, which may be determined using an RNA aptamer.

[0043] The measuring step (b) may comprise measuring the quantitative or qualitative level of mRAGE on sperm cells from the semen sample, which may be determined using a peptide aptamer.

[0044] The assessing step (c) may include assessing sperm health based on quantitative or qualitative levels of mRAGE on sperm cells from the semen sample.

[0045] The assessing step (c) may include assessing sperm health based on quantitative or qualitative levels of mRAGE on sperm cells from a semen sample, wherein the presence of mRAGE is defined as mRAGE positive.

[0046] The evaluating step (c) may include determining whether the patient is mRAGE negative or mRAGE positive by assessing the presence or absence of quantifiable levels of mRAGE on sperm cells from a semen sample.

[0047] The presence of quantifiable levels of mRAGE on sperm cells from the semen sample may define the patient as mRAGE positive, and the absence of quantifiable levels of mRAGE on sperm cells from the semen sample may define the patient as mRAGE negative.

[0048] The assessing step (c) may comprise comparing the amount of sperm expressing a quantifiable level of mRAGE with the amount of sperm not expressing a quantifiable level of mRAGE.

[0049] The amount of sperm expressing quantifiable levels of mRAGE may deviate by at least a factor of 1 from the amount of sperm not expressing quantifiable levels of mRAGE.

[0050] The amount of sperm expressing quantifiable levels of mRAGE may deviate by at least a factor of two from the amount of sperm not expressing quantifiable levels of mRAGE.

[0051] The amount of sperm expressing quantifiable levels of mRAGE may deviate by at least a factor of three from the amount of sperm not expressing quantifiable levels of mRAGE.

[0052] The amount of sperm expressing quantifiable levels of mRAGE may deviate by at least four-fold from the amount of sperm not expressing quantifiable levels of mRAGE.

[0053] The amount of sperm expressing quantifiable levels of mRAGE may deviate by at least 5-fold from the amount of sperm not expressing quantifiable levels of mRAGE.

[0054] The amount of sperm expressing quantifiable levels of mRAGE may deviate by at least 6-fold from the amount of sperm not expressing quantifiable levels of mRAGE.

[0055] The amount of sperm expressing quantifiable levels of mRAGE may deviate by at least 7-fold from the amount of sperm not expressing quantifiable levels of mRAGE.

[0056] The amount of sperm expressing quantifiable levels of mRAGE may deviate by at least 8-fold from the amount of sperm not expressing quantifiable levels of mRAGE.

[0057] The amount of sperm expressing quantifiable levels of mRAGE may deviate by at least 9-fold from the amount of sperm not expressing quantifiable levels of mRAGE.

[0058] The amount of sperm expressing quantifiable levels of mRAGE may deviate by at least 10-fold from the amount of sperm not expressing quantifiable levels of mRAGE.

[0059] The assessing step (c) may comprise assessing the proportion of sperm expressing quantifiable levels of mRAGE on sperm cells from the semen sample.

[0060] The evaluating step (c) may include assessing the percentage of sperm expressing quantifiable levels of mRAGE on sperm cells from a semen sample to define the patient as mRAGE negative or mRAGE positive.

[0061] The evaluating step (c) may comprise evaluating the proportion of sperm expressing quantifiable levels of mRAGE on sperm cells from the semen sample, wherein mRAGE-negative is defined as a mean fluorescence intensity (MFI, geometric mean) value that is less than 10-fold higher than that of mRAGE-positive sperm cells.

[0062] The evaluating step (c) may comprise evaluating the proportion of sperm expressing quantifiable levels of mRAGE on sperm cells from the semen sample, wherein mRAGE positivity is defined as a mean fluorescence intensity (MFI, geometric mean) value that is at least 10 times higher than mRAGE negative sperm cells.

[0063] The evaluating step (c) may comprise evaluating the proportion of sperm expressing quantifiable levels of mRAGE on sperm cells from the semen sample, wherein mRAGE positivity is defined as a mean fluorescence intensity (MFI, geometric mean) value that is at least 20 times higher than mRAGE negative sperm cells.

[0064] The evaluating step (c) may comprise evaluating the proportion of sperm expressing quantifiable levels of mRAGE on sperm cells from the semen sample, wherein mRAGE positivity is defined as a mean fluorescence intensity (MFI, geometric mean) value that is at least 30 times higher than mRAGE negative sperm cells.

[0065] Mean fluorescence intensity values ​​representative of quantifiable levels of mRAGE may deviate by less than a factor of 10 from mean fluorescence intensity values ​​not representative of quantifiable levels of mRAGE.

[0066] Mean fluorescence intensity values ​​representative of quantifiable levels of mRAGE may deviate by at least a factor of 10 from mean fluorescence intensity values ​​not representative of quantifiable levels of mRAGE.

[0067] The assessing step (c) may include assessing sperm health based on quantitative or qualitative levels of mRAGE on sperm cells from the semen sample, wherein mRAGE positivity indicates poor sperm health.

[0068] The evaluating step (c) may include assessing sperm health based on quantitative or qualitative levels of mRAGE on sperm cells from the semen sample, wherein mRAGE negativity indicates healthy and fertile sperm.

[0069] Further, the evaluating step (c) may include evaluating sperm health based on quantitative or qualitative levels of mRAGE on sperm cells from the semen sample, wherein a positive mRAGE result indicates an abnormality in the sperm.

[0070] Further, the evaluating step (c) may include evaluating sperm health based on quantitative or qualitative levels of mRAGE on sperm cells from the semen sample, wherein mRAGE negativity indicates normal sperm.

[0071] Further, the evaluating step (c) may include evaluating sperm health based on quantitative or qualitative levels of mRAGE on sperm cells from the semen sample, wherein mRAGE negativity indicates good sperm health.

[0072] Further, the evaluating step (c) may include evaluating sperm health based on quantitative or qualitative levels of mRAGE on sperm cells from the semen sample, wherein mRAGE negativity indicates one or more of normal sperm and good sperm health.

[0073] The evaluating step (c) may include assessing sperm health based on quantitative or qualitative levels of mRAGE on sperm cells from the semen sample, wherein a positive mRAGE result indicates one or more of poor sperm health and sperm abnormalities.

[0074] Further, the evaluating step (c) may include assessing sperm health based on quantitative or qualitative levels of mRAGE on sperm cells from the semen sample, wherein mRAGE positivity indicates poor sperm motility.

[0075] The assessing step (c) may include assessing sperm health based on quantitative or qualitative levels of mRAGE on sperm cells from the semen sample, wherein a positive mRAGE result indicates one or more of poor sperm health, sperm abnormalities, and low sperm motility.

[0076] Further, the evaluating step (c) may include evaluating sperm health based on quantitative or qualitative levels of mRAGE on sperm cells from the semen sample, wherein a positive mRAGE level indicates infertility.

[0077] The evaluating step (c) may include assessing sperm health based on quantitative or qualitative levels of mRAGE on sperm cells from the semen sample, wherein a positive mRAGE result indicates one or more of poor sperm health, sperm abnormalities, low sperm motility, and infertility.

[0078] In the method for assessing sperm health, the patient may be a mammal.

[0079] In the methods for assessing sperm health, the patient may be non-human.

[0080] In the method of assessing sperm health, the subject may be a non-human animal, such as a cow, horse, dog, cat, pig, sheep, or bear.

[0081] In the method for assessing sperm health, the patient is preferably a human.

[0082] In the method for assessing sperm health, the patient may be a non-diabetic human.

[0083] In the method for assessing sperm health, the patient may be a non-diabetic, non-smoking human.

[0084] In the method for assessing sperm health, the patient may be a non-diabetic, non-smoking, non-obese human.

[0085] In the method of assessing sperm health, the patient may be a human who is non-diabetic, non-smoking, and non-obese (BMI<29.9).

[0086] In the method of assessing sperm health, the patient may be a male who is non-diabetic, non-smoking, and non-obese (BMI<29.9).

[0087] The method of assessing sperm health may further comprise assessing sperm motility.

[0088] The method for assessing sperm health may further include assessing sperm motility, whereby the sperm are classified as immotile (IM), non-progressively motile (NP), or progressively motile (PR).

[0089] The method of assessing sperm health may further comprise assessing sperm motility, wherein sperm classified as IM are non-motile.

[0090] The method for assessing sperm health may further include assessing sperm motility, wherein sperm classified as IM are non-motile, indicating poor sperm health.

[0091] The method for assessing sperm health may further include assessing sperm motility, wherein sperm classified as IM are non-motile, indicating poor sperm health and low sperm motility.

[0092] The method for assessing sperm health may further include assessing sperm motility, wherein sperm classified as IM are non-motile, as defined in the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen (5th ed., 2010).

[0093] The method for assessing sperm health may further comprise assessing sperm motility, wherein sperm classified as IM are indicative of poor sperm health, as defined in the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen (5th ed., 2010).

[0094] The method for assessing sperm health may further include assessing sperm motility, where sperm classified as NP are motile but do not move in a generally linear direction.

[0095] The method for assessing sperm health may further include assessing sperm motility, where sperm classified as NP, as defined in the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen (5th ed., 2010), are motile but do not move in a generally linear direction.

[0096] The method of assessing sperm health may further include assessing sperm motility, where sperm classified as PR move in a generally straight line direction.

[0097] The method for assessing sperm health may further include assessing sperm motility, where sperm classified as PR move in a generally straight line, as defined in the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen (5th ed., 2010).

