WO2025104680A1

WO2025104680A1 - Dna accessibility for the assessment of male fertility

Info

Publication number: WO2025104680A1
Application number: PCT/IB2024/061390
Authority: WO
Inventors: Christian M.H.R. DE GEYTER; Mark E. GILL; Antoine Hendrik Felix Marie PETERS
Original assignee: Universitaet Basel; Novartis Forschungsstiftung Zweigniederlassung Friedrich Miescher Institute for Biomedical Research
Current assignee: Universitaet Basel; Novartis Forschungsstiftung Zweigniederlassung Friedrich Miescher Institute for Biomedical Research
Priority date: 2023-11-17
Filing date: 2024-11-15
Publication date: 2025-05-22
Anticipated expiration: 2026-05-17

Abstract

The present invention provides a method for assessing male fertility by assessing the DNA accessibility in the spermatozoa of a population of spermatozoa from mammalian semen from a subject. The present invention also provides a method of selecting a population of spermatozoa from a subject, wherein the fertility of the population is assessed by assessing the DNA accessibility in the spermatozoa of the population. In addition, the present invention provides a method of in vitro fertilization comprising the step of selecting a population of spermatozoa from a subject by assessing the DNA accessibility in the spermatozoa of spermatozoa populations from the subject.

Description

DNA accessibility for the assessment of male fertility

FIELD OF THE INVENTION

The present invention relates to a method of assessing male fertility by investigating a population of spermatozoa from mammalian semen.

BACKGROUND OF THE INVENTION

Unintended childlessness is a widespread phenomenon in modern societies. Male infertility is a global population health problem and around 48.5 million couples suffer from infertility worldwide. Around 30 million men worldwide are infertile with the highest rates in Africa and Eastern Europe. Infertility is defined by the World Health Organization (WHO) as a disease of the male or female reproductive system characterized by the failure to achieve a pregnancy within 12 months of regular unprotected sexual intercourse. Investigations have shown that male factors that drive childlessness are often a consequence of decreased functioning of spermatozoa.

Several different causes of infertility in men exist, including gonadal disorders (30-40%), disorders of sperm transport (10-20%) or hypothalamic or pituitary disorders (1 -2%), but most commonly the cause is unknown (40-50% of cases). Sperm can be abnormal for several different reasons; the most common reasons are unusually short life span of the sperm or low mobility, or both in combination. Sperm abnormalities can be caused by a variety of factors including e.g., inflammation of the testis, varicoceles (swollen veins in the scrotum), abnormally developed testis, genetic disorders, or hormonal problems.

Assessment of semen quality is the major way in which male fertility potential is currently evaluated. Semen quality is a measure of the ability of the semen to accomplish fertilization, and a decrease in semen quality is associated with male infertility. A semen analysis evaluates certain characteristics of a male's semen and the spermatozoa contained within. The most common and conventional variables measured to evaluate sperm quality are sperm count, motility and morphology. The cutoff levels of all these conventional parameters have been determined and revised repeatedly based on population surveys in various parts of the world. Additional variables such as volume, DNA fragmentation index, fructose level and pH are also sometimes measured. The characteristics measured by the current tests for semen analysis offer some prediction for male fertility but do not full predict fertility outcomes, most particularly in assisted reproductive technology. Ideally, novel tests systems might be tailored based on identifiable steps of the process of fertilization and early embryonic development, such as capacitation, oocyte penetration, pronucleus formation and embryonic development. Thus, a decreased level fertility of the spermatozoa plays a significant role when considering means for further therapeutic or non-medicinal steps. Unintendedly childless couples are often interested to know whether their infertility is caused by male and/or female factors in order to consider suitable treatments including sperm or egg donation. Accordingly, it is of particular interest to improve the determination of the fertility of spermatozoa. Likewise, the determination of the fertility capacity of spermatozoa is also of interest for sperm banks in order to eliminate donor samples with low chances for reproductive success.

Additionally, assessment of the fertility of spermatozoa also plays a significant role when breeding animals. The vast majority of larger sized farm animals, such as bovines, are currently bred by means of artificial insemination of the female animals. In this context, numerous aliquots of sperm donations are provided to farmers. Farmers interested in both good breeding success and highly fertile progeny. Likewise, also in projects for conserving biodiversity, an assessment of the fertility of spermatozoa of the animals to be protected is of considerable interest, e.g., in the breeding of endangered species in zoos.

In summary, for humans interested in reproducing as well as for the breeding of non-human animals, an assessment of the fertility of spermatozoa is of considerable interest.

Chromatin refers to the complex of DNA and set of packaging proteins known as histones, which form a structural unit known as the nucleosome, that is present in the nuclei of almost all eukaryotic cells. Unlike in somatic cells the genome of mammalian sperm is packaged in a tightly compacted structure utilizing spermatogenic-specific packaging proteins known as protamines (Gill and Peters 2018). The sperm genome does, however, contain some regions with canonical nucleosomal chromatin structure (Hammoud et al. 2009; Brykczynska et al. 2010). Decreased levels of sperm DNA packaging have been associated with decreased fertility (Erenpreiss et al. 2006), but DNA packaging has not been standardly accepted as a diagnostic feature for human male infertility.

At present two assays are most often used to examine chromatin/DNA packaging in human sperm: Acidic aniline blue (AAB) staining and Chromomycin A3 (CMA3) staining. The AAB assay is simple and straightforward, employing a classical histological stain that binds to the amino acid lysine (Auger et al. 1990). Lysine residues are much more common in the protein sequences of histones than they are in mammalian protamines. The AAB assay makes two major assumptions: (1) that increased lysine signal in the heads of human sperm quantitatively relates to the amount of histone protein found there and (2) that regions of the genome that lack protamine would instead be packaged by histones (which leave the DNA more accessible). Both of these assumptions have not been directly assessed, leaving this assay as an indirect measurement of the chromatin status of human sperm.

