Methods of detecting DNA damage in sperm cells

Abstract

A method of detecting damage to sperm cell DNA molecules is disclosed. The method can be used for assessing whether sperm cells are a candidate for in-vitro fertilization, for diagnosing a disease associated with sperm cell damage or for detecting the effect of an agent on sperm cells.

Description

[0001] METHODS OF DETECTING DNA DAMAGE IN SPERM CELLS

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of priority of U.S. Patent Application No. 63 / 626,549 filed January 30, 2024, the contents of which are incorporated herein by reference in their entirety.

[0004] FIELD AND BACKGROUND OF THE INVENTION

[0005] The present invention, in some embodiments thereof, relates to a method of detecting DNA damage in sperm cells and more particularly to a method of detecting, on a molecular level, single strand breaks in sperm cells.

[0006] Various stressors, originating from both external factors and cellular processes, pose a continuous threat to the integrity and functionality of the human DNA within the genome. This constant exposure increases the risk of DNA damage, potentially leading to detrimental effects on its structure and proper function. Failure to repair this damage can lead to mutations, genomic instability, and the development of diseases. Single-strand lesions represent the most prevalent form of DNA damage and range from physical rupture of the strands to chemical modification of the affected base. These lesions can be induced by external radiation or toxins, as well as by normal metabolic processes that result in the production of reactive oxygen species (ROS) that oxidize DNA, or by directly triggering the formation of specific DNA structures like cyclobutane pyrimidine dimers (CPDs) and 6—4 photoproducts.

[0007] In normal cellular conditions, highly efficient repair enzymes address DNA damage and prevent single-strand breaks (SSBs) from progressing into double-strand breaks (DSBs). Single strand damage is usually repaired by nucleotide excision repair (NER) or base excision repair (BER) processes. These mechanisms involve the excision of damaged DNA bases, creating a gap that is subsequently filled by DNA polymerase, using the complementary strand as a template.

[0008] Detection and quantification of DNA damage are essential for elucidating the underlying mechanisms of DNA repair, assessing the impact of environmental exposures and toxins, enabling early clinical diagnosis, and evaluating the response to therapy. Various techniques have emerged in recent years to address these needs, often relying on the use of lesion- specific antibodies or DNA integrity detection methods. SSBs and DSBs are commonly measured indirectly by unwinding of the DNA. This includes the comet assay and other electrophoresis-based techniques. Alternatively, immunological labeling of damage sites using fluorescent-based detection assays such as Enzyme-Linked Immunosorbent Assay (ELISA), Dot-blot, flow cytometry, and immunohistochemistry, have also been utilized. However, these techniques have limitations, including poor sensitivity and constrains in detecting multiple types of damage. To overcome these limitations, the single-molecule approach has emerged as a powerful tool for sensitive detection and quantification of various types of DNA damage. This approach directly detects damaged lesions within single DNA molecules, allowing for the precise localization and quantification of damage that may be masked in other large-scale assays.

[0009] Zur et al., (DNA Repair 129 (2023) 103533) discloses single-molecule, in vitro Repair assisted damage detection (RADD) assays that rely on enzymatic repair. This multi-step technique utilizes specific repair enzymes to remove damaged bases and incorporate fluorescently labeled nucleotides, enabling precise visualization and quantification of DNA lesions. Firstly, specific repair enzymes, such as glycosylases or endonucleases recognize and excise damaged bases from the DNA molecule. These enzymes possess high specificity, targeting particular types of damage, including oxidized lesions or base dimers. Following base excision, the resulting gaps are filled in by DNA polymerases using fluorescently labeled nucleotides as building blocks. The incorporated fluorescent nucleotides are detectable under fluorescence microscopy, allowing for the precise localization and quantification of the damaged DNA regions.

[0010] An abstract published October 14, 2023 ASRM, New Orleans, Louisiana teaches analysis of sperm cell single-strand DNA breaks.

[0011] SUMMARY OF THE INVENTION

[0012] According to an aspect of the invention, there is provided a method of detecting damage to sperm cell DNA molecules comprising:

[0013] (a) labeling the DNA molecules with a detectable moiety which is capable of being incorporated specifically at a damaged site of the DNA molecules, wherein the DNA molecules are double-stranded; and

[0014] (b) detecting the detectable moiety, thereby detecting damage to the sperm cell DNA molecules.

[0015] According to embodiments of the invention, the labeling is effected ex vivo.

[0016] According to embodiments of the invention, the damage comprises a breakage of a single strand of the double- stranded DNA molecule.

[0017] According to embodiments of the invention, the labeling is effected by:

[0018] (i) contacting the DNA molecules with at least one DNA repair enzyme under conditions that the DNA repair enzyme removes at least one base at the breakage site of a broken strand of the DNA; and subsequently (ii) contacting the DNA molecules with a DNA polymerase and dNTPs, the dNTPs being attached to the detectable moiety, under conditions that at least one dNTP is incorporated into the DNA molecules at the breakage site.

[0019] According to embodiments of the invention, the at least one DNA repair enzyme is DNA Polymerases Lambda (X) and / or DNA polymerase MB (p).