[0098] The method for assessing sperm health may further include assessing sperm motility, wherein at least 10% of sperm classified as PR in a semen sample are considered normal.

[0099] The method for assessing sperm health may further include assessing sperm motility, wherein at least 20% of sperm classified as PR in a semen sample are considered normal.

[0100] The method for assessing sperm health may further include assessing sperm motility, wherein at least 30% of sperm classified as PR in a semen sample are considered normal.

[0101] The method for assessing sperm health may further include assessing sperm motility, wherein at least 32% of sperm classified as PR in a semen sample are considered normal.

[0102] The method for assessing sperm health may further include assessing sperm motility, wherein at least 32% of sperm classified as PR in a semen sample are considered normal, as defined in the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen (5th Edition, 2010).

[0103] The method for assessing sperm health may further include assessing sperm motility, wherein at least 10% of the combined NP and PR classified sperm in the semen sample are considered normal.

[0104] The method for assessing sperm health may further include assessing sperm motility, wherein at least 20% of the combined NP and PR classified sperm in a semen sample are considered normal.

[0105] The method for assessing sperm health may further include assessing sperm motility, wherein at least 30% of the combined NP and PR classified sperm in a semen sample are considered normal.

[0106] The method for assessing sperm health may further include assessing sperm motility, wherein at least 40% of the combined NP and PR classified sperm in a semen sample are considered normal.

[0107] The method for assessing sperm health may further include assessing sperm motility, wherein at least 40% of the combined NP and PR sperm in a semen sample are considered normal, as defined in the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen (5th ed., 2010).

[0108] The method for assessing sperm health may further include assessing sperm motility, wherein the percentage of mRAGE-positive sperm is inversely correlated with the percentage of PR sperm, and wherein PR sperm are defined as sperm with a spatial gain of 5 μm / sec or greater.

[0109] The method of assessing sperm health may further comprise assessing sperm morphology.

[0110] The method of assessing sperm health may further include assessing sperm morphology, whereby the sperm are classified as normal or abnormal.

[0111] The method for assessing sperm health may further include assessing sperm morphology, whereby sperm are classified as normal or abnormal, as defined in the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen (5th ed., 2010).

[0112] The method for assessing sperm health may further include assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, well-defined, and generally oval.

[0113] The method for assessing sperm health may further include assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, well-defined, and generally ovoid, as defined in the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen (5th ed., 2010).

[0114] The method for assessing sperm health may further include assessing sperm morphology, wherein sperm are classified as normal if the sperm head is smooth, well-defined, approximately ovoid, and has a distinct acrosomal region that occupies 40-70% of the sperm head area.

[0115] The method for assessing sperm health may further include assessing sperm morphology, wherein sperm are classified as normal when the sperm head is smooth, well-defined, approximately ovoid, and has a distinct acrosomal region that occupies 40-70% of the sperm head area, as defined in the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen (5th ed., 2010).

[0116] The method for assessing sperm health may further include assessing sperm morphology, wherein sperm are classified as normal if the sperm head is smooth, well-defined, and generally ovoid, with a distinct acrosomal region that occupies 40-70% of the sperm head area, with no large vacuoles in the acrosomal region, no more than two small vacuoles, and the vacuoles do not occupy more than 20% of the sperm head.

[0117] The method for assessing sperm health may further include assessing sperm morphology, where sperm are classified as normal when the sperm head has a smooth, well-defined, roughly ovoid shape, a clear acrosomal region that occupies 40-70% of the sperm head area, no large vacuoles, no more than two small vacuoles, and the vacuole does not occupy more than 20% of the sperm head, as defined in the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen (5th ed., 2010).

[0118] The method for assessing sperm health may further include assessing sperm morphology, wherein sperm are classified as normal if the sperm head has a smooth, well-defined, and generally ovoid shape, a distinct acrosomal region that occupies 40-70% of the sperm head area, the acrosomal region has no large vacuoles, no more than two small vacuoles, and the vacuoles do not occupy more than 20% of the sperm head, and the sperm midpiece is elongated, well-defined, and approximately the same length as the sperm head.

[0119] The method for assessing sperm health may further include assessing sperm morphology, where sperm are classified as normal if, as defined in the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen (5th ed., 2010), the sperm head is smooth, well-defined, and approximately ovoid, with a distinct acrosomal region that occupies 40-70% of the sperm head area, the acrosomal region lacks a large vacuole, lacks two or more small vacuoles, and the vacuole does not occupy more than 20% of the sperm head, and the midpiece is elongated, well-defined, and approximately the same length as the sperm head.

[0120] The method for assessing sperm health may further include assessing sperm morphology, wherein sperm are classified as normal if the sperm head has a smooth, well-defined, and generally ovoid shape, a distinct acrosomal region that occupies 40-70% of the sperm head area, the acrosomal region has no large vacuoles, no more than two small vacuoles, and the vacuoles do not occupy more than 20% of the sperm head, the sperm midpiece is elongated, well-defined, and approximately the same length as the sperm head, and the long axis of the sperm midpiece is aligned with the long axis of the sperm head.

[0121] The method for assessing sperm health may further include assessing sperm morphology, where sperm are classified as normal when, as defined in the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen (5th ed., 2010), the sperm have a smooth, well-defined, and approximately ovoid sperm head, a distinct acrosomal region that occupies 40-70% of the sperm head area, the acrosomal region lacks a large vacuole, lacks two or more small vacuoles, and the vacuole does not occupy more than 20% of the sperm head, the sperm midpiece is elongated, well-defined, and approximately the same length as the sperm head, and the long axis of the sperm midpiece is aligned with the long axis of the sperm head.

[0122] The method for assessing sperm health may further include assessing sperm morphology, wherein sperm are classified as normal if the sperm head has a smooth, well-defined, approximately ovoid sperm head, a distinct acrosomal region that occupies 40-70% of the sperm head area, no large vacuoles in the acrosomal region, no more than two small vacuoles, and the vacuoles do not occupy more than 20% of the sperm head, the sperm midpiece is elongated, well-defined, and approximately the same length as the sperm head, the long axis of the midpiece is aligned with the long axis of the sperm head, the main portion is of constant thickness along its length, is thinner than the midpiece, and is approximately 45 μm in length.

[0123] The method for assessing sperm health may further include assessing sperm morphology, where sperm are classified as normal if, as defined in the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen (5th ed., 2010), the sperm have a smooth, well-defined, and approximately ovoid sperm head, a distinct acrosomal region that occupies 40-70% of the sperm head area, the acrosomal region lacks a large vacuole, and the vacuole does not contain more than two small vacuoles, and the vacuole does not occupy more than 20% of the sperm head, the sperm midpiece is elongated, well-defined, and approximately the same length as the sperm head, the long axis of the midpiece is aligned with the long axis of the sperm head, the main portion is of constant thickness along its length, is thinner than the midpiece, and is approximately 45 μm in length.

[0124] The method of assessing sperm health may further include assessing sperm morphology, wherein sperm are classified as abnormal when there is a deviation from normal.

[0125] The method for assessing sperm health may further include assessing sperm morphology, where sperm are classified as abnormal when there is a deviation from normal, as defined in the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen (5th Edition, 2010).

[0126] The method for assessing sperm health may further include assessing sperm morphology, wherein sperm are classified as abnormal when there is a deviation from normal, which is indicative of poor sperm health.

[0127] The method for assessing sperm health may further include assessing sperm morphology, whereby sperm are classified as abnormal when there is a deviation from normal, as defined in the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen (5th Edition, 2010), which indicates poor sperm health.

[0128] The method for assessing sperm health may further comprise assessing mitochondrial membrane potential (MMP).

[0129] The method for assessing sperm health may further comprise assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros.

[0130] The method for assessing sperm health may further include assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros, wherein MitoTracker CMX Ros does not stain cells with low MMP.

[0131] The method for assessing sperm health may further include assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros, wherein MitoTracker CMX Ros does not stain cells with low MMP, indicating poor sperm health.

[0132] The method for assessing sperm health may further include assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros, wherein MitoTracker CMX Ros does not stain cells with low MMP, defined as a mean fluorescence intensity (MFI, geometric mean) value of 10-fold or less compared to mRAGE-negative sperm cells, indicating poor sperm health.

[0133] The method for assessing sperm health may further include assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros, wherein MitoTracker CMX Ros stains cells with high MMP.

[0134] The method for assessing sperm health may further include assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros, wherein MitoTracker CMX Ros stains cells with high MMP, indicating good sperm health.

[0135] The method for assessing sperm health may further include assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros, and mRAGE-positive sperm are associated with an increase in sperm with low MMP.

[0136] The method for assessing sperm health may further include assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros, and an increase in mRAGE-positive sperm is observed in sperm with low MMP, indicating poor sperm health.

[0137] The method for assessing sperm health may further include assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros, and mRAGE-negative sperm are found to have an increased number of sperm with high MMP.

[0138] The method for assessing sperm health may further include assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros, and an increase in sperm with high MMP is observed among mRAGE-negative sperm, indicating good sperm health.

[0139] The method for assessing sperm health may further include assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros, and mRAGE-negative sperm show an increase in sperm with high MMP compared to mRAGE-positive sperm.

[0140] The method for assessing sperm health may further include assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros, and mRAGE-negative sperm show at least a two-fold increase in high MMP compared to RAGE-positive sperm.