CMA3 is a naturally occurring antibiotic compound produced by the soil bacteria Streptomyces griseus. This compound binds to DNA via the minor groove with a high affinity for GC-rich DNA sequences (Hou et al. 2004). CMA3 becomes fluorescent upon binding the DNA in the presence of divalent cations. In somatic cells CMA3 fluorescence shows relatively low intercellular variation, while in human sperm variation in fluorescence is substantial and patients with increased numbers of sperm with high CMA3 levels tend to have decreased rates of success following in vitro fertilization (IVF) (Esterhuizen et al. 2000). Given that the GO percentage of different sperm is identical, the variation in CMA3 levels must reflect variation in DNA structure or packaging. One study has shown that pre-incubating human sperm samples with protamine proteins extracted from salmon sperm is capable of quenching CMA3 fluorescence, leading to the idea that under-protaminated DNA is the source of increased CMA3 levels in sperm (Bianchi et al. 1993). However, treatment of sperm with reducing agents (such as dithiothreitol) leads to global increases in CMA3 staining levels, while protamine remains bound to the genome. These results suggest that CMA3 indeed recognizes something variable in sperm DNA packaging or structure, but what exactly remains unclear. Thus, a more direct, quantitative assay for the accessibility of DNA in human sperm is needed to enable more direct interpretation of the role of chromatin in human male infertility.

A recent advance in the assessment of DNA accessibility in somatic cells was developed by the New England Biolabs (Ponnaluri et al. 2017)(European Patent: EP 3 507 382 B1). This method utilizes an enzyme capable of nicking the DNA backbone at a specific sequence, in a specific chromatin context. The regions surrounding these nicks can then be labelled using a DNA polymerase enzyme and labelled dNTPs. The NEB patent discusses two forms of labels: fluorescence and affinity tags. With fluorescent labels, microscopy or flow cytometry can be used to visualize or quantify levels of labelled DNA in individual cells. With affinity tag labelling, fragments of labelled DNA can be enriched by affinity purification and then subjected to sequencing, allowing determination of which regions of the genome are packaged in a particular way.

SUMMARY OF THE INVENTION

The present inventors have serendipitously found that DNA accessibility as measured by the methods described herein can be used to assess the fertility of spermatozoa of a population. The present invention hence encompasses a method for assessing male fertility by assessing the DNA accessibility in the spermatozoa of a population of spermatozoa from mammalian semen from a subject, said method comprising the steps of isolating a population of spermatozoa from total mammalian semen, permeabilizing the isolated population of spermatozoa using freezing and/or chemical pre-treatment, contacting the permeabilized population of spermatozoa with a composition comprising at least a DNA nicking-inducing agent, a DNA polymerase, four dNTPs and at least one labelled dNTP, incubating the mixture under conditions suitable for the DNA nicking agent to cut one strand of the double-stranded DNA of the permeabilized spermatozoa at several sites, thereby producing single-stranded DNA nicks, and for the DNA polymerase to initiate synthesis of new DNA from said singlestranded DNA nicks using the dNTPs, thereby labelling the newly synthesized DNA by introduction of at least one labelled dNTP into the DNA of the permeabilized population of spermatozoa, and quantifying the presence of labelled dNTP in the labelled population of spermatozoa of step d after at least one washing step, wherein the presence of a significantly higher content of labelled dNTP in the labelled population of spermatozoa from the subject, as compared to the content of labelled dNTP in a comparable sample of spermatozoa obtained from normally fertile control individuals and treated using the same steps as above, is indicative of a poor fertility for said subject. In some embodiments, the isolation of the spermatozoa involves the preparation of spermatozoa via selection of motile spermatozoa capable of swimming through a column of liquid, wherein only the spermatozoa that have been able to reach the upper part of the column are isolated, or the selection of spermatozoa that have been able to swim through a microfluidic chip. In some embodiments, the isolation of the spermatozoa involves the preparation of spermatozoa through selection of spermatozoa via centrifugation through a gradient, for instance a gradient composed of two concentrations of colloidal silica particles. In some embodiments, the isolation of the spermatozoa involves the preparation of spermatozoa via Fluorescence Activated Cell Sorting (FACS) using at least one dye, wherein the spermatozoa are selected based on their uptake of the dye. In some embodiments, the permeabilization of the isolated population of spermatozoa involves the treatment of the isolated population of spermatozoa with DTT and/or other reducing agents. In some embodiments, a step of pre-treatment with a DNA ligase is performed after permeabilization and before the nicking and labelling reaction. This pre-treatment with a DNA ligase, for instance T4 DNA ligase, ensures that most of the labelling will occur in regions of open chromatin. In some embodiments, the DNA nicking- inducing agent used is a chemical agent, such as hydrogen peroxide. In some embodiments, the DNA nicking-inducing agent used is a sequence specific nicking endonuclease, such as Alwl, or a sequence non-specific endonuclease, such as DNAse I. In some embodiments, the labelled dNTP comprises dNTP labelled with an optically detectable label, for instance a fluorophore. In some embodiments, the labelled dNTP comprises dNTP labelled with an affinity tag, such as biotin, for example for subsequent isolation of labelled DNA. In some embodiments, the labelled dNTP comprises dNTP labelled with a chemically modified DNA base, for instance a methylated DNA base, wherein said chemically modified DNA base can be detected by, for instance, DNA sequencing and/or mass spectrometry. In some embodiments, the subject is human, bovine, porcine, ovine, equine or camelid.

The present invention also encompasses a method of selecting a population of spermatozoa from a subject, wherein the fertility of at least two different populations of spermatozoa from said subject is assessed by a method as described in the previous paragraph, and wherein said population is selected based on its fertility as assessed using a method as described in the previous paragraph. In some embodiments, the population of spermatozoa is a population selected from the group of motile spermatozoa, spermatozoa of a specific density, spermatozoa able, or not able, to uptake a specific dye, spermatozoa demonstrating thermotaxis, or testicular spermatozoa.