[0020] According to embodiments of the invention, the detectable moiety is a fluorescent moiety, a phosphorescent moiety or a radioactive moiety.

[0021] According to embodiments of the invention, the method further comprises contacting the DNA molecules with an intercalating agent prior to the detecting.

[0022] According to embodiments of the invention, the intercalating agent is selected from the group consisting of SYBR Green I, SYBR Gold, YoYo, EvaGreen and LCGreen.

[0023] According to embodiments of the invention, the method further comprises immobilizing the DNA molecules on a solid surface following step (a) and prior to step (b).

[0024] According to embodiments of the invention, the solid surface is positively charged.

[0025] According to embodiments of the invention, the solid surface is coated with poly-L-lysine.

[0026] According to embodiments of the invention, the DNA molecules are longer than 20,000 base pairs.

[0027] According to embodiments of the invention, the method further comprises isolating DNA from sperm cells prior to the labeling under conditions that prevents damage to the DNA molecules.

[0028] According to embodiments of the invention, the isolating comprises contacting the sperm cells with a buffer comprising an anionic detergent capable of lysing the sperm cells and denaturing protamines of the sperm cells.

[0029] According to embodiments of the invention, the anionic detergent comprises SDS.

[0030] According to embodiments of the invention, the concentration of the SDS in the composition is between 2-5 %.

[0031] According to embodiments of the invention, the buffer further comprises a reducing agent.

[0032] According to embodiments of the invention, the reducing agent comprises DTT or P- mercaptoethanol.

[0033] According to embodiments of the invention, the reducing agent is DTT.

[0034] According to embodiments of the invention, the concentration of the DTT in the composition is between 60-100 mM.

[0035] According to embodiments of the invention, the method further comprises quantifying a number of damage sites per DNA molecule. According to an aspect of the invention, there is provided a method of assessing whether sperm cells are a candidate for in-vitro fertilization comprising detecting damage to the sperm cells DNA molecules according to the method described herein, wherein an extent of the damage is indicative whether sperm cells are a candidate for in-vitro fertilization.

[0036] According to an aspect of the invention, there is provided a method of diagnosing a disease associated with sperm cell damage comprising detecting damage to the sperm cell DNA molecules according to the method described herein, wherein an extent of the damage above a predetermined threshold is indicative of a disease associated with sperm cell damage.

[0037] According to an aspect of the invention, there is provided a method of detecting the effect of an agent on sperm cells comprising:

[0038] (a) contacting sperm cells with the agent; and

[0039] (b) detecting damage to the sperm cell DNA molecules according to the method described herein, wherein a level of damage above a predetermined amount is indicative that the agent is toxic to the sperm cells.

[0040] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0041] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0042] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0043] In the drawings:

[0044] FIGs. 1A-F depict an overview of the method for identifying DNA lesions in sperm cells according to embodiments of the invention. (A) DNA lesions are identified by specific repair enzymes. (B) The repair enzyme removes the damaged lesion, resulting in a gap in the DNA. (C) DNA polymerase and fluorescent nucleotides are introduced into the tube to fill the created gap. (D) The labeled DNA is loaded onto a glass slide with a positive charge. (E) A slide scanner is utilized to capture images of the slide, which are then subjected to image analysis. (F) Calculations are performed based on the obtained images.

[0045] FIG. 2 shows results depicting oxidative damage induced by H2O2 (n=10). Sperm cells wereincubated for two hours in increasing concentrations of H2O2, followed by DNA extraction. The DNA samples were labeled using PreCR repair mix and the damage level was quantified. Data represent mean ± ste.

[0046] FIGs. 3A-D depict results measuring the amount of DNA damage by DNA break inducer (H2O2 (ImM)) on sperm samples using three different assays (paired control and treated sperm samples (n=25), (A) Repair Assisted Damage Detection (RADD) (B) sperm chromatin dispersion (SCD) and (C) Terminal deoxy nucleotidyl transferase dUTP Nick End Labeling (TUNEL)). (D) DNA damage level ratio comparing H2O2 treated over the controls (n=25), accessed by RADD, SCD and TUNEL.

[0047] FIG. 4 is a graph illustrating results of a correlation analysis between RADD assay and SCD (DFI%). The grey band denoted 95% confidence band. The relative DNA damage level of control and DNA break inducer (H2O2 (ImM)) measured by RADD assay and DFI% measured by SCD (n=25). Pearson’s correlation for control samples r=0.55, p<0.01 and H2O2 treated samples- pearson’s correlation r=0.59, p<0.01.

[0048] FIG. 5 is a graph illustrating results of correlation within paired samples measured with RADD assay between H2O2 and control sperm samples (n=25).

[0049] FIGs. 6A-B. Fig. 6A Relative DNA damage levels in each of 40 sperm samples from healthy males who donated sperm at the sperm bank. Fig. 6B Correlation between RADD and sperm quality measures.

[0050] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0051] The present invention, in some embodiments thereof, relates to a method of detecting DNA damage in sperm cells and more particularly to a method of detecting, on a molecular level, single strand breaks in sperm cells.