[0141] The method for assessing sperm health may further include assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros, and mRAGE-negative sperm show at least a two-fold increase in high MMP compared to RAGE-positive sperm, indicating good sperm health.

[0142] The method for assessing sperm health may further include assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros, and mRAGE-negative sperm show at least a five-fold increase in high MMP sperm compared to RAGE-positive sperm.

[0143] The method for assessing sperm health may further include assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros, and mRAGE-negative sperm show at least a five-fold increase in high MMP sperm compared to RAGE-positive sperm, indicating good sperm health.

[0144] The method for assessing sperm health may further include assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros, and wherein mRAGE-negative sperm show at least a 10-fold increase in high MMP sperm compared to mRAGE-positive sperm.

[0145] The method for assessing sperm health may further include assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros, and wherein mRAGE-negative sperm show at least a 10-fold increase in high MMP sperm compared to mRAGE-positive sperm, indicating good sperm health.

[0146] The method for assessing sperm health may further include assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros, and mRAGE-positive sperm show at least a two-fold reduction in high MMP compared to RAGE-negative sperm.

[0147] The method for assessing sperm health may further include assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros, and mRAGE-positive sperm show at least a two-fold decrease in high MMP sperm compared to RAGE-negative sperm, indicating poor sperm health.

[0148] The method for assessing sperm health may further include assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros, and mRAGE-positive sperm show at least a five-fold reduction in high MMP sperm compared to RAGE-negative sperm.

[0149] The method for assessing sperm health may further include assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros, and mRAGE-positive sperm show at least a five-fold decrease in high MMP sperm compared to RAGE-negative sperm, indicating poor sperm health.

[0150] The method for assessing sperm health may further include assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros, and mRAGE-positive sperm show at least a 10-fold decrease in high MMP sperm compared to mRAGE-negative sperm, indicating poor sperm health.

[0151] The method for assessing sperm health may further include assessing MMP, wherein MMP is assessed by staining with MitoTracker CMX Ros, and mRAGE-positive sperm show at least a 10-fold reduction in high MMP sperm compared to mRAGE-negative sperm.

[0152] Quantifiable levels of MMP may indicate mRAGE-negative sperm, and the absence of quantifiable levels of MMP may indicate mRAGE-positive sperm.

[0153] The method for assessing sperm health may further comprise assessing cell permeability.

[0154] The method of assessing sperm health may further comprise assessing cell permeability, wherein cell permeability is assessed by staining with DAPI.

[0155] The method for assessing sperm health may further include assessing cell permeability, wherein cell permeability is assessed by staining with DAPI, wherein positive staining with DAPI indicates non-viable cells.

[0156] The method for assessing sperm health may further comprise assessing cell permeability, wherein cell permeability is assessed by staining with DAPI, where positive staining with DAPI indicates non-viable cells, and where mRAGE-positive sperm exhibit an increase in non-viable cells.

[0157] The method for assessing sperm health may further comprise assessing cell permeability, wherein cell permeability is assessed by staining with DAPI, where positive staining with DAPI indicates non-viable cells, and mRAGE-positive sperm show an increase in non-viable cells, indicating poor sperm health.

[0158] The method for assessing sperm health may further comprise assessing cell permeability, wherein cell permeability is assessed by staining with DAPI, where positive staining with DAPI indicates non-viable cells, and where mRAGE-positive sperm show an increase in non-viable cells compared to mRAGE-negative sperm, indicating poor sperm health.

[0159] The method for assessing sperm health may further include assessing cell permeability, wherein cell permeability is assessed by staining with DAPI, wherein positive staining with DAPI indicates non-viable cells, and wherein mRAGE-positive sperm show at least a two-fold increase in non-viable cells compared to mRAGE-negative sperm, indicating poor sperm health.

[0160] The method for assessing sperm health may further include assessing cell permeability, wherein cell permeability is assessed by staining with DAPI, wherein positive staining with DAPI indicates non-viable cells, and wherein mRAGE-positive sperm show at least a three-fold increase in non-viable cells compared to mRAGE-negative sperm, indicating poor sperm health.

[0161] mRAGE-positive sperm may have at least a two-fold reduction in non-permeable cells compared to mRAGE-negative sperm, indicating poor sperm health.

[0162] The method for assessing sperm health may further include a procedure for assessing apoptosis.

[0163] The method of assessing sperm health may further comprise assessing apoptosis, wherein apoptosis is assessed by staining with Annexin V.

[0164] The method for assessing sperm health may further comprise assessing apoptosis, wherein apoptosis is assessed by staining with Annexin V, and positive staining with Annexin V indicates poor sperm health.

[0165] The method for assessing sperm health may further comprise assessing apoptosis, wherein apoptosis is assessed by staining with a combination of Annexin V and DAPI.

[0166] The method for assessing sperm health may further comprise assessing apoptosis, wherein apoptosis is assessed by staining with a combination of Annexin V and DAPI to allow simultaneous assessment of apoptosis and necrosis.

[0167] The method for assessing sperm health may further comprise assessing apoptosis, wherein apoptosis is assessed by staining with a combination of Annexin V and DAPI to allow simultaneous assessment of apoptosis and necrosis, and positive staining with the combination of Annexin V and DAPI indicates poor sperm health.

[0168] The method for assessing sperm health may further include assessing apoptosis, wherein apoptosis is assessed by staining with a combination of Annexin V and DAPI to enable simultaneous assessment of apoptosis and necrosis, and wherein mRAGE-positive sperm exhibit increased apoptosis and necrosis.

[0169] The method for assessing sperm health may further include assessing apoptosis, wherein apoptosis is assessed by staining with a combination of Annexin V and DAPI to enable simultaneous assessment of apoptosis and necrosis, and wherein mRAGE-positive sperm show increased apoptosis and necrosis, indicating poor sperm health.

[0170] The method for assessing sperm health may further include assessing apoptosis, wherein apoptosis is assessed by staining with a combination of Annexin V and DAPI to enable simultaneous assessment of apoptosis and necrosis, and wherein mRAGE-positive sperm show increased apoptosis and necrosis compared to mRAGE-negative sperm, indicating poor sperm health.

[0171] The method for assessing sperm health may further include assessing apoptosis, wherein apoptosis is assessed by staining with a combination of Annexin V and DAPI to enable simultaneous assessment of apoptosis and necrosis, and wherein mRAGE-positive sperm exhibit at least a two-fold increase in apoptosis and necrosis compared to mRAGE-negative sperm, indicating poor sperm health.

[0172] The method for assessing sperm health may further comprise assessing apoptosis, wherein apoptosis is assessed by staining with a combination of Annexin V and DAPI to enable simultaneous assessment of apoptosis and necrosis, and wherein mRAGE-positive sperm exhibit at least a four-fold increase in apoptosis and necrosis compared to mRAGE-negative sperm, indicating poor sperm health.

[0173] The method for assessing sperm health may further include assessing apoptosis, wherein apoptosis is assessed by staining with a combination of Annexin V and DAPI to enable simultaneous assessment of apoptosis and necrosis, and wherein mRAGE-positive sperm show at least a four-fold increase in apoptosis and necrosis compared to mRAGE-negative sperm, indicating poor sperm health.

[0174] The method for assessing sperm health may further comprise assessing DNA fragmentation.

[0175] The method for assessing sperm health may further comprise assessing DNA fragmentation by ApoBrdU terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis.

[0176] The method for assessing sperm health may further include assessing DNA fragmentation by ApoBrdU terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis, wherein mRAGE-positive sperm exhibit increased DNA fragmentation, indicating poor sperm health.

[0177] The method for assessing sperm health may further include assessing DNA fragmentation by ApoBrdU terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis, wherein mRAGE-positive sperm exhibit increased DNA fragmentation compared to mRAGE-negative sperm, indicating poor sperm health.

[0178] The method for assessing sperm health may further include assessing DNA fragmentation by ApoBrdU terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis, wherein mRAGE-positive sperm exhibit at least a two-fold increase in DNA fragmentation compared to mRAGE-negative sperm, indicating poor sperm health.

[0179] The method for assessing sperm health may further include assessing DNA fragmentation by ApoBrdU terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis, wherein mRAGE-positive sperm exhibit at least a three-fold increase in DNA fragmentation compared to mRAGE-negative sperm, indicating poor sperm health.

[0180] The method for assessing sperm health may further include a procedure for identifying sperm that can be used in assisted reproductive technologies (ART).

[0181] The method of assessing sperm health may further include identifying sperm that can be used in assisted reproductive technologies (ART), including intrauterine insemination (IUI).

[0182] The method of assessing sperm health may further include identifying sperm that can be used in assisted reproductive technologies (ART), including one or more of intrauterine insemination (IUI) and intracytoplasmic sperm injection (ICSI).

[0183] The method of assessing sperm health may further include identifying sperm that can be used in assisted reproductive technologies (ART), including one or more of intrauterine insemination (IUI), intracytoplasmic sperm injection (ICSI), and in vitro fertilization (IVF).

[0184] The method of assessing sperm health may further include identifying sperm that can be used in assisted reproductive technologies (ART), including one or more of intrauterine insemination (IUI), intracytoplasmic sperm injection (ICSI), in vitro fertilization (IVF), and artificial insemination (AI).

[0185] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed.

[0186] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques.

[0187] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques including density gradient centrifugation (DGC).