The present invention also encompasses a method of in vitro fertilization comprising the step of selecting a population of spermatozoa from a subject according to a method as described in the previous paragraph. The present invention also encompasses a kit comprising the reagents necessary to perform the methods described in the preceding paragraphs.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Representative images of NicE-view and T4 NicE-view assay on human sperm using confocal fluorescent microscopy. Left panels show DAPI signal indicating all sperm heads, right panels show NicE-view and T4 NicE-view signal.

Figure 2: Summary of quantification of NicE-view and T4 NicE-view signals from 114 sperm samples derived from 57 individuals. Labels represent study participants with differing reproductive outcomes. Black signals (left) represent quantification of total sperm, while grey signals (right) indicate quantification of swim-up sperm. Grey lines represent median values. 1000 sperm were randomly selected from each sperm sample to equalize signals between each participant.

DETAILED DESCRIPTION OF THE INVENTION

The present invention also encompasses a method of in vitro fertilization comprising the step of selecting a population of spermatozoa from a subject according to a method as described in the previous paragraph.

The present invention also encompasses a kit comprising the reagents necessary to perform the methods described in the preceding paragraphs.

As used herein, the term "animal" is used herein to include all animals having spermatozoa. In some embodiments of the invention, the animal is a vertebrate. Examples of animals are human, mice, rats, cows, pigs, horses, chickens, ducks, geese, cats, dogs, etc. The term "animal" also includes an individual animal in all stages of development, including embryonic and fetal stages. The animal can be a genetically modified animal. A "genetically-modified animal" is any animal containing one or more cells bearing genetic information altered or received, directly or indirectly, by deliberate genetic manipulation at a sub-cellular level, such as by targeted recombination, microinjection or infection with recombinant virus.

In the present invention, "isolated" refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered "by the hand of man" from its natural state.

"Label" refers to agents that are capable of providing a detectable signal, either directly or through interaction with one or more additional members of a signal producing system. Labels that are directly detectable and may find use in the invention include fluorescent labels. Specific fluorophores include fluorescein, rhodamine, BODIPY, cyanine dyes and the like.

A "fluorescent label" refers to any label with the ability to emit light of a certain wavelength when activated by light of another wavelength.

"Fluorescence” refers to any detectable characteristic of a fluorescent signal, including intensity, spectrum, wavelength, intracellular distribution, etc.

"Detecting" fluorescence refers to assessing the fluorescence of a cell using qualitative or quantitative methods. In some of the embodiments of the present invention, fluorescence will be detected in a qualitative manner. In other words, either the fluorescent marker is present, indicating that the label is present, or not. For other instances, the fluorescence can be determined using quantitative means, e. g., measuring the fluorescence intensity, spectrum, or intracellular distribution, allowing the statistical comparison of values obtained under different conditions. The level can also be determined using qualitative methods, such as the visual analysis and comparison by a human of multiple samples, e. g., samples detected using a fluorescent microscope or other optical detector (e. g., image analysis system, etc.). An "alteration" or "modulation" in fluorescence refers to any detectable difference in the intensity, intracellular distribution, spectrum, wavelength, or other aspect of fluorescence under a particular condition as compared to another condition. For example, an "alteration" or "modulation" is detected quantitatively, and the difference is a statistically significant difference. Any "alterations" or "modulations" in fluorescence can be detected using standard instrumentation, such as a fluorescent microscope, CCD, or any other fluorescent detector, and can be detected using an automated system, such as the integrated systems, or can reflect a subjective detection of an alteration by a human observer. As used herein, the singular forms "a", "an," and "the" include plural reference unless the context clearly dictates otherwise.

As used herein, the term "disorder" refers to an ailment, disease, illness, clinical condition, or pathological condition.

The terms "dNTP mixture" and "four dNTPs" are intended to refer to mixture of deoxyribonucleotides that correspond to G, A, T and C that can be incorporated by a polymerase into a growing polynucleotide strand. A dNTP mix may contain dGTP, dATP, dTTP and dCTP as well as other deoxyribonucleotides, e.g., a labeled dNTP. In one embodiment, the composition of the invention comprises all four d NTPs dGTP, dATP, dTTP and dCTP, and also a labelled dNTP. The method of the invention uses all four dNTPs dGTP, dATP, dTTP and dCTP, and also a labeled dNTP. In use, the dNTPs may each be at a working concentration of 50 pM to 1 mM (e.g., 100 pM to 500 pM, or 150 pM to 300 pM).

The term "nucleotide" includes dNTPs (also referred to as nucleoside triphosphates) as well as nucleic acid residues that are in a polynucleotide. "Nucleotides" include those moieties that contain not only the known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acetylated purines or pyrimidines, alkylated riboses or other heterocycles. In addition, the term "nucleotide" includes those moieties that contain hapten or fluorescent labels and may contain not only conventional ribose and deoxyribose sugars, but other sugars as well. Modified nucleosides or nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, or are functionalized as ethers, amines, or the like.

The term "methylation-dependent" is intended to refer to an enzyme that only cleaves at, adjacent or proximate to a recognition site in DNA that contains at least one methylated nucleotide, e.g., methylcytosine. These enzymes cleave single stranded or double stranded DNA depending on whether a subset or all nucleotides (e.g. cytosines) in the recognition site are methylated, and do not cleave DNA if the recognition sequence is unmethylated. Some methylation-dependent nicking enzymes recognize methylated CpGs.

The term "methylation-sensitive" is intended to refer to an enzyme that only nicks at or adjacent to a recognition site that contains one or more unmethylated nucleotides, e.g., one or more unmethylated cytosines. These enzymes nick DNA if one or more nucleotides in the recognition site are unmethylated and do not nick DNA if all the nucleotides in the recognition site are methylated.

The term "methylation-insensitive" is intended to refer to an enzyme that nicks at or adjacent to a recognition site that contains methylated or unmethylated nucleotides (e.g. cytosine or methylcytosine). These enzymes nick DNA regardless of whether any nucleotides in the recognition site are methylated. A "plurality" contains at least 2 members. For example, a plurality of labeled nucleotides means 2 or more labeled nucleotides. In certain cases, a plurality may have at least 2, at least 5, at least 10, at least 100, at least 1000, at least 10,000, at least 100,000, at least 10⁶, at least 10⁷, at least 10⁸ or at least 10⁹ or more members.