[0052] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0053] Single strand DNA (ssDNA) damage is the most common form of induced genetic damage, and if not efficiently repaired, can lead to the generation of non-lethal mutations which can interfere in normal cell functioning. Detection and quantification of ssDNA damage is therefore of major importance in the fields of diagnostics, therapeutics and toxicology. However, existing DNA damage detection assays are limited in their sensitivity and reproducibility. Spermatozoa are particularly susceptible to DNA damage due to their loss of DNA repair capability after spermatogenesis. This vulnerability makes them more prone to oxidative stress, potentially leading to the formation of adducts and eventual DNA fragmentation.

[0054] Repair Assisted Damage Detection (RADD) is a single-molecule technique that employs specific repair enzymes to excise damaged bases and incorporates fluorescently labeled nucleotides to visualize the damage (Zur et al., DNA Repair 129 (2023) 103533). An exemplary RADD assay workflow for the analysis of ssDNA in sperm cells is depicted in Figures 1A-F. Following extraction from sperm cells, DNA is treated with a specific cocktail of DNA repair enzymes which identify and remove DNA damage lesions. Subsequently, fluorescent nucleotides and DNA polymerase are introduced, leading to the incorporation of fluorescent nucleotides into the DNA gaps. The sample is added to a glass slide treated with poly-L-lysine. It is then imaged using a standard fluorescence imager, and the extent of DNA damage is quantified.

[0055] The present inventors have now shown that the RADD assay can be used as a technique for quantifying single- stranded DNA damage in sperm cells. In particular, the present inventors showed that the RADD assay effectively identified DNA damage in a dose-dependent manner. H2O2 was used as an effective inducer of oxidative stress (Figures 2).

[0056] In comparison with established methods, such as SCD and TUNEL, RADD consistently detected significantly higher levels of DNA damage in sperm cells (Figures 3A-D). Unlike other methods, RADD quantitatively detects both DNA adducts and single strand breaks (SSB). Moreover, both RADD and SCD detected more DNA damage than TUNEL, which mainly captured later-stage DNA damage.

[0057] Thus, according to a first aspect of the present invention, there is provided a method of detecting damage to sperm cell DNA molecules comprising:

[0058] (a) labeling the DNA molecules with a detectable moiety which is capable of being incorporated specifically at a damaged site of the DNA molecules, wherein the DNA molecules are double-stranded; and

[0059] (b) detecting the detectable moiety, thereby detecting damage to the sperm cell DNA molecules.

[0060] Generally, the term “sperm cells” refers to mature ejaculated male gametes, which are able to fertilize the female gamete counterpart. The term “sperm cells” is used interchangeably with the term “spermatozoa” herein. In addition to that, the term “sperm” refers to spermatozoa and seminal plasma. The term “sperm” also encompasses subpopulations of sperm (e.g., sorted sperm, marked sperm, frozen sperm, purified sperm, motile sperm subpopulation, etc.). Said spermatozoa are comprised in a sperm sample obtained from a subject, which is commonly male. The subject of the present invention may be a mammal, bird or fish.

[0061] The DNA molecules which are analyzed are typically genomic DNA retrieved from sperm cell samples. Sperm cell samples may be frozen or fresh. Cellular matter may be removed from seminal plasma by centrifugation. The cells may then be rinsed in an appropriate buffer (e.g. phosphate buffered saline (PBS)). Optionally, non-sperm cells may be removed using methods known in the art including, but not limited to density gradient centrifugation (e.g. Percoll gradients) to purify the sperm.

[0062] Sperm cells have highly compacted chromatin and high levels of DNA-protective proteins such as protamines. Disulfide cross-links in protamines require reduction for efficient DNA extraction. These unique characteristics dictate specific conditions for isolating genomic DNA therefrom, which ensure further prevention of damage to the DNA molecules.

[0063] The sperm cell membrane is lysed using anionic detergents which are capable of lysing the sperm cells and denaturing the protamines of the sperm cells.

[0064] Examples of such anionic detergents include but are not limited to sodium dodecyl sulfate SDS) or Triton X-100.

[0065] According to a particular embodiment, the anionic detergent is SDS (e.g. at a concentration of between 2-5 %.

[0066] Lysis of the sperm cell membrane may optionally be accompanied by mechanical disruption, such as vortexing with glass beads or sonication to enhance membrane breakage.

[0067] In order to break the disulfide bonds in protamines, typically a reducing agent is added to the extraction buffer - for example Dithiothreitol (DTT) or P-mercaptoethanol.

[0068] In one embodiment, the reducing agent is DTT.

[0069] The concentration of DTT in the extraction composition is typically between 60-100 mM, 70-90 mM.

[0070] The extraction buffer may further comprise Proteinase K in order to digest residual proteins. The buffer also typically comprises sufficient EDTA to chelate magnesium and calcium ions and inhibit nucleases (e.g. about 20 mM).

[0071] Optionally, the extraction buffer may also comprise RNAse A to digest RNA contaminants. Once isolated, the sperm cell DNA molecules are labeled with a detectable moiety which is capable of being incorporated specifically at a damaged site of the DNA molecules, wherein the DNA molecules are double-stranded.