[0188] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including sperm swim-up.

[0189] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques, including sperm swim-up, as defined in the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen (5th ed., 2010).

[0190] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up.

[0191] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, where DGC separates sperm based on their density.

[0192] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, where DGC separates sperm based on sperm density, as morphologically normal sperm are denser than abnormal sperm.

[0193] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, where DGC separates sperm based on sperm density since morphologically normal sperm are denser than abnormal sperm, and swim-up relies on sperm motility.

[0194] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC), which separates morphologically normal sperm based on sperm density since morphologically normal sperm are denser than abnormal sperm, and swim-up, which relies on sperm motility to separate PR sperm from IM and NP sperm.

[0195] The method for assessing sperm health may further include identifying sperm suitable for use in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, and the DGC method results in a reduction in mRAGE-positive sperm.

[0196] The method for assessing sperm health may further include identifying sperm suitable for use in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, where the swim-up results in a reduction in mRAGE-positive sperm.

[0197] The method for assessing sperm health may further include identifying sperm suitable for use in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, and the DGC and swim-up methods result in a reduction in mRAGE-positive sperm.

[0198] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, and the DGC and swim-up methods result in a reduction in mRAGE-positive sperm, indicating good sperm health.

[0199] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, and the DGC and swim-up results in at least a two-fold reduction in mRAGE-positive sperm compared to mRAGE-negative sperm.

[0200] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, and the DGC method results in at least a two-fold reduction in mRAGE-positive sperm compared to mRAGE-negative sperm, and the swim-up method results in at least a two-fold reduction in mRAGE-positive sperm compared to mRAGE-negative sperm.

[0201] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, and the DGC method results in at least a two-fold reduction in processed mRAGE-positive sperm compared to processed mRAGE-negative sperm, and the swim-up method results in at least a two-fold reduction in processed mRAGE-positive sperm compared to processed mRAGE-negative sperm.

[0202] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, and the DGC method results in at least a three-fold reduction in processed mRAGE-positive sperm compared to processed mRAGE-negative sperm, and the swim-up method results in at least a three-fold reduction in processed mRAGE-positive sperm compared to processed mRAGE-negative sperm.

[0203] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, and the DGC method results in at least a four-fold reduction in processed mRAGE-positive sperm compared to processed mRAGE-negative sperm, and the swim-up method results in at least a four-fold reduction in processed mRAGE-positive sperm compared to processed mRAGE-negative sperm.

[0204] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, and the DGC method results in at least a four-fold reduction in processed mRAGE-positive sperm compared to processed mRAGE-negative sperm, and the swim-up method results in at least a five-fold reduction in processed mRAGE-positive sperm compared to processed mRAGE-negative sperm.

[0205] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, and the DGC method results in an increase in PR sperm.

[0206] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, where the swim-up results in an increase in PR sperm.

[0207] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, and the DGC and swim-up methods result in an increase in PR sperm.

[0208] The method for assessing sperm health may further include identifying sperm suitable for use in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, and the DGC and swim-up methods result in at least a two-fold increase in PR sperm.

[0209] The method for assessing sperm health may further include identifying sperm suitable for use in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, and wherein the DGC results in at least a two-fold increase in treated PR sperm compared to untreated sperm, and the swim-up results in at least a two-fold increase in treated PR sperm compared to untreated sperm.

[0210] The method for assessing sperm health may further include identifying sperm suitable for use in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, and wherein the DGC results in at least a three-fold increase in treated PR sperm compared to untreated sperm, and the swim-up results in at least a three-fold increase in treated PR sperm compared to untreated sperm.

[0211] The method for assessing sperm health may further include identifying sperm suitable for use in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, and wherein the DGC method results in at least a 3.5-fold increase in treated PR sperm compared to untreated sperm, and the swim-up method results in at least a 3.4-fold increase in treated PR sperm compared to untreated sperm.

[0212] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, where the DGC method results in reduced sperm cellular permeability.

[0213] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, where the swim-up results in reduced sperm cellular permeability.

[0214] The method for assessing sperm health may further include identifying sperm suitable for use in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, where the DGC and swim-up result in reduced sperm cellular permeability.

[0215] The method for assessing sperm health may further include identifying sperm suitable for use in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, and the DGC and swim-up result in at least a two-fold decrease in sperm cellular permeability.

[0216] The method for assessing sperm health may further include identifying sperm suitable for use in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, and the DGC and swim-up result in a decrease in sperm cellular permeability of at least 2.75-fold.

[0217] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technologies (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, where the DGC method results in a reduction in apoptotic sperm.

[0218] The method for assessing sperm health may further include identifying sperm suitable for use in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, where the swim-up results in a reduction of apoptotic sperm.

[0219] The method for assessing sperm health may further include identifying sperm suitable for use in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, where the DGC and swim-up methods result in a reduction of apoptotic sperm.

[0220] The method for assessing sperm health may further include identifying sperm suitable for use in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, and wherein the DGC and swim-up methods result in at least a two-fold reduction in apoptotic sperm.

[0221] The method for assessing sperm health may further include identifying sperm suitable for use in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, and the DGC and swim-up methods result in at least a two-fold reduction in treated apoptotic sperm compared to untreated sperm.

[0222] The method for assessing sperm health may further include identifying sperm suitable for use in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques including one or more of density gradient centrifugation (DGC) and sperm swim-up, and the DGC and swim-up methods result in at least a 2.75-fold reduction in treated apoptotic sperm compared to untreated sperm.

[0223] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques such as density gradient centrifugation (DGC) or sperm swim-up, and the DGC method results in increased sperm MMP.

[0224] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques such as density gradient centrifugation (DGC) or sperm swim-up, where the swim-up results in increased sperm MMPs.

[0225] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques such as density gradient centrifugation (DGC) or sperm swim-up, and the DGC and swim-up methods result in increased sperm MMPs.

[0226] The method for assessing sperm health may further include identifying sperm that can be used in assisted reproductive technology (ART), wherein the semen sample is processed using separation techniques such as density gradient centrifugation (DGC) or sperm swim-up, and the DGC and swim-up methods result in at least a 1.5-fold increase in sperm MMP.

[0227] The method for assessing sperm health may further include identifying sperm suitable for use in assisted reproductive technology (ART), wherein the semen sample is treated using a separation technique such as density gradient centrifugation (DGC) or sperm swim-up, and the DGC and swim-up result in at least a 1.5-fold increase in MMPs in the treated sperm compared to untreated sperm.

[0228] The method may be a method for diagnosing infertility.

[0229] The method may be a method for diagnosing infertility, with poor sperm health being indicative of infertility.

[0230] In the method for diagnosing infertility, the patient may be a mammal.

[0231] In the method for diagnosing infertility, the patient may be a non-human.

[0232] In the method for diagnosing infertility, the patient may be an animal other than a human, such as a cow, a horse, a dog, a cat, a pig, a sheep, or a bear.

[0233] In the method for diagnosing infertility, the patient is preferably a human.

[0234] In the method for diagnosing infertility, the patient may be a human who does not have diabetes.

[0235] In the method for diagnosing infertility, the patient may be a non-diabetic, non-smoking human.

[0236] In the method for diagnosing infertility, the patient may be a non-diabetic, non-smoking, non-obese human.

[0237] In the method for diagnosing infertility, the patient may be a human who is not diabetic, does not smoke, and is not obese (BMI<29.9).

[0238] In the method for diagnosing infertility, the patient may be a male who is not diabetic, does not smoke, and is not obese (BMI<29.9).

[0239] The method may be a method for diagnosing infertility, the method comprising providing a biological sample from a patient, measuring a quantitative level of a biomarker in the sample, and diagnosing infertility based on the quantitative level of the biomarker, wherein the biomarker is membrane-bound receptor for advanced glycation end products (mRAGE).

[0240] Preferably, the method is a method for diagnosing infertility, the method comprising providing a semen sample from a patient, measuring a quantitative level of a biomarker in the sample, and diagnosing infertility based on the quantitative level of the biomarker, wherein the biomarker is membrane-bound receptor for advanced glycation end products (mRAGE).