The term "strand" as used herein refers to a nucleic acid made up of nucleotides covalently linked together by covalent bonds, e.g., phosphodiester bonds. In a cell, DNA usually exists in a double-stranded form, and as such, has two complementary strands of nucleic acid referred to herein as the "top" and "bottom" strands. In certain cases, complementary strands of a chromosomal region may be referred to as "plus" and "minus" strands, the "first" and "second" strands, the "coding" and "noncoding" strands, the "Watson" and "Crick" strands or the "sense" and "antisense" strands. The assignment of a strand as being a top or bottom strand is arbitrary and does not imply any particular orientation, function or structure.

The term "sequencing", as used herein, refers to a method by which the identity of at least 10 consecutive nucleotides (e.g., the identity of at least 20, at least 50, at least 100 or at least 200 or more consecutive nucleotides) of a polynucleotide are obtained.

The term "next-generation sequencing" refers to the so-called parallel sequencing-by- synthesis or sequencing-by-ligation platforms currently employed by Illumina, Life Technologies, Pacific Biosciences and Roche etc. Next-generation sequencing methods may also include nanopore sequencing methods or electronic-detection based methods such as Ion Torrent technology commercialized by Life Technologies.

The term "extending", as used herein, refers to the extension of a nucleic acid strand by the addition of one or more nucleotides using a polymerase. A polymerase may generate an oligonucleotide flap at a nick site in a double stranded DNA where all of one or two types of nucleotides in the flap are labeled. The flap may be a plurality of nucleotides, having a length ranging from 2 nucleotides to several hundred nucleotides. If a primer that is annealed to a nucleic acid is extended, the nucleic acid acts as a template for extension reaction.

The term "in vitro" refers to a reaction that occurs in a vessel with isolated components, not in live cells. The term "ex vivo" refers to a reaction or method that is not performed on the living human or animal body. For example, an ex vivo method may be performed outside the living human or animal body on a sample (e.g. a cell or tissue sample, such as a clinical sample) that has previously been obtained from the human or animal body. In some embodiments, the methods of the present invention are performed in vitro or ex vivo.

The term "non-naturally occurring" refers to a composition that does not exist in nature. Any protein described herein may be non-naturally occurring, where the term "non-naturally occurring" refers to a protein that has an amino acid sequence and/or a post-translational modification pattern that is different to the protein in its natural state. For example, a non- naturally occurring protein may have one or more amino acid substitutions, deletions or insertions at the N-terminus, the C-terminus and/or between the N- and C- termini of the protein. A "non-naturally occurring" protein may have an amino acid sequence that is different to a naturally occurring amino acid sequence (i.e., having less than 100% sequence identity to the amino acid sequence of a naturally occurring protein) but that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the naturally occurring amino acid sequence. In certain cases, a non-naturally occurring protein may contain an N-terminal methionine or may lack one or more post-translational modifications (e.g., glycosylation, phosphorylation, etc.) if it is produced by a different (e.g., bacterial) cell. A "mutant" or "variant" protein may have one or more amino acid substitutions relative to a wild-type protein and may include a "fusion" protein. The term "fusion protein" refers to a protein composed of a plurality of polypeptide components that are unjoined in their native state. Fusion proteins may be a combination of two, three or even four or more different proteins. The term polypeptide includes fusion proteins, including, but not limited to, a fusion of two or more heterologous amino acid sequences, a fusion of a polypeptide with: a heterologous targeting sequence, a linker, an immunologically tag, a detectable fusion partner, such as a fluorescent protein, p-galactosidase, luciferase, etc., and the like. A fusion protein may have one or more heterologous domains added to the N-terminus, C- terminus, and or the middle portion of the protein. If two parts of a fusion protein are "heterologous", they are not part of the same protein in its natural state. In the context of a nucleic acid, the term "non-naturally occurring" refers to a nucleic acid that contains: a) a sequence of nucleotides that is different to a nucleic acid in its natural state (i.e. having less than 100% sequence identity to a naturally occurring nucleic acid sequence), b) one or more non- naturally occurring nucleotide monomers (which may result in a non-natural backbone or sugar that is not G, A, T or C) and/or c) may contain one or more other modifications (e.g., an added label or other moiety) to the 5'- end, the 3' end, and/or between the 5'- and 3'-ends of the nucleic acid.

In the context of a preparation, the term "non-naturally occurring" refers to: a) a combination of components that are not combined by nature, e.g., because they are at different locations, in different cells or different cell compartments; b) a combination of components that have relative concentrations that are not found in nature; c) a combination that lacks something that is usually associated with one of the components in nature; d) a combination that is in a form that is not found in nature, e.g., dried, freeze dried, crystalline, aqueous; and/or e) a combination that contains a component that is not found in nature. For example, a preparation may contain a "non-naturally occurring" buffering agent (e.g., Tris, HEPES, TAPS, MOPS, tricine or MES), a detergent, a dye, a reaction enhancer or inhibitor, an oxidizing agent, a reducing agent, a solvent or a preservative that is not found in nature.

The term "nicking", as used herein, refers to a reaction that breaks the phosphodiester bond between two nucleotides in one strand of a double-stranded DNA molecule to produce a 3' hydroxyl group and a 5' phosphate group. The term "nick site," as used herein, refers to the site at which a double-stranded DNA molecule has been nicked.