[0072] It will be appreciated that the DNA which is analyzed may be fully double stranded or at least partially double stranded. In one embodiment, the DNA is double stranded except at, or near to (e.g. within 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides of) the break site. In one embodiment, at least one, two, three, four, five, six, seven, eight, nine or more bases of a first strand of the DNA is not base paired with its corresponding base of the second strand of the DNA. In still another embodiment, at least one, two, three, four, five, six, seven, eight, nine or more bases of a first strand of the DNA is not base-paired with its corresponding base of the second strand of the DNA at a single breakage site.

[0073] In another embodiment, the DNA comprises at least one, two, three, four five, six, seven, eight, nine, or more breaks in one strand (e.g. in the same strand) of the DNA.

[0074] In one embodiment, the DNA molecule is at least 10,000 nucleotides in length, at least 20,000 nucleotides in length, at least 30,000 nucleotides in length, at least 40,000 nucleotides in length and even at least 50,000 nucleotides in length. In one embodiment, the DNA molecule is between 20,000 - 60,000 nucleotides in length.

[0075] In order to label the DNA molecules at the damaged site, DNA repair enzymes (including glycosylases and endonucleases) that are capable of incorporating the labeled dNTPs (deoxynucleotide triphosphates) into the DNA are incubated with the isolated sperm DNA in the presence of a ligase enzyme and a polymerase enzyme. Incubation is carried out under conditions that allow the DNA repair enzyme to incorporate the labelled dNTP at the damaged site.

[0076] Examples of DNA repair enzymes include DNA Polymerases Lambda (X) and / or DNA polymerase Mu (p).

[0077] Particular examples include formamidopyrimidine DNA glycosylase (FPG, 8-oxoguanine DNA glycosylase), endonuclease VIII, endonuclease IV, endonuclease V and uracil-DNA glycosylase (UDG). The enzymes are all commercially available and exist also as a premixed cocktail known as PreCR repair mix (New England Biolabs).

[0078] The above mentioned DNA repair enzymes are specialized enzymes which recognize specific damage lesions and catalyze the formation of apurinic / apyrimidinic (AP) sites (also known as abasic sites) by hydrolysis of the N-glycosydic bond. These AP sites are converted to nicks that leave behind a 3-OH end and a 5-phosphate, making them readily available for nucleotide incorporation by the DNA polymerase. Examples of damaged sites which may be labeled include single- strand breaks (which are caused by oxidative stress, radiation, or certain chemicals); adducts (i.e. segments of DNA bound to a cancer-causing chemical. This process can create a site of DNA damage that can interfere with replication and transcription); damage caused by oxidation; and damage caused by UV (formation of pyrimidine dimers, which are covalent bonds between adjacent pyrimidine bases (thymine or cytosine).

[0079] According to a particular embodiment, the damage is a single-strand break.

[0080] In one embodiment, the labeled dNTP includes at least a labeled UTP.

[0081] In one embodiment, the labeled dNTP includes at least a labeled GTP.

[0082] In one embodiment, the labeled dNTP includes at least a labeled ATP.

[0083] In one embodiment, the labeled dNTP includes at least a labeled CTP.

[0084] The detectable signal which is used to label the dNTP may be directly detectable such as for example a fluorescent signal, a phosphorescent signal, a radioactive signal or a colour signal (such as emitted by a chromophore).

[0085] In some embodiments, the labeling agent is an agent that is detectable by spectrophotometric measurements, and / or which can be utilized to produce optical imaging. Such agents include, for example, chromophores, fluorescent agents, phosphorescent agents, and heavy metal clusters.

[0086] As used herein, the term “chromophore” refers to a chemical moiety that, when attached to another molecule, renders the latter colored and thus visible when various spectrophotometric measurements are applied.

[0087] The phrase “fluorescent agent” refers to a compound that emits light at a specific wavelength during exposure to radiation from an external source.

[0088] The phrase “phosphorescent agent” refers to a compound emitting light without appreciable heat or external excitation as by slow oxidation of phosphorous.

[0089] A heavy metal cluster can be for example a cluster of gold atoms used, for example, for labeling in electron microscopy techniques (e.g., AFM).

[0090] The term "bioluminescent agent" describes a substance which emits light by a biochemical process.

[0091] The term "chemiluminescent agent" describes a substance which emits light as the result of a chemical reaction.

[0092] According to some embodiments of the invention, the labeling agent is a fluorescent labeling agent. Synthetic fluorophores that can be used to label the dNTPs (deoxynucleotide triphosphates) so as to label DNA molecules include fluorescein derivatives (e.g. fluorescein, 6-FAM, HEX or TET); cyanine dyes (e.g. Cy3, Cy5, Cy7); Alexa Fluor dyes (Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647); Bodipy dyes (Bodipy FE, BODIPY TMR); Rhodamine derivatives (e.g. TAMRA, ROX); Atto dyes (Atoo488, Atto 550, Atto 647N); sulfo-cyanine dyes; IR dyes (e.g. IRDye 680, 700, 800).