[0241] According to another aspect of the present invention, there is provided a method for treating infertility, the method comprising: (a) a procedure for providing a biological sample from a patient; (b) determining the presence or absence of a quantitative level of membrane-bound receptor for advanced glycation end products (mRAGE) in the sample to define the patient as mRAGE negative or mRAGE positive; and (c) A procedure for treating infertility by selecting mRAGE-negative samples and fertilizing eggs. Includes:

[0242] The method for treating infertility comprises: (a) Procedures for identifying sperm that can be used in assisted reproductive technologies (ART); and (b) a procedure for treating infertility by selecting sperm that can be used for said ART and fertilizing eggs; may also include:

[0243] The method for treating infertility comprises: (a) A procedure for identifying sperm that can be used in assisted reproductive technologies (ART) by providing a semen sample from a patient; (b) determining the presence or absence of quantitative levels of membrane-bound receptor for advanced glycation end products (mRAGE) in the semen sample to define the patient as mRAGE negative or mRAGE positive; and (c) treating infertility by selecting mRAGE-negative sperm and fertilizing an egg with the selected sperm; may also include:

[0244] Embodiments of the present invention will now be described with reference to the accompanying drawings and the following non-limiting examples. [Brief explanation of the drawings]

[0245] [Figure 1] Graph showing that RAGE was found in the acrosome and equatorial regions of the plasma membrane of human sperm. [Figure 2] Diagram showing flow cytometry gating strategy for analyzing membrane RAGE protein expression and molecular markers of sperm cell health. [Figure 3] Diagram showing validation of sperm molecular health analysis by flow cytometry. [Figure 4] Diagram showing flow cytometry and CASA evaluation of direct swim-up specimen analysis for the separation of PR motile sperm from washed sperm. [Figure 5] Figure showing that RAGE expression follows a Gaussian distribution in sperm from a population of non-diabetic, non-smoking, non-obese men. [Figure 6a] Diagram showing that RAGE is a stably expressed protein on sperm that is associated with the immotile sperm population. [Figure 6b] Diagram showing that RAGE is a stably expressed protein on sperm that is associated with the immotile sperm population. [Figure 6c] Diagram showing that RAGE is a stably expressed protein on sperm that is associated with the immotile sperm population. [Figure 6d] Diagram showing that RAGE is a stably expressed protein on sperm that is associated with the immotile sperm population. [Figure 6e] Diagram showing that RAGE is a stably expressed protein on sperm that is associated with the immotile sperm population. [Figure 6f] Diagram showing that RAGE is a stably expressed protein on sperm that is associated with the immotile sperm population. [Figure 7a] Flow cytometry quantification of sperm cell health in washed and purified sperm. [Figure 7b] Flow cytometry quantification of sperm cell health in washed and purified sperm. [Figure 8] FIG. 1 shows that RAGE expression on human sperm is associated with sperm with low mitochondrial membrane potential, dying and dead sperm with permeable plasma membranes. [Figure 8c] FIG. 1 shows that RAGE expression on human sperm is associated with sperm with low mitochondrial membrane potential, dying and dead sperm with permeable plasma membranes. [Figure 9] Figure 1 shows that RAGE expression in human sperm correlates with the degree of DNA fragmentation. [Figure 10] Diagram showing assessment of sperm health and RAGE protein expression in human sperm. [Figure 10c] Diagram showing assessment of sperm health and RAGE protein expression in human sperm. [Figure 11] Figure 1 shows the expression of RAGE protein in untreated and treated human sperm and assessment of sperm cell health. [Figure 11d] Figure 1 shows the expression of RAGE protein in untreated and treated human sperm and assessment of sperm cell health. [Figure 12] FIG. 1 shows an evaluation of the correlation between RAGE protein expression and sperm motility after treatment with the DGC method or the swim-up method. [Figure 12c] FIG. 1 shows an evaluation of the correlation between RAGE protein expression and sperm motility after treatment with the DGC method or the swim-up method. [Figure 13] Figure 1 shows an evaluation of two motility assessment methods and their correlation with sperm morphology. [Figure 14] 1A-1C show assessment of mRAGE protein expression, sperm health, and sperm morphology. DETAILED DESCRIPTION OF THE INVENTION

[0246] Materials and Methods

[0247] Ethics approval

[0248] The study was conducted using semen samples from anonymous patients treated at the Merrion Fertility Clinic, with donor permission and ethical approval from the Faculty of Medicine Research Ethics Committee, Trinity College Dublin, and the National Maternity Hospital Ethics Committee, Holles Street. The study was conducted at the Trinity Institute for Biomedical Sciences, Trinity College Dublin.

[0249] Study population and participants

[0250] Men receiving treatment at Merrison Fertility Clinic between August 2017 and July 2018 were invited to participate in the study, and written consent was obtained from all participants. Inclusion criteria were samples collected from normal-sperm patients undergoing fertility evaluation. Exclusion criteria included participants with type 1 or type 2 diabetes, smokers, and / or samples collected from individuals with a BMI > 29.9.

[0251] Sample collection and analysis

[0252] Semen samples were collected after 2-5 days of sexual abstinence. Samples were collected in sterile plastic containers (Starstedt, 75-562-105) and liquefied at 37°C for 30 minutes. Semen analysis was performed within 1 hour of ejaculation. All samples were subjected to conventional analysis to determine semen volume (ml), sperm concentration (×10), and sperm count (×10) according to World Health Organization (WHO) recommendations (WHO, 5th edition). 6 / ml), sperm morphology and motility were determined.

[0253] Assessment of sperm morphology

[0254] Sperm morphology was assessed using a simple normal / abnormal classification. Prestained slides (TestSimplet, Waldeck) were used according to the strict Kruger (Tygerberg) criteria. Briefly, a small drop of semen was added to the center of a coverslip, carefully placed on the prestained area of ​​the slide, and incubated for 15 minutes at room temperature. A small drop of oil was placed on the center of the coverslip, and morphology was assessed using a 100x oil immersion lens mounted on an Eclipse E400 microscope. Approximately 200 sperm were graded to determine the percentage of normal (score >4% normal) or abnormal (score <4% normal) morphology.

[0255] Sperm motility assessment

[0256] Sperm motility was assessed by CASA using Hamilton Thorne software and a Zeiss microscope, following WHO and manufacturer guidelines. Briefly, semen samples were resuspended with a Pasteur pipette and diluted 1 part in 3 parts sperm wash medium. 3 μl of diluted semen was added to a CASA microscope slide, placed on a preheated stage (37°C), and allowed to settle until no drift was visible on the screen. Motility was assessed by capturing the tracks of at least 1000 sperm cells per slide for untreated samples and at least 300 sperm cells per slide for treated samples. The total number of cells (×10 6 / ml), the percentage of immotile sperm (IM), non-progressively motile sperm (NP) and progressively motile sperm (PR), and the mean path velocity (VAP) of sperm were recorded for each sample.

[0257] Sperm washing

[0258] Seminal plasma was removed by centrifugation at 1500 × g for 1 minute at room temperature, followed by pouring the sperm into 0.5 ml of Sperm Washing Medium (Irvine Scientific, 9983) twice. The final sperm pellet was resuspended in 0.1 ml of Sperm Washing Medium prior to further experiments. This sample is hereafter referred to as "washed sperm."

[0259] Sperm preparation

[0260] Semen samples were purified by density gradient centrifugation (DGC) or direct swim-up. For DGC, puresperm 40 / 80 Nidacon PS40-100 / PS80-100 was used as the separation medium. 1.5 ml of 40% bottom separation medium was added to a 15 ml Falcon tube, followed by 1.5 ml of 80% top separation medium. A minimum of 1 ml of semen sample was loaded onto the gradient and centrifuged at 400 × g for 20 minutes at room temperature. Approximately 2.5 ml of the top layer was gently removed with a sterile Pasteur pipette, and the sperm pellet was washed twice with 2 ml of sperm wash medium and centrifuged at 380 × g for 5 minutes at room temperature. This washed pellet, containing highly motile purified sperm, was resuspended in 0.5 ml of sperm wash medium. For direct swim-up, a minimum of 1 ml of seminal plasma was washed twice by centrifuging 2 ml of sperm wash medium at 380 x g for 5 minutes at room temperature. The washed sperm pellet was gently resuspended and layered under 1 ml of multi-purpose treatment medium (Irvine Scientific, 90166), pre-equilibrated at 37°C, and incubated at room temperature for 30 minutes. Approximately 0.8 ml of the top layer, containing highly motile purified sperm, was removed.

[0261] Quantitative assessment of sperm cell health

[0262] Mitochondrial membrane potential (MMP), cell permeability, and cell death were measured using MitoTracker CMX Ros (ThermoFisher), DAPI (4',6-diamidino-2-phenylindole; ThermoFisher), and Annexin V-PeCy7 (ThermoFisher), respectively. 6Sperm were washed in 0.5 ml of sperm washing medium and then washed in two steps in 0.5 ml of PBS containing 1% FBS. MMP was assessed by incubation with 6.25 nM MitoTracker CMX Ros (Invitrogen; M7512) for 15 min at 37°C. Cell permeability was assessed by incubation with 35 ng / ml DAPI (Invitrogen, D1306) in 0.1 ml of PBS containing 1% FBS for 5 min at 37°C, protected from light. Samples were washed twice with 0.5 ml of PBS containing 1% FBS before flow cytometry analysis. 1 × 10 6 Sperm were poured into 0.5 ml of 1x Annexin V binding buffer and then incubated with 5 μl of Annexin V-PE-Cy7 (Invitrogen; 8007-72) in 0.1 ml of 1x binding buffer for 15 min at room temperature to assess apoptosis and necrosis.

[0263] Quantitative assessment of RAGE protein expression

[0264] RAGE protein expression was measured using the monoclonal mouse anti-human mRAGE antibody AF647 (Santa Cruz; 80652). Briefly, 1 × 10 cells were cultured in PBS containing 1% FBS. 6 After injecting sperm, the cells were incubated with 2 μg / ml of RAGE antibody at 37°C for 1 hour in the dark. After washing the samples twice with 0.5 ml of PBS containing 1% FBS, flow cytometry analysis was performed.

[0265] Quantitative assessment of DNA fragmentation

[0266] DNA fragmentation was measured using the ApoBrdU terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay according to the manufacturer's instructions (Invitrogen; A23210) with slight modifications. Briefly, after washing in PBS containing 1% FBS, 5 × 10 6Washed sperm cells were fixed with 4% PFA for 30 minutes and washed twice with 1% FBS in PBS. Fixed cells were permeabilized by incubation in 0.1 ml of 0.1% Triton-X, 1% sodium citrate in PBS for 2 minutes on ice and then washed twice with 0.5 ml of the wash buffer provided with the kit. Fixed and permeabilized cells were incubated with the TdT TUNEL reaction (TdT enzyme, BrdU, reaction buffer) for 1 hour at 37°C, washed twice with 0.5 ml of rinse buffer, and incubated with mouse anti-BrdU-AF488 antibody (Invitrogen) for 30 minutes at room temperature. Cells were washed twice with 0.5 ml of PBS containing 1% FBS and counterstained with 10 μl of PI in the RNase A buffer provided with the kit.