As used herein, the term "nicking enzyme" refers to an enzyme that cleaves (e.g. nicks) one strand (either the top or bottom strands, but not both strands) of a double-stranded nucleic acid at a nonrandom position in the DNA. In some embodiments, the nicking enzyme will be site-specific. In some cases, a nicking enzyme will nick the bottom or top strand at a specific sequence on the nucleic acid. Nicking enzymes useful in the compositions and methods of the invention, which may be methylation-dependent, methylation-sensitive, or methylationinsensitive, are known in the art. Nb.Bsml, Nb.BbvCI, Nb.BsrDI, Nb.BtsI, Nt.BbvCI, Nt.Alwl, Nt. CviPII, Nt. BsmAI, Nt. Alwl and Nt.BstNBI are examples of naturally occurring nicking enzymes that are not 5-methylcytosine dependent. Nicking enzymes that have been engineered from Type Ils restriction enzymes (e.g., Alwl, BpulOI, BbvCI, Bsal, BsmBI, BsmAI, Bsml, BspOJ, Mlyl, Mval269l and Sapl, etc.) and methods of making nicking enzymes can be found in references for example, US 7,081 ,358; US 7,011 ,966; US 7,943,303; US 7,820,424. Labeling of closed chromatin may occur using methylation dependent nicking enzymes that preferably favor 5mCpG sites. Selective labeling of open chromatin preferentially uses methylation independent or methylation sensitive nicking enzymes, such as exemplified herein. Nicking enzymes that are methylation-dependent include, but are not limited to: Nhol (G5mCNG5mC); Bisl (G5mCNG5mC) (Chmuzh, et al., Biotekhnologiya 3: 22-26 (2005); Pam 79021 (G5mCNNG5mC); N.BceSVIll; and Nb.LpnPI (C5mCDG(N)io/GGHmC(N)i4, nicking of the bottom strand) (Cohen Kami, et al., Proc. Natl. Acad. Sci. U. S. A.108: 11040-11045 (2011); Xu et al, Sci. Rep.6:28579 (2016)). Methylation-dependent nicking enzymes can be produced using the methods described in, Gutjahr, et al., Nucleic Acids Res.42:e77 (2014) and Xu, et al., Sci. Rep.6:28579 (2016). N. Gamma is a strand-specific and site-specific DNA nicking enzyme that cleaves at (YCGsL-GT or AC ^ACGR). Nb.LpnPI can be made by making an R335A mutation in the sequence LpnPI sequence defined by Genbank accession number AAU27318.1 . Other nicking enzymes can be made by making an Arg to Ala substitution at the position corresponding to position 335 in LpnPI. Such enzymes may cleave at a methylated CpG.

A description of nicking enzymes can be found in a variety of publications (e.g., Bellamy, et a I. J. Mol. Biol. 2005 345, 641-653; Heiter, et al., J . Mol. Biol. 2005 348, 631-640; Xu, et al., Proc. Natl. Acad. Sci. USA 2001 98, 12990-12995; Samuelson, et a I., Nucl. Acids Res. 2004 32, 3661-3671 ; Zhu, et al., J. Mol. Biol. 2004 337, 573-583; Morgan, et a I., Biol. Chem. 2000 381 , 1123-1125; Chan, Nucl. Acids Res. 2004 32, 6187-6199; Sasnauskas, Proc. Natl. Acad. Sci. USA 2003 100, 6410-6415; Jo, et a I., PNAS 2007 104:2673-2678; Xiao, et al., Nucleic Acids Res. 2007 35:el6; US 7,081 ,358; US 6,191267, US 2005/0136462, US 7,943,303, US 8,163,529, WO 2006/047183 and WO 2008/0268507. DNase I cleaves DNA at random positions.

A nicking enzyme can also be made by inactivating one of the catalytic domains. For example, see US 7,081 ,358. Another type of example is a programmable endonuclease, e.g., Cas9 or a functional equivalent thereof (such as Argonaute or Cpfl). For example, Cas9 contains two catalytic domains, RuvC and H N H. Inactivating one of those domains will generate a nicking enzyme. In Cas9, the RuvC domain can be inactivated by an amino acid substitution at position D10 (e.g., D10A) and the H NH domain can be inactivated by an amino acid substitution at position H840 (e.g., H840A), or at a position corresponding to those amino acids in other proteins. Such endonucleases may be Argonaute or Type II CRISPR/Cas endonucleases that are composed of two components: a nuclease (e.g., a Cas9 or Cpfl endonuclease or variant or ortholog thereof) that cleaves the target DNA and a guide nucleic acid e.g., a guide DNA or RNA that targets the nuclease to a specific site in the target DNA (see, e.g., Hsu, et al., Nature Biotechnology 2013 31 : 827-832). A nicking enzyme can also be made by fusing a site-specific DNA binding domain such as the DNA binding domain of a DNA binding protein (e.g., a restriction endonuclease, a transcription factor, or another doma in that binds to DNA at non-random positions) with a nuclease or deaminase so that it acts on a non-random site. In these embodiments, the deaminase can introduce a uracil, and a nick can be created by removing the uracil using a deglycosylase and treating the abasic site with an AP endonuclease. It will be understood from the foregoing that non-random cleavage by a nicking enzyme results from recognition sites within the nicking enzyme or from guide molecules that direct the nicking enzyme to a non-random site or optionally by inherent defined bias of the enzyme for a plurality of nucleotides that may be preferentially As and Ts or Gc and Cs.

As used herein, the term "chromatin" refers to a complex of molecules including proteins and genomic DNA as found in a nucleus of a eukaryotic cell. Chromatin is composed in part of histone proteins that form nucleosomes, genomic DNA, and other DNA binding proteins (e.g., transcription factors) that are bound to the genomic DNA. Chromatin in mammalian sperm additionally contains protamine proteins as major constituents. Chromatin is therefore distinct from purified genomic DNA. Chromatin is available in permeabilized cells, in isolated nuclei, and as well as in isolated chromatin.

As used herein, the terms "open chromatin" and "closed chromatin" refer to the level of the accessibility of genomic DNA in a sample that contains chromatin. Open chromatin (or "euchromatin") is not densely packaged into nucleosomes and can be accessed by a nicking enzyme; it is accessible chromatin. In contrast, closed chromatin (or "heterochromatin") is densely packaged into nucleosomes and not accessible by a nick endonuclease.

As used herein, the term "isolated nucleus" refers to a nucleus that has been isolated from other components of a cell, e.g., from the cytoplasm and plasma membrane, by centrifugation or another technique.