[0093] These fluorophores can be attached to dNTPs through various chemical modifications, for example targeting the base or sugar moiety. The choice of fluorophore depends on the experimental requirements, including excitation / emission spectra, photostability, and compatibility with detection systems.

[0094] Exemplary synthetic fluorophores used in the present invention may include, but are not limited to generic or proprietary fluorophores listed in Table 1 below:

[0095] Table 1. Generic or proprietary exemplary fluorophores suitable for use in the present invention

[0096] Examples of donor fluorophores that can be used to label the DNA include but are not limited to: CAL Fluor® Gold 540, CAL Fluor® Orange 560, Quasar® 670, Quasar® 705, 5-FAM (also called 5-carboxyfluorescein; also called Spiro(isobenzofuran-1(3H), 9'-(9H)xanthene)-5-carboxylic acid,3',6'-dihydroxy-3-oxo-6-carboxyfluorescein); 5-Hexachloro-Fluorescein ([4, 7, 2', 4', 5', 7'- hexachloro-(3',6'-dipivaloyl-fluoresceinyl)-6-carboxylic acid]); 6-Hexachloro-Fluorescein ([4,7,2',4',5',7'-hexachloro-(3',6'-dipivaloylfluoresceinyl)-5-carboxylic acid]); 5-Tetrachloro-

[0097] Fluorescein ([4,7,2',7'-tetra-chloro-(3',6'-dipivaloylfluoresceinyl)-5-carboxylic acid]); 6- Tetrachloro-Fluorescein ([4,7,2',7'-tetrachloro-(3',6'-dipivaloylfluoresceinyl)-6-carboxylic acid]); 5- TAMRA (5- carboxytetramethylrhodamine; Xanthylium, 9-(2,4-dicarboxyphenyl)-3,6- bis(dimethyl-amino); 6-TAMRA (6-carboxytetramethylrhodamine; Xanthylium, 9-(2,5- dicarboxyphenyl)-3, 6-bis(dimethylamino); EDANS (5-((2-aminoethyl) amino)naphthalene-l- sulfonic acid); 1,5-IAEDANS (5-((((2- iodoacetyl)amino)ethyl) amino)naphthalene-l- sulfonic acid); DABCYL (4-((4-(dimethylamino)phenyl) azo)benzoic acid) Cy5 (Indodicarbocyanine-5) Cy3 (Indo-dicarbocyanine-3); and BODIPY FL (2,6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a- diaza-s-indacene-3-proprionic acid), ROX, as well as suitable derivatives thereof. Additional examples include, but are not limited to fluorescein, fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, FITC, Oregon green, Alexa Fluor (e.g. AF488), FAM, JOE, HEX, Texas Red, TET, TRITC, cyanine-based dye and thiadicarbocyanine dye. According to a particular embodiment, the fluorophore is AF488 or EDANS.

[0098] Besides synthetic fluorophores, several other types of labels can be used to label dNTPs. These include radiolabels, biotin, digoxigenin, enzyme-based labels, gold nanoparticles, quantum dots, chemiluminescent labels, halogenated nucleotides, click chemistry tags, heavy metals, magnetic labels, photoactivatable labels.

[0099] Methods of detecting the detectable moiety are known in the art and are dependent upon the type of detectable moiety used in the assay. In one embodiment, the detectable moiety is a fluorescent moiety and the method of detecting is by fluorescent imaging.

[0100] According to some embodiments of the invention, detection ofr the presence of the detectable moiety is carried out without subjecting the DNA molecule to fragmentation.

[0101] In one embodiment, prior to detecting, the labeled DNA molecule is immobilized on a solid phase (i.e. solid surface).

[0102] According to another embodiment, the labeled DNA is extended on the solid phase.

[0103] According to some embodiments of the invention, the extending is linearly extending.

[0104] According to some embodiments of the invention, the extending is effected by depositing the DNA molecule on a surface or extending the DNA molecule in a nanochannel.

[0105] As used herein “extended DNA molecule” or “elongated DNA molecule” which is interchangeably used herein refers to a single or plurality elongated and fixed (i.e., immobilized) DNA.

[0106] According to some embodiments of the invention, the extended DNA molecules are elongated and fixed in a controllable manner directly onto a solid, planar surface. According to a specific embodiment, this solid, planar surface contains a positive charge density which has been controllably modified such that the single nucleic acid molecules will exhibit an optimal balance between the critical parameters of nucleic acid elongation state, degree of relaxation stability and biological activity. Further, methods, compositions and assays are described by which such an optimal balance can precisely and reproducibly be achieved.

[0107] The solid surface typically is positively charged.

[0108] In one embodiment, the solid surface is coated with a positively charged polymer (e.g. Poly-L- lysine (PLL) or chitosan).

[0109] In another embodiment, the solid surface is chemically functionalized e.g. by silane chemistry (e.g. using aminosilanes such as 3-aminopropyltriethoxysilane (APTES) to introduce amine groups).