[0267] Analyzing sperm cell health by flow cytometry

[0268] For flow cytometry data collection, a BD LSR Fortessa flow cytometer was used, recording a minimum of 10,000 single events per sample at a flow rate of 200 events / second. DAPI was measured using a 450 / 50 bandpass filter, CMX Ros using a 610 / 20 filter, PE-Cy7 using a 780 / 60 filter, AF647 using a 670 / 14 filter, and AF488 using a 530 / 30 filter. Sperm were gated on the flame area using FSC-A and SSC-A to remove non-sperm debris and cells. Doublets were excluded using a dot plot of FSC-A and FSC-W. Negative and positive populations were selected by gating on each fluorophore using a rectangular gate on FSC-A. Quadrant plots were used to analyze apoptotic and necrotic spermatids.

[0269] Confocal analysis of RAGE and mitochondrial localization

[0270] For microscopic evaluation of RAGE and mitochondrial localization on sperm, each slide was observed using a Leica SP8 Gated STED confocal microscope. 6Washed, untreated human sperm cells were incubated with 4 μg / ml mouse anti-human RAGE primary antibody (Santa Cruz; sc-80652) at 37°C for 1 hour, followed by incubation with 2 μg / ml goat anti-mouse AF647 secondary antibody (ThermoFisher; A28181) at 37°C for 30 minutes. For mitochondrial localization, 1 × 10 6 Purified human sperm cells (by DGC) were incubated with 0.5 μM MitoTracker CMX Ros for 15 minutes at 37°C. Stained cells were fixed with 0.5 ml of 4% PFA pH 7.2 for 20 minutes at room temperature and incubated with 70 ng / ml DAPI. 20 μl of stained sperm cells were placed on a poly-L-lysine microscope slide and air-dried at 37°C. Slides were then prepared by adding mounting medium and a coverslip.

[0271] statistical analysis

[0272] Statistical analysis was performed using Prism 6.0 software (GraphPad). Morphology and mRAGE protein expression were compared using an unpaired parametric t-test. Motility and mRAGE protein expression were compared using a paired parametric t-test. Flow cytometry markers were compared using a paired parametric t-test. All data were assumed to be normally distributed. Values ​​are expressed as ±SD. P values ​​less than 0.05 were considered statistically significant. Pearson's correlation test was performed to analyze the correlation between RAGE protein expression and motility. One-way ANOVA was performed to compare flow cytometry marker expression across untreated and treated samples using a two-tailed P value. P values ​​less than 0.05 were considered statistically significant.

[0273] Example 1

[0274] mRAGE is present in the acrosomal and equatorial membrane regions of human spermatids

[0275] Although RAGE exists in numerous forms (membrane-bound, soluble, or secreted), the detection of membrane-bound RAGE (mRAGE) on human sperm was important for this study.

[0276] Figure 1(a) is a bar graph showing mRAGE-positive fluorescence in untransfected and transfected HEK293T cells analyzed by flow cytometry using a monoclonal RAGE antibody (n=2 biological replicates, SD). Figure 1(b) is a Western blot analysis of RAGE expression in total, cytosolic, and membrane protein lysates from transfected and untransfected HEK293T cells using a monoclonal mouse anti-human RAGE primary antibody and a mouse anti-HRP secondary antibody. Figure 1(c) is a grayscale confocal image showing mRAGE localization in the acrosome and equatorial region on human sperm cells.

[0277] In summary, using a transiently transfected HEK293T cell model, we screened a number of antibodies to detect mRAGE by flow cytometry (Fig. 1(a)) and demonstrated mRAGE detection by Western blot (Fig. 1(b)). Mouse anti-human mRAGE antibodies also detected mRAGE in the midequatorial part and acrosome of sperm cell heads (Fig. 1(c)).

[0278] Example 2

[0279] Validation of a flow cytometry gating method for analyzing mRAGE protein expression and molecular markers of sperm cell health, and analysis of sperm molecular health by flow cytometry

[0280] Figure 2 shows dot plots of gating strategies for analyzing mRAGE protein expression and sperm health in human sperm samples by flow cytometry: (i) a polygonal gate drawn around the sperm cell population; (ii) a rectangular gate drawn around single cells excluding doublets; (iii) rectangular gates drawn around mRAGE-positive and -negative single cell populations, respectively; (iv) rectangular gates drawn around DAPI-negative, dim, and bright single cell populations, respectively; (v) rectangular gates drawn around MitoTracker-positive (high MMP) and -negative (low MMP) single cell populations, respectively; and (vi) a quadrant plot for analyzing apoptosis and necrosis: Q1 is AnV-DAPI+ (necrosis), Q2 is AnV+DAPI+ (apoptosis), Q3 is AnV+DAPI- (early apoptosis), and Q4 is AnV-DAPI- (vital).

[0281] Figure 3 shows validation of sperm molecular health analysis by flow cytometry. Figure 3(a) is a line graph of human sperm incubated with increasing concentrations of DAPI (n=3). The final concentration selected for further study is indicated by the boxed region. Figure 3(b) is a bar graph showing nuclear staining in unfixed and fixed human sperm incubated with 35 ng / ml DAPI. Figure 3(c) is a line graph of human sperm incubated with increasing concentrations of MitoTracker CMX Ros (n=3). The final concentration selected for further study is indicated by the boxed region. Figure 3(d) is a bar graph showing the mean fluorescence intensity (MFI) of MitoTracker CMX Ros-positive human sperm in the presence or absence of mitochondrial electron transport chain (ETC) inhibitors (FCCP, rotenone, and malonate). Figure 3(e) shows a representative dot plot (top) illustrating the gating strategy for human sperm for TUNEL analysis, along with corresponding flow cytometry dot plots for TUNEL-negative and -positive control reactions (bottom) as indicated. Figure 3(f) is a bar graph showing TUNEL-positive sperm in negative (-TdT enzyme) and positive (+DNase I) control reactions for analyzing DNA fragmentation in human sperm.

[0282] Example 3

[0283] CASA evaluation of flow cytometry and direct swim-up specimen analysis for the isolation of PR motile sperm from washed sperm

[0284] CASA evaluation of flow cytometry and direct swim-up preparation analysis for the isolation of PR motile sperm from washed sperm. Figure 4(a) shows mRAGE protein expression, Figure 4(b) shows PR motility, Figure 4(c) shows cell permeability, Figure 4(d) shows MMP, and Figure 4(e) shows apoptosis rates compared between washed sperm and pellet and supernatant samples obtained during the direct swim-up preparation (n=8; % mean ± SEM). By one-way ANOVA, ****, P < 0.0001, **, P < 0.005, *, P < 0.05.

[0285] Example 4

[0286] In sperm from a population of non-diabetic, non-smoking, non-obese men, mRAGE expression follows a Gaussian distribution.

[0287] Figure 5 shows a histogram of the frequency distribution of mRAGE expression in n = 60 washed sperm samples measured by flow cytometry.

[0288] Overall, mRAGE expression on human sperm was found to be normally distributed in a population of patients being treated at a fertility clinic, in the absence of medical or "self-inflicted" conditions such as diabetes, smoking, or obesity.

[0289] Example 5

[0290] mRAGE is a protein stably expressed on sperm that is associated with the immotile sperm population.

[0291] Figure 6a is a line graph showing the percentage of immotile sperm and mRAGE expression in DGC-purified sperm measured over time (0-72 h) and analyzed by CASA and FC (n=6, % mean ± SEM). Figure 6b is a dot plot showing mRAGE expression in DGC-purified sperm over time (0-72 h). Figure 6c is a comparison of the percentage of PR motility in washed and purified sperm samples (n=15; % mean ± SEM). ****P < 0.0001 by one-way ANOVA. Figure 6d is a comparison of the percentage of mRAGE protein expression in washed and purified sperm samples (n=15; % mean ± SEM). ****P < 0.0001 by one-way ANOVA. Figure 6e is a dot plot showing mRAGE expression in washed sperm samples purified by DGC and direct swim-up. Figure 6f shows the correlation between the % total of IM sperm in washed sperm and the percentage of mRAGE protein expression (n=35). Pearson's R 2 Values ​​are shown in the graphs, with error bars within the 95% confidence interval indicated by dashed lines.

[0292] A time-course analysis revealed that mRAGE expression did not change significantly over time (from 4.6% ± 1.3 (SD) to 14.7% ± 7.3), whereas the percentage of IM sperm in purified samples increased from 7.7 ± 1.9% (SD) to 91.3 ± 5.1% (SD) over 72 hours (Fig. 6a and 6b, P < 0.0001). This suggests that mRAGE expression on sperm is directly related to the health status of individuals and does not change after semen sample collection. Notably, the percentage of PR sperm increased, on average, from 20.5 ± 10.9% (SD) in washed samples to 69.1 ± 11.9% in purified samples for both methods (Fig. 6c, P < 0.0001). In contrast, the percentage of mRAGE-expressing sperm decreased, on average, from 22.1 ± 10.1% (SD) in washed samples to 5.5 ± 4.8% (SD) in purified samples (Figures 6d and 6e, P < 0.0001). In a larger sample group, we also observed that mRAGE expression was associated with reduced sperm motility (Figure 6f). This suggests that mRAGE expression may be used to distinguish normal from abnormal sperm at the cellular level. These data indicate that the absence of mRAGE in purified samples is associated with improved PR sperm and cellular health.