As used herein, the term "permeabilized cell" refers to a cell that has a cell plasma membrane and, in some cases a nuclear membrane, that have been permeabilized, e.g., by a detergent. As used herein, the term "fixed cell" refers to a cell that has been treated with a crosslinking or non- crosslinking fixative, e.g., formaldehyde or paraformaldehyde, acetone, or methanol or the like. In some embodiments, a fixed cell may be "formalin fixed", in which case it may be fixed in formaldehyde (e.g., 3%- 5% formaldehyde in phosphate buffered saline) or Bouin solution. An "unfixed" cell refers to a cell that has not been treated by such an agent.

As used herein, the term "labeled nucleotide" refers to a modified nucleotide that has an optically detectable label or an affinity tag attached thereto.

As used herein, the term "optically detectible label" refers to a light-emitting or fluorescent label that can be detected using a light detector, e.g., a microscope. Light emitting labels include fluorophores, although others are known.

As used herein, the term "affinity tag" refers to a tag that can be used to separate a molecule to which the affinity tag is attached from other molecules that do not contain the affinity tag. In many cases, an affinity tag is a member of a specific binding pair, i.e. two molecules where one of the molecules through chemical or physical means specifically binds to the other molecule. The complementary member of the specific binding pair, which can be referred to herein as a "capture agent" may be immobilized (e.g., to a chromatography support, a bead or a planar surface) to produce an affinity chromatography support that specifically binds the affinity tag. Affinity tags include a biotin moiety (e.g., biotin, desthibiotin, oxybiotin, 2- iminobiotin, diaminobiotin, biotin sulfoxide, biocytin, etc.) which can bind to streptavidin.

Affinity tags also include chemoselective groups such as azido and alkynyl groups, which can participate in a copper-free cycloaddition reaction (see, e.g., Kolb, et al., Drug Disco Today 2003 8: 1128-113 and Baskin, et al., Proc. Natl. Acad. Sci. 2007 104: 16793-16797).

As used herein, the term "enriching" refers to a method step in which some components of a sample (e.g., components that are labelled) are separated from other components in the sample (e.g., components that are not labelled).

The term "barcode sequence", "molecular barcode" or "index", as used herein, refers to a unique sequence of nucleotides used to (a) identify and/or track the source of a polynucleotide in a reaction and/or (b) count how many times an initial molecule is sequenced (e.g., in cases where substantially every molecule in a sample is tagged with a different sequence, and then the sample is amplified). A barcode sequence may be at the 5'-end, the 3'-end or in the middle of an oligonucleotide, or both the 5' end and the 3' end.

Barcode sequences may vary widely in size and composition; the following references provide guidance for selecting sets of barcode sequences appropriate for particular embodiments: Brenner, US 5,635,400; Brenner et al, Proc. Natl. Acad. Sci., 97: 1665-1670 (2000); Shoemaker, et al, Nature Genetics, 14: 450-456 (1996); Morris et al, European patent publication 0799897A1 ; Wallace, US 5,981 ,179; and the like. In particular embodiments, a barcode sequence may have a length in range of from 4 to 36 nucleotides, or from 6 to 30 nucleotides, or from 8 to 20 nucleotides. The term "reacting," as used herein, refers to combining under conditions (e.g., a suitable temperature, time and conditions) that result in a reaction, e.g., nicking and/or strand extension by a polymerase.

Determining the fertility of spermatozoa may be understood in the broadest sense as any assessment of the degree of suitability of the spermatozoa for sexual reproduction by natural or artificial insemination. The determination of spermatozoa as being infertile or less fertile does not exclude sexual reproductive capability by fusing a spermatozoon with an ovule artificially in vitro, in particular by injecting the genetic material into the ovule (in vitro fertilization in a test tube) because in such process no mobility and viability of the latter is required.

The term "spermatozoon", "sperm", "spermatozoon cell", "sperm cell" and the like may be understood interchangeably in the common sense in the art, i.e., as a, preferably matured, male gamete.

A negative control sample of spermatozoa of low fertility may be obtained from an individual of the same species suffering from oligozoospermia, asthenozoospermia and/or teratozoospermia, or oligo-astheno-teratozoospermia (OAT syndrome) and/or azoospermia and/or any other pathologic state accompanied by low fertility of the spermatozoa, in particular oligozoospermia, asthenozoospermia and/or teratozoospermia.

As used herein, a total content per spermatozoon may be understood in the broadest sense as the content of the respective label in the cells. It will be understood that typically not the amount of label comprised in one spermatozoon is determined, but typically the average of a larger number of spermatozoa. As it is well-known that all spermatozoa of a sample will generally each have (essentially) the same weight, the determination of the total content per spermatozoon is equivalent to the total content per spermatozoon weight. The reference "per spermatozoon" or "per spermatozoa weight" or the like normalizes different samples and makes them comparable with another, independent on the total number or concentration of spermatozoa comprised in the respective sample. Alternatively, also the content of label per milliliter of the sample or per milligram of the sample can be determined, when the spermatozoa content is comparable in the different samples to be compared with another.

As used herein, "decreased amount" may be understood in the broadest sense as lower degree. Typically, a decreased amount is a degree that is at least 10%, at least 20%, at least 50%, at least 75%, at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold lower than the value it is compared with (e.g., that of the positive control sample). As used herein, "not higher than" in the context of comparison with control sample may be understood in the broadest sense as not being significantly higher, i.e., not 10% higher, in particular equal or lower than the value it is compared with. When exemplarily fluorescence detection is used, the decreased amount may refer to the decrease of the intensity of the fluorescence signal (detectable under the same measurement conditions). When exemplarily fluorescence microscopy is used, the decreased amount may refer to the decrease of the intensity of the local fluorescence signal (detectable under the same measurement conditions). When exemplarily flow cytometry (also designated as fluorescence activated cell sorting (FACS), herein understood interchangeably) is used, the decreased amount may refer to the decrease of the intensity of the mean fluorescence signal (detectable under the same measurement conditions for the spermatozoa).