[0110] According to alternative or additional embodiments, the single nucleic acid molecules are elongated via flow-based techniques. In such an embodiment, a single nucleic acid molecule is elongated, manipulated (via, for example, a regio- specific restriction digestion), and / or analyzed in a laminar flow elongation device. Such a laminar flow elongation devices and methods of elongating or extending DNA are described in U.S. Patent Application 20030124611, which is hereby incorporated by reference in its entirety.

[0111] The elongated, individual labeled DNA molecules can then be detected.

[0112] Additionally, methods are also presented for the imaging and sizing of the elongated single nucleic acid molecules. These imaging techniques may, for example, include the use of fluorochromes, microscopy and / or image processing computer software and hardware.

[0113] According to some embodiments of the invention, the extending of the DNA is effected following the labeling with the detectable moiety. However, it will be appreciated that extending the DNA molecule can be done prior to labeling with the detectable moiety.

[0114] According to some embodiments of the invention, the method further comprises attaching to the DNA molecule an additional labeling agent distinct of the labeling of the damaged site of the DNA molecules.

[0115] According to some embodiments of the invention, the additional labeling agent is a non- epigenetic modification specific labeling agent. Examples of such stains and dyes include DNA fluorescent dyes such as cyanine nucleic acid stains, which are essentially nonfluorescent in the absence of nucleic acids and exhibit significant fluorescence enhancements upon DNA binding. The stain may be cell permeant or impermeant.

[0116] Such stains include intercalating agents which dye the DNA include SYBR Green I, SYBR Gold, YoYo, EvaGreen and LCGreen. Such agents are not sequence specific and color the entire length of the DNA molecule.

[0117] As mentioned, once the damaged site on the DNA is labeled according to the methods described herein, the detectable moiety is detected.

[0118] In one embodiment, the DNA molecule is subjected to to an imaging method suitable for detecting the detectable moiety.

[0119] According to some embodiments of the invention, the labeling agent is a fluorescent agent, as described herein, and the imaging method is a fluorescence imaging.

[0120] Other labeling agents, as described herein, are also contemplated and respective imaging methods are utilized accordingly.

[0121] According to some embodiments of the invention, the method further comprises generating an optical image of the DNA molecule following the imaging.

[0122] In one embodiment, the number of sites which are labeled along the extended DNA are counted. The number of damaged sites per bp of DNA may be calculated.

[0123] The agents used to carry out the methods described herein may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The kit may comprise at least one agent for isolating sperm cells (such as those described herein above) and at least one agent which is used to detect DNA damage to the sperm cells. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for carrying out the method. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.

[0124] The methods of detecting DNA damage to sperm cells described herein may be used for assessing whether sperm cells are suitable candidates for in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). The method allows for the determination of defined sperm categories and the prediction whether or not the sperm cells will be functional. Further the results may be indicative whether sperm cells are a suitable candidate for in-vitro fertilization.

[0125] Upon determination of the amount of damage present in the sperm cells, the cells may be subject to one or more viability tests. This includes another aliquot of the same semen sample, which was analyzed by the method of the present invention as well as another semen sample from the same e.g. and identical subject. “More viability tests” means, for example, a spermatozoa motility test, an analysis of the acrosome status, morphology and morphometry analysis of the spermatozoa as well as an analysis of the sperm heads, which are known to a person skilled in the art. It further includes the use of a sperm sample for the purposes of research, or for the use in assisted reproductive technology (ART) such as in vitro fertilization (IVF), artificial insemination (Al), intracytoplasmic sperm injection (ICSI, as well as other techniques using enucleated cells), and multiple ovulation and embryo transfer (MOET, as well as other embryo transfer techniques).

[0126] The methods described herein may be used for diagnosing diseases associated with sperm cell damage. Thus, the method may be used to improve the diagnostic of male infertility and guide the treatment efficiently.

[0127] The methods described herein may further be used in order to determine the effect of an agent on sperm cells. According to this aspect, the method includes:

[0128] (a) contacting sperm cells with the agent; and

[0129] (b) detecting damage to the sperm cell DNA molecules as described herein. A level of damage above a predetermined amount is indicative that the agent is toxic to the sperm cells. A level of damage below a predetermined amount is indicative that the agent is non-toxic to the sperm cells.

[0130] As used herein the term “about” refers to ± 10 %

[0131] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0132] The term “consisting of’ means “including and limited to”.

[0133] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0134] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0135] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0136] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0137] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0138] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0139] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0140] EXAMPLES

[0141] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0142] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); “Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et ah, "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

[0143] MATERIALS AND METHODS

[0144] Semen samples: 40 samples of normozoospermic (aligned with the 2010 WHO reference values) semen were obtained after 2-3 days of sexual abstinence. Fresh ejaculate was allowed to liquefy and then cryopreserved in the test yolk buffer. All experimental protocols were approved and performed in accordance with relevant guidelines and regulations of Sheba Hospital Helsinki Committee, and informed consent was obtained from all the participants.

[0145] Sperm preparation: The samples were thawed at room temperature and then washed twice in Dulbecco’s phosphate- buffered saline (PBS). Following the washing steps, the semen samples were diluted in MHM-C (Multipurpose Handling Medium-Complete, purchased from FUJIFILM) to achieve a concentration of 2.5 x 106.