[0293] Example 6

[0294] Flow cytometric quantification of sperm cell health in washed and purified sperm

[0295] Figure 7a shows a comparison of cell permeability, apoptotic cell percentage, and MMP percentage in washed, DGC, and swim-up specimens (n ​​= 15; % mean ± SEM). ****P < 0.0001 by one-way ANOVA. Figure 7b shows representative dot plots showing DAPI, MitoTracker CMXRos, and Annexin V fluorescence in washed sperm and in DGC and swim-up specimens.

[0296] Simultaneous analysis of molecular parameters of sperm cell health by flow cytometry revealed significantly improved sperm mitochondrial function, levels of cell death (by apoptosis and necrosis), and membrane integrity in purified samples compared to washed samples (Figures 7a and 7b, P < 0.0001).

[0297] Example 7

[0298] Expression of mRAGE on human sperm is associated with sperm with low mitochondrial membrane potential, dying and dead sperm with permeable plasma membranes.

[0299] Figure 8(a) shows a typical dot plot showing the mRAGE-positive and -negative subgroups in washed sperm, as well as the fluorescence of MitoTracker (MMP), AnnexinV-DAPI (apoptosis or necrosis), and DAPI (cell permeability) in each subgroup of mRAGE-positive and -negative sperm. Figure 8(b) shows a comparison of the percentage of MitoTracker-positive sperm, AnnexinV-DAPI-positive or -negative sperm, and DAPI-positive sperm in each subgroup of mRAGE-positive and -negative sperm (n=35; % mean ± SEM). **** indicates P < 0.0001 by paired t-test. Figure 8c shows an evaluation of the correlation between sperm health markers and mRAGE protein expression in washed samples (n=35). Pearson's R 2 Values ​​are shown in the graphs, with error bars within the 95% confidence interval indicated by dashed lines. ****: P < 0.0001, **: P < 0.005, *: P < 0.05.

[0300] DNA integrity, mitochondrial function, and cell death levels were all used to analyze the molecular health of sperm. The data above indicate improved cell health and reduced mRAGE-expressing sperm in purified samples. In a larger sample set, flow cytometry revealed reduced mitochondrial function (10-fold), increased levels of apoptosis (6-fold) and necrosis (2.5-fold), and reduced membrane integrity (3-fold) in the mRAGE-positive population compared with the mRAGE-negative population, demonstrating that mRAGE expression on sperm correlates with unhealthy sperm (Figures 8 and 8c, P < 0.0001). This data supports the use of mRAGE as a diagnostic biomarker for unhealthy sperm.

[0301] In summary, the data generated support the finding that mRAGE protein expression is elevated in (i) dead or dying cells, (ii) spermatids with low mitochondrial membrane potential, (iii) spermatids dying by apoptosis, and (iv) spermatids dying by necrosis, which may suggest that the mRAGE membrane receptor plays a role in cell surface markers of unhealthy sperm cells.

[0302] Example 8

[0303] Expression of mRAGE in human sperm correlates with the degree of DNA fragmentation.

[0304] Figure 9(a) shows a comparison of the percentage of TUNEL-positive sperm in each mRAGE-positive and -negative subgroup (n=8; % mean ± SEM). Paired t-test, *** indicates P < 0.005. Figure 9(b) shows an evaluation of the correlation between the percentage of TUNEL-positive sperm and the percentage of mRAGE-positive sperm (n=8). Pearson's R 2 Values ​​are shown, with error bars within the 95% confidence interval indicated by dashed lines.

[0305] In non-diabetic men, we observed that the level of DNA fragmentation analyzed by TUNEL analysis was significantly higher (2.5-fold) in mRAGE-positive sperm compared to mRAGE-negative sperm (Figure 9(a) , P < 0.001). The data also indicate that mRAGE expression on sperm correlates with sperm DNA damage (Figure 9(b) , P < 0.05) and may serve as an indirect marker of DNA integrity and sperm cell health.

[0306] Example 9

[0307] Assessment of sperm health and mRAGE protein expression in human sperm

[0308] Assessment of sperm cell health in mRAGE-positive and mRAGE-negative cell populations in unprocessed sperm. mRAGE protein expression was assessed using mouse anti-human mRAGE AF647 antibody. Cell permeability was assessed with DAPI. MMP was assessed with MitoTracker CM Ros. Apoptosis / necrosis was assessed with Annexin V and DAPI. Flow cytometry data were obtained using an LSR Fortessa flow cytometer. Figure 10(a) shows representative dot plots (n=1) of mRAGE-positive and -negative sperm populations, along with the corresponding subpopulations of sperm stained with DAPI, MitoTracker, and Annexin V. Figure 10(b) shows assessment of cell permeability (DAPI-positive cells) and MMP (low-MMP cells) in RAGE-positive and -negative sperm populations (n=35; % mean ± SEM). **** indicates P<0.0001 by paired t-test. Figure 10c: Assessment of apoptotic, early apoptotic, and necrotic spermatids in RAGE-positive and -negative sperm populations (n=35; % mean±SEM). Paired t-test, ****, P<0.0001.

[0309] Flow cytometry dot plot analysis revealed clear differences in cell permeability, MMP, and apoptosis and necrosis between the mRAGE-positive and mRAGE-negative cell populations (Figure 10(a)). Statistical analysis revealed a 3-fold increase in DAPI-stained, nonviable cells in the mRAGE-positive cell population compared to the mRAGE-negative cell population (Figure 10(b); P<0.0001). There was also a 10-fold increase in high MMP cells in the mRAGE-negative cell population compared to the mRAGE-positive cell population (Figure 10(b); P<0.0001). There was also a 6-fold and 2.5-fold increase in apoptotic and necrotic cells, respectively, in the mRAGE-positive cell population compared to the mRAGE-negative cell population (Figure 10(c); P<0.0001).

[0310] Example 10

[0311] Expression of mRAGE protein in unprocessed and processed human sperm and assessment of sperm cell health

[0312] The next set of experiments was designed to determine whether there were differences in mRAGE protein expression after highly motile sperm were isolated from unprocessed semen samples. Highly motile sperm can be isolated from the total sperm population by swim-up purification and / or DGC purification.

[0313] Figure 11(a) shows a representative dot plot (n=1) depicting the populations of mRAGE-positive and -negative sperm in untreated (unadulterated) sperm and sperm treated with DGC and swim-up. Figure 11(b) shows the assessment of mRAGE-expressing sperm cells (mRAGE-positive sperm cells) in untreated sperm and sperm treated with DGC and swim-up (n=15; % mean ± SEM). The corresponding untreated (unadulterated) fractions were included for each treatment method. ****P<0.0001 by paired one-way ANOVA. Figure 11(c) shows the assessment of forward motility in untreated sperm and sperm treated with DGC and swim-up (n=15; % mean ± SEM). The corresponding untreated (unadulterated) fractions were included for each treatment method. ****P<0.0001 by paired one-way ANOVA. Figure 11d shows the assessment of cell permeability (DAPI-positive cells), high MMP cells, and apoptotic sperm in untreated and DGC- and swim-up-treated stained sperm (n=15; % mean ± SEM). A corresponding untreated (unadulterated) fraction was included for each treatment method. ****P<0.0001 by paired one-way ANOVA.

[0314] A 5-fold and 4-fold decrease in RAGE protein expression was observed in DGC- and swim-up-purified sperm (Fig. 11(b); P<0.0001). Concurrently, a 3.5-fold and 3.4-fold increase in progressively motile sperm was observed in DGC- and swim-up-purified sperm (Fig. 11(c); P<0.0001). This was accompanied by an average 2.75-fold decrease in cell permeability impairment, a 2.75-fold decrease in apoptotic sperm, and a 1.5-fold increase in MMP expression in DGC- and swim-up-purified sperm (Fig. 11d; P<0.0001).

[0315] In summary, both methods of sperm purification discussed here resulted in sperm with high forward motility and vitality, impermeable membranes, and highly active mitochondria. In addition, the reduced expression of mRAGE protein in the purified fraction suggests that mRAGE-expressing sperm are removed during the purification process.

[0316] Example 11

[0317] Evaluation of the correlation between mRAGE protein expression and sperm motility after treatment with DGC or swim-up method

[0318] Figure 12(a) shows the correlation between mRAGE-expressing sperm (mRAGE-positive cells) and immotile cells present after DGC and swim-up treatment (n=15; % mean ± SEM). Figure 12(b) shows the correlation between mRAGE-non-expressing sperm (mRAGE-negative cells) and motile cells present after DGC and swim-up treatment (n=15). Figure 12c shows the correlation between mRAGE-non-expressing sperm (mRAGE-negative cells) and motile sperm present after DGC or swim-up treatment (n=15). Pearson's R correlation 2 Values ​​are shown on the graph, with error bars within the 95% confidence interval indicated by dashed lines. **** indicates P < 0.0001.