Exemplarily, the sample may be an ejaculate or a processed ejaculate or an aliquot thereof. A processed ejaculate may optionally be diluted in an aqueous buffer and/or in an organic liquid.

Alternatively, the sample may be a testis biopsy (e.g., obtained in the context of testicular sperm extraction (TESE)). The experimenter may compare the signal intensity found (in the relevant parts containing spermatozoa and precursors thereof) of the testis biopsy of a patient of interest with a comparable testis sample of a well- fertile individual of the same species.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspects or embodiments of the invention. Accordingly, the terms defined herein are more fully defined by reference to the specification as a whole.

EXAMPLES

Participant selection and sperm preparation:

Participants were selected based on previous reproductive outcomes. 19 participants were known fertile (having generated a pregnancy without the use of assisted reproductive technologies), the remaining 38 participants were infertile. Infertile participants were further sub-divided based upon embryo development rates following assisted reproduction: 20 participants had a pre-implantation embryo development rate of greater than 50%, while 18 participants generated maximally one blastocyst following fertilization.

Sperm preparation: Semen samples were collected from participants following 2-5 days of abstinence and allowed to liquefy for 30 minutes at 37°C. Sperm was isolated from semen using two methods: total sperm was isolated by washing semen with media and motile sperm was isolated via a swim-up protocol.

NicE-view staining of human sperm samples:

10-well slides of total and swim-up sperm were thawed at room temperature and washed once with phosphate buffered saline (PBS, 50 pl per well). Sperm were then fixed with 1% paraformaldehyde (Electron Microscopy Sciences, Hatfield USA, #15713) in PBS for 10 minutes at room temperature, followed by two washes in PBS. Sperm were permeabilized using Omni ATAC permeabilization buffer¹ (10 mM Tris, pH 7.5, 10 mM NaCI, 3 mM MgCh, 0.1% Igepal, 0.1% Tween-20, 0.01% digitonin) for 15 minutes at room temperature and then washed once with PBS. For T4 NicE-view, samples were incubated for 15 minutes at room temperature with 30 pl per well of T4 DNA ligase (ThermoFisher Scientific, Waltham USA, #EL0012) in 1x T4 DNA ligase buffer (40 mM Tris-HCI, 10 mM MgCI₂, 10 mM DTT, 0.5 mM ATP, pH 7.8) and subsequently washed once with 50 pl per well PBS. Samples were then labelled for 2 hours at 37°C in 30 pl per well labelling mix. The NicE-view labelling mix was based on previously published protocols^{2 4} and consisted of 1x NEBuffer 2 (NEB, Ipswich USA, #B7002S), 30 pM dGTP (Thermo Fisher Scientific, #R0181), 30 pM dTTP (Thermo Fisher Scientific, #R0181) and 30 pM dmCTP (Jena Bioscience, Jena Germany, #NU-1125L), 24 pM dATP (Thermo Fisher Scientific, #R0181), 6 pM Fluorescein-dATP (Jena Biosciences, #NU-1611-FAMX), 3.2 U/ml Nt.CviPI I (NEB #R0626S) and 64 U/ml E. coli DNA Polymerase I (NEB #M0209L). For Nick translation the nicking endonuclease Nt.CviPII was excluded, while for negative control reactions both Nt.CviPII and DNA Polymerase I were excluded. Following labelling, samples were washed 3 times with PBS + 0.01% SDS + 50 mM EDTA, pre-heated to 55°C, for 15 minutes each. Samples were counter-stained with DAPI (10 pg/ml in PBS, Sigma Aldrich, St. Louis USA, #D9542-10MG), mounted with VectaShield (Vector Laboratories, Newark USA, #H-1000) and covered with 50 mm coverslips. Slides were then sealed with nail polish. Slides were stored at 4°C until imaging (maximally 2 days).

NicE-view imaging:

Stained slides were imaged on a Visitron Spinning Disk W1 confocal microscope (Visitron Systems GmbH, Puchheim Germany). Images were acquired with a 40x oil objective (NA: 1 .3, WD 0.21) using a 405 nm laser with a 460/50 filter set for DAPI and a 488 nm laser with a 525/50 filter set for NicE-view and ATAC signal. For double labelling experiments a 561 nm laser with a 609/54 filter set was also utilized. For each well, 2 separate 5x5 tiles were acquired including a 6 pm Z-stack for each image (with 1 pm step size). For Nick translation, NicE-view and T4 NicE-view, 2-wells per slide were independently stained resulting in 100 image stacks per slide for these assays. For negative control and T4 DNA ligase treatment of Nick translation, 1 well per slide was stained resulting in 50 image stacks per slide for these controls.

NicE-view image processing:

Maximum intensity projections were generated for each stack in VisiView (Visitron Systems, Puchheim Germany). Projections were processed with Fiji⁵ and Photoshop (Adobe Systems, San Jose, USA) to generate images. Projections were further processed using CellProfiler⁶’⁷. Nuclear segmentation was performed on DAPI images (Segmentation Parameters: 25-50 pixel diameter, Adaptive Sauvola thresholding with no smoothing scale, a threshold correction factor of 1 .0, and an adaptive window size of 800. Clumped objects were distinguished by shape with intensity used to draw dividing lines between clumped objects). Segmentation masks were inspected visually to confirm reasonable segmentation. Intensity values and size and shape parameters were then calculated for each segmented nucleus.

Imaging data analysis:

All data analyses were performed in R. Comma separated value (csv) files generated by CellProfiler were loaded into R and pre-processed using the tidyverse suite of packages⁸. Plots were generated using ggplot2⁹, for plots showing distributions of values between individuals, 1000 sperm were randomly sub-sampled (using sample_n()⁸) from each sperm sample (total and swim-up) to generate distributions equally weighted for each individual.