[0146] Incubation ofDNA breaks

[0147] H2O2 treatment: Sperm samples were divided into aliquots and diluted in MHM-C medium. This division allowed for the control group to be compared to the treated cells. Spermatozoa were incubated for 2 hours at 37 °C with hydrogen peroxide (Sigma Aldrich).

[0148] DNA extraction: DNA was extracted using the following buffer:

[0149] 20 mM Tris- Cl (pH 8.0)

[0150] 20 mM EDTA

[0151] 200 mM NaCl

[0152] 80 mM DTT

[0153] 4% SDS

[0154] 250 pg / ml Proteinase K

[0155] Other components which were used were obtained from the QIAamp® DNA Mini Kit.

[0156] Labeling DNA damage: DNA samples were labeled for oxidation damage. In the first step, each reaction tube contained 500ng ofDNA sample, 3 pl lOx thermopol buffer, 0.3pl xlOO NAD+, 0.6pl preCR™ repair mix (an enzymatic cocktail that includes repair enzymes, DNA polymerase and DNA ligase) topped to 30pl total reaction volume with ultrapure water. The samples were incubated for 30 min at 37°C. Next, deoxynucleotides (A, G, C) and fluorescent Atto550 UTP were added. The reaction mixture was incubated for 30 minutes at 65°C. The labeled DNA samples were purified from excess fluorophores using Oligo Clean & Concentrator columns (Zymo research), according to manufacturer's recommendations, with two washing steps for optimal results.

[0157] Slide preparation -. Teflon coated microscope slides (Tekdon, customized well formation, 2 mm diameter wells, 90 wells per slide) were immersed in 0.005% poly-L- lysine solution in water (Sigma), in order to positively charge the surface. The immersed slides were incubated for one hour at 37°C with mild shaking (25rpm) and then incubated overnight at 4°C. The following day a blocking step was performed. Slides were washed twice with PBST solution, twice with PBS (Sigma), and immersed in a 5% w / v bovine serum albumin (Sigma) solution in PBS. The immersed slides were incubated for one hour at 37°C with mild shaking (25 rpm) and then incubated overnight at 4°C. Lastly, slides were washed with water and dried under a flow of nitrogen gas. Slides were used immediately upon drying.

[0158] Applying DNA samples onto the activated slides'. Labeled-DNA samples (1 pl) were placed in each well. The optimal DNA concentration for attachment is 10-20 ng per well. Three to five replicates of each sample were placed on the slide. Slides were incubated for 14 minutes at 42°C and then for 24 minutes at 30°C, in humid conditions to avoid rapid drying of the wells (Thermo shaker, Eppendorf). The slides were then washed with water and dried under a flow of nitrogen gas.

[0159] Total DNA staining-. Total DNA was stained with EvaGreen DNA binding dye (Biotium). Ipl of 1.25 pM dye (90% water, 10% DMSO) was added to each well containing the bound DNA. Wells containing only water and no DNA were also stained, in order to obtain the background signal of the EvaGreen dye in the absence of DNA. Slides were incubated for 30 minutes at room temperature in the dark. Slides were then washed with water and dried under a flow of nitrogen gas.

[0160] Slide imaging: Slides were imaged using InnoScanl lOO slide scanner (Innopsys). A 532nm green laser was used to image the ATTO-550 fluorophore / TAMRA fluorophore. A 488nm blue laser was used to image the EvaGreen stain.

[0161] Data analysis: Images were analyzed using ImageJ 30. The mean fluorescence intensity inside each well in both channels was extracted. The background signal was determined from the control replicates and subtracted from the ATTO-550 fluorescence signal (DNA damage labels) in each sample's well. To account for background noise in the EvaGreen signal (total DNA), a mean fluorescence signal of all wells containing EvaGreen and no DNA was calculated and subtracted from the EvaGreen signal for each sample well. We divided the calculated ATTO-550 signal in each well by the fluorescence intensity calculated in the EvaGreen channel of the same well, in order to normalize the signal to the actual amount of DNA in the well. Next, the average and standard deviation for each sample were calculated over three to five replicates.

[0162] Sperm chromatin dispersion (SCI)): The assay was carried out using a commercially, validated, available kit Halosperm G2 kit, and it has been described in detail elsewhere (Femadez et al., 2005). Briefly, an aliquot of the semen sample was diluted to 20million / ml. Eppendorf tubes were placed in a water bath at 90-100 °C for 5 min. After incubation, we transfer the diluted semen sample to the melted agarose tube. Next, we placed 8ul of the cell suspension onto the sample well (provided by the kit) and covered with a coverslip. Slides were then placed on a plate in the refrigerator (4°C) for 5 min. After taking out the slides from the fridge, the cover-slips were gently removed. The denaturing agent was applied for 7 min. Subsequently, the slides were immersed in a lysis solution and incubated for 20 min. After washing with abundant distilled water for 5 min, the slides were dehydrated in increasing concentrations of ethanol (70% and 100% for 2 min each) and were left to dry. Lastly, the slides were covered with a mix of Wright’s staining solution for 8 min. A minimum of 200 spermatozoa were assessed per patient.

[0163] Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (Tunel): TUNEL assay has been previously described by Palermo (O’ Neill et al., 2018). Briefly, the assay was carried out using a commercially available kit (in-situ cell death detection kit; Roche Diagnostics, Rotkreuz, Switzerland). Semen samples were applied to glass slides using 8 pL of the semen sample, which were then left to dry Fixation was carried out by placing slides in 4% paraformaldehyde for Ih. Slides were then washed in PBS and left to dry. Permeabilization was performed by exposing slides to 0.1% Triton X-100 and 0.1% sodium citrate in PBS for 2 min at 4°C. Slides were washed in PBS and left to dry. The kit reagent was applied to the slides and left to incubate with coverslips added in a humidified chamber at 37 °C for Ih. Slides were washed thrice in PBS and DAPI Antifade was added in order to visualize the spermatozoa's nuclei, which were then viewed under a fluorescent microscope. Thus, to detect signal indicating DNA breakage. A minimum of 300 spermatozoa were assessed per patient.

[0164] RESULTS

[0165] Sperm cells were exposed to increasing concentrations of H2O2 (0.5, 1, 1.5 mM). As illustrated in Figure 2, RADD could effectively detect DNA damage in a dose-dependent manner after exposure to H2O2 at concentrations with ratios of 1.06, 2.16, and 4.83 compared to controls, respectively (Figure 2). As shown in Figure 2, the strength of the fluorescent signal positively correlates with increasing concentration of H2O2. Furthermore, the results from Figure 5 indicate a significant correlation between the baseline level of DNA damage in each sample and the subsequent DNA damage values after H2O2 treatment. Notably, sperm with lower baseline DNA damage displayed a reduced susceptibility to H2O2- induced DNA damage, suggesting a potential protective effect. Conversely, sperm with higher baseline DNA damage exhibited an increased vulnerability to DNA insult upon exposure to FhCh-

[0166] Next, a parallel analysis was carried out in the presence and absence of H2O2. Levels of DNA damage were analyzed using three different assays - RADD, SCD, and TUNEL. As depicted in Figure 3A, RADD detected significantly higher levels of DNA damage as compared to control group. Similarly, SCD and TUNEL assays also detected a significant level of DNA damage.

[0167] However, there are differences in the capabilities of SCD and TUNEL methods compared to RADD. SCD examines DNA damage indirectly through chromatin changes, while TUNEL directly marks the free 3 ’-OH ends in damaged DNA, detecting DNA breaks (ss and ds) in a binary way. In sharp contrast, RADD is capable of quantitatively detecting both DNA adducts and singlestrand DNA breaks. To compare the results of these different assays, the damaged samples were normalized to the control group. Figure 3D illustrates that there were no significant differences between RADD and SCD in detecting DNA damage. However, both RADD and SCD detected significantly more DNA damage than TUNEL.

[0168] Next, the present inventors analyzed how each method scored the semen samples (Figure 4). An increase in RADD detection was associated with a corresponding increase in SCD, both in the control and treated samples (r>0.55, p<0.01). Additionally, the correlation between treated and control was examined within RADD to determine how each sample's level of DNA damage changed.

[0169] Lastly, DNA damage levels was investigated in a healthy population. No significant correlation between DNA damage and sperm count, volume, or motility was found (Figure 6B). Hence, the commonly used WHO criteria for sperm evaluation may not be adequate for predicting ssDNA damage. Furthermore, a nomogram curve was created for DNA damage in healthy population (Figure 6A). The present samples ranged from 0.005 to 0.1, with a median of 0.026 and 25% quantile of 0.015 and 75% quantile of 0.055. As such, despite the good sperm parameters, there was a wide range of DNA damage levels.

[0170] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. It is the intent of the applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety

Claims

WHAT IS CLAIMED IS:

1. A method of detecting damage to sperm cell DNA molecules comprising:(a) labeling the DNA molecules with a detectable moiety which is capable of being incorporated specifically at a damaged site of the DNA molecules, wherein said DNA molecules are double-stranded; and(b) detecting the detectable moiety, thereby detecting damage to the sperm cell DNA molecules.

2. The method of claim 1, wherein said labeling is effected ex vivo.

3. The method of claim 1, wherein said damage comprises a breakage of a single strand of said double-stranded DNA molecule.

4. The method of claim 3, wherein said labeling is effected by:(i) contacting the DNA molecules with at least one DNA repair enzyme under conditions that said DNA repair enzyme removes at least one base at the breakage site of a broken strand of said DNA; and subsequently(ii) contacting the DNA molecules with a DNA polymerase and dNTPs, said dNTPs being attached to said detectable moiety, under conditions that at least one dNTP is incorporated into said DNA molecules at said breakage site.

5. The method of claim 4, wherein said at least one DNA repair enzyme is DNA Polymerases Lambda (X) and / or DNA polymerase Mu (p).

6. The method of any one of claims 1-4, wherein said detectable moiety is a fluorescent