[0319] Further analysis of the data revealed that there was a statistically significant correlation between mRAGE protein expression and sperm motility in the treated samples and generated a correlation plot. The statistically significant correlation was between mRAGE-positive cells and immotile sperm (Figure 12(a); R 2 >0.84), between mRAGE-negative cells and motile sperm (Figure 12(b); R 2 >0.82), and between mRAGE-negative cells and progressively motile sperm (Fig. 12c; R 2 >0.39).

[0320] This data confirms that the swim-up and DGC purification method results in a highly motile sperm fraction that is very depleted in mRAGE-expressing sperm. The mRAGE-expressing sperm remaining in the purified fraction may be related to the low percentage of non-motile / slow sperm remaining after the processing event.

[0321] Altogether, the results of these experiments indicate that mRAGE protein expression is associated with immotile sperm, and conversely, sperm cells that do not express mRAGE represent healthy, highly motile sperm.

[0322] Example 12

[0323] Evaluation of two motility assessment methods and their correlation with sperm morphology

[0324] Evaluation of two sperm motility assessment methods, CASA and manual motility assessment, and correlation between normal and abnormal morphology in unprocessed semen samples. Sperm morphology was assessed using Test-Simplet slides and the strict Kruger criteria. CASA motility assessment was performed using Hamilton Thorne's CASA software. Manual sperm motility assessment and morphology assessment were performed at Merrison Fertility Clinic as part of their routine semen analysis procedures. Figure 13(a) shows sperm motility (non-motile, motile, and progressive) measured by CASA in patients with normal and abnormal morphology (n=35; mean SEM). ***, P<0.0005; **, P<0.005 by unpaired t-test. Figure 13(b) shows sperm motility (non-motile, motile, and progressive) measured by manual motility assessment in patients with normal and abnormal morphology (n=35; mean SEM). *** indicates P<0.0005 by unpaired t-test.

[0325] In sperm samples with abnormal morphology, a statistically significant increase was observed in immotile sperm measured by the CASA (Figure 13(a)) and manual (Figure 13(b)) methods (P = 0.0048 and P = 0.0007, respectively). Corresponding results were observed when comparing total motility and forward motility with sperm morphology (Figures 13(a) and (b); P values ​​ranged from 0.0030 to 0.007). Although there was a difference in statistical significance between the two motility assessment methods, the trends between the two groups were similar. This data demonstrates the known phenomenon that motility and morphology are correlated and indicate sperm health.

[0326] Example 13

[0327] Assessment of RAGE protein expression, sperm health, and sperm morphology

[0328] Figure 14(a) shows the evaluation of RAGE protein expression, cell permeability (DAPI-positive cells), and MMP (low MMP cells) in patients with normal and abnormal morphology (n=35; % mean ± SEM). ***, P<0.0005, *, P<0.005 by unpaired t-test. Figure 14(b) shows the evaluation of apoptotic cells, early apoptotic cells, and necrotic cells in patients with normal and abnormal morphology (n=35; % mean ± SEM).

[0329] Subsequent analysis of the flow cytometry data and morphology data using a paired t-test revealed a statistically significant difference between RAGE protein expression and MMP in patients with normal and abnormal morphology (Figure 14(a); P=0.0002 and P=0.0236, respectively). This data complements the previously measured data and further supports our hypothesis that mRAGE protein expression is associated with unhealthy, immotile sperm, and now reveals a new association with morphologically abnormal sperm.

[0330] Example 14

[0331] Quantification of mRAGE-positive and mRAGE-negative sperm cells and mitochondrial membrane potential (MMP)

[0332] Table 1 shows a comparison of pure samples from treated and untreated experiments (N=15). The mean fluorescence intensity (MFI, geometric mean) was analyzed for mRAGE-positive and mRAGE-negative sperm cells, and the MFI for high-MMP and low-MMP sperm cells was analyzed.

[0333] Table 1 shows the percentage of mRAGE-positive and mRAGE-negative sperm cells. It also shows the MFI (geometric mean) of mRAGE-positive and mRAGE-negative sperm cells. It also shows the fold difference between the MFI of mRAGE-positive and mRAGE-negative sperm cells. The average fold difference in MFI between mRAGE-positive and mRAGE-negative sperm cells is 21.1% ± 13.8%.

[0334] Table 1 shows the percentage of high-MMP and low-MMP sperm cells. It also shows the MFI (geometric mean) of high-MMP and low-MMP sperm cells. It also shows the fold difference between the MFI of high-MMP and low-MMP sperm cells. The average fold difference in MFI between high-MMP and low-MMP cells is 25.3% ± 10.1%.

[0335] [Table 1]

Claims

1. In an in vitro fertilization method to evaluate sperm health, (a) a procedure for measuring the quantitative or qualitative level of a biomarker in a previously obtained semen sample from a patient; and (b) assessing sperm health based on the quantitative or qualitative levels of said biomarkers; wherein the biomarker is membrane-associated receptor for advanced glycation end products (mRAGE).

2. 2. The method of claim 1, wherein the evaluating step (b) comprises determining whether the patient is mRAGE negative or mRAGE positive by assessing the presence or absence of a quantifiable level of mRAGE on sperm cells from the sample.

3. The method of claim 2, wherein a positive mRAGE result indicates one or more of poor sperm health, abnormal sperm, low sperm motility, and infertility, and a negative mRAGE result indicates one or more of normal sperm and good sperm health.

4. The method of claim 2 or 3, wherein the presence of a quantifiable level of mRAGE on sperm cells from the sample defines the patient as mRAGE positive, and the absence of a quantifiable level of mRAGE on sperm cells from the sample defines the patient as mRAGE negative.

5. 5. The method of any one of claims 1 to 4, wherein the evaluating step (b) comprises comparing the amount of sperm expressing a quantifiable level of mRAGE with the amount of sperm not expressing a quantifiable level of mRAGE.

6. 6. The method of claim 5, wherein the amount of sperm expressing a quantifiable level of mRAGE deviates by at least 1-fold from the amount of sperm not expressing a quantifiable level of mRAGE.

7. The method of claim 6, wherein the patient is determined to be mRAGE positive if the amount of sperm expressing a quantifiable level of mRAGE deviates by at least one-fold from the amount of sperm not expressing a quantifiable level of mRAGE, and / or the patient is determined to be mRAGE negative if the amount of sperm not expressing a quantifiable level of mRAGE deviates by at least one-fold from the amount of sperm expressing a quantifiable level of mRAGE.

8. 8. The method of claim 1, wherein the patient is determined to be mRAGE positive if the mean fluorescence intensity value of sperm expressing a quantifiable level of mRAGE deviates by at least 10-fold from the mean fluorescence intensity value of sperm not expressing a quantifiable level of mRAGE, and / or the patient is determined to be mRAGE negative if the mean fluorescence intensity value of sperm not expressing a quantifiable level of mRAGE deviates by at least 10-fold from the mean fluorescence intensity value of sperm expressing a quantifiable level of mRAGE.

9. 9. The method of any one of claims 1 to 8, wherein the method for assessing sperm health further comprises the step of rating sperm motility as IM, NP or PR as defined in the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen (5th edition, 2010), wherein sperm classified as IM indicate poor sperm health, and sperm classified as NP and / or PR indicate good sperm health.

10. 10. The method of claim 9, wherein the amount of mRAGE-positive sperm is inversely correlated with the amount of PR sperm.

11. 11. The method of any one of claims 1 to 10, wherein the method for assessing sperm health further comprises assessing sperm morphology, wherein sperm are classified as normal when the sperm heads are smooth, well-defined and generally oval, and sperm are classified as abnormal when there is a deviation from normal, which is indicative of poor sperm health.

12. 12. The method of any one of claims 1 to 11, wherein the method for assessing sperm health further comprises assessing mitochondrial membrane potential (MMP), wherein a quantifiable level of MMP indicates mRAGE-positive sperm and the absence of a quantifiable level of MMP indicates mRAGE-negative sperm.

13. 13. The method of any one of claims 1 to 12, wherein the method for assessing sperm health further comprises assessing cell permeability, wherein a decrease in cell permeability of at least two-fold in mRAGE-positive sperm compared to mRAGE-negative sperm indicates poor sperm health; and / or the method for assessing sperm health further comprises assessing apoptosis and necrosis, wherein a decrease in apoptosis and necrosis of at least two-fold in mRAGE-positive sperm compared to mRAGE-negative sperm indicates poor sperm health; and / or the method for assessing sperm health further comprises assessing DNA fragmentation, wherein a decrease in DNA fragmentation of at least two-fold in mRAGE-positive sperm compared to mRAGE-negative sperm indicates poor sperm health.

14. 14. The method of any one of claims 1 to 13, wherein the method for assessing sperm health further comprises processing the sample using a separation technique comprising one or more of density gradient centrifugation (DGC) and sperm swim-up.

15. As a result of the DGC method and the swim-up method, (a) at least a two-fold reduction in mRAGE-positive sperm compared to mRAGE-negative sperm; and / or (b) at least a two-fold increase in PR sperm; and / or (c) at least a two-fold decrease in cell permeability in sperm; and / or (d) At least a two-fold decrease in apoptotic spermatozoa 15. The method of claim 14, wherein the sperm are indicative of sperm that can be used in assisted reproductive technologies (ART), including one or more of intrauterine insemination (IUI), intracytoplasmic sperm injection (ICSI), and in vitro fertilization (IVF).

16. The method of any of claims 1 to 15, wherein the patient is one or more of: non-diabetic, non-smoking, and non-obese.

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