Results of NicE-view and T4 NicE-view on human sperm of differing fertility:

NicE-view and T4 NicE-view were performed on 114 sperm samples derived from 57 individuals. Sperm with highly different labelling intensities were clearly visible in all samples. Quantification of signal intensities revealed that sperm were largely present in two populations with varying DNA accessibility levels. Frequencies of NicE-view^h'9^h and T4 NicE-view^h'9^h sperm were generally greater in swim-up samples than total sperm samples. The number of individuals with high frequencies of NicE-view^h'9^h and T4 NicE-view^h'9^h sperm was also found to be greater within the infertile patient categories, particularly within infertile participants with a history of poor pre-implantation development. As such the NicE-view and the T4 NicE-view tests can be used to predict poor or absent embryonic development as caused by defective sperm quality.

References:

1 Corces, M. R. et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat Methods 14, 959-962, doi:10.1038/nmeth.4396 (2017).

2 Chin, H. G. et al. Universal NicE-seq for high-resolution accessible chromatin profiling for formaldehyde- fixed and FFPE tissues. Clin Epigenetics 12, 143, doi:10.1186/s13148-020-00921 -6 (2020).

3 Chin, H. G. et al. Universal NicE-Seq: A Simple and Quick Method for Accessible Chromatin Detection in Fixed Cells. Methods Mol Biol 2611, 39-52, doi:10.1007/978-1-0716-2899-7_3 (2023).

4 Ponnaluri, V. K. C. et al. NicE-seq: high resolution open chromatin profiling. Genome Biol S, 122, doi: 10.1186/s13059-017-1247-6 (2017). 5 Sch indelin , J. et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676-682, doi:10.1038/nmeth.2019 (2012).

6 Carpenter, A. E. et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome BioH , R100, doi:10.1186/gb-2006-7-10-r100 (2006).

7 Stirling, D. R. et al. CellProfiler 4: improvements in speed, utility and usability. BMC Bioinformatics 22, 433, doi: 10.1186/s12859-021 -04344-9 (2021 ).

8 Wickham, H. et al. Welcome to the Tidyverse. Journal of open source software 4, 1686 (2019).

9 Wickham, H. ggplot2: Elegant Graphics for Data Analysis. (Springer, 2016).

Claims

1 . A method for assessing male fertility by assessing the DNA accessibility in the spermatozoa of a population of spermatozoa from mammalian semen from a subject, said method comprising the following steps: a) isolating a population of spermatozoa from total mammalian semen, b) permeabilizing the isolated population of spermatozoa using freezing and/or chemical pre-treatment, c) contacting the permeabilized population of spermatozoa with a composition comprising at least a DNA nicking-inducing agent, a DNA polymerase, four dNTPs and at least one labelled dNTP, d) incubating the mixture of step c under conditions suitable for the DNA nicking agent to cut one strand of the double-stranded DNA of the permeabilized spermatozoa at several sites, thereby producing single-stranded DNA nicks, and for the DNA polymerase to initiate synthesis of new DNA from said singlestranded DNA nicks using the dNTPs, thereby labelling the newly synthesized DNA by introduction of at least one labelled dNTP into the DNA of the permeabilized population of spermatozoa, e) quantifying the presence of labelled dNTP in the labelled population of spermatozoa of step d after at least one washing step, wherein the presence of a significantly higher content of labelled dNTP in the labelled population of spermatozoa from the subject, as compared to the content of labelled dNTP in a comparable sample of spermatozoa obtained from normally fertile control individuals and treated using the same steps as above, is indicative of a poor fertility for said subject.

2. The method of claim 1 wherein step a) involves the preparation of spermatozoa via selection of motile spermatozoa capable of swimming through a column of liquid, wherein only the spermatozoa that have been able to reach the upper part of the column are isolated, or the selection of spermatozoa that have been able to swim through a microfluidic chip.

3. The method of claims 1 to 2 wherein step a involves the preparation of spermatozoa through selection of spermatozoa on the basis of their density via centrifugation through at least one dense medium or density gradient.

4. The method of claims 1 to 3 wherein step a) involves the preparation of spermatozoa via Fluorescence Activated Cell Sorting (FACS) using at least one dye, wherein the spermatozoa are selected based on their uptake of the dye.

5. The method of claims 1 to 4 wherein step b) involves the treatment of the isolated population of spermatozoa with DTT and/or other reducing agents.

6. The method of claims 1 to 5 wherein after step b) and before step c) the isolated population of spermatozoa are pre-treated with a DNA ligase, for instance T4 DNA ligase, under conditions suitable for the repair of endogenous single stranded DNA.

7. The method of claims 1 to 6 wherein the DNA nicking-inducing agent is a chemical agent, such as hydrogen peroxide.

8. The method of claims 1 to 6 wherein the DNA nicking-inducing agent is a sequence specific nicking endonuclease, such as Alwl, or a sequence non-specific endonuclease, such as DNAse I.

9. The method of claims 1 to 8 wherein the labelled dNTP comprises dNTP labelled with an optically detectable label, for instance a fluorophore.

10. The method of claims 1 to 9 wherein the labelled dNTP comprises dNTP labelled with an affinity tag, such as biotin.

11 . The method of claims 1 to 10 wherein the labelled dNTP comprises dNTP labelled with a chemically modified DNA base, for instance a methylated DNA base, wherein said chemically modified DNA base can be detected by, for instance, DNA sequencing and/or mass spectrometry.

12. The method of any of the preceding claims, wherein the subject is human, bovine, porcine, ovine, equine or camelid.

13. A method of selecting a population of spermatozoa from a subject, wherein the fertility of at least two different populations of spermatozoa from said subject is assessed by a method according to claims 1 to 12, and wherein said population is selected based on its assessed fertility.

14. The method of claim 13 wherein the population of spermatozoa is a population selected from the group of motile spermatozoa, spermatozoa of a specific density, spermatozoa able, or not able, to uptake a specific dye, spermatozoa demonstrating thermotaxis, or testicular spermatozoa.

15. A method of in vitro fertilization comprising the step of selecting a population of spermatozoa from a subject according to a method of claims 13 or 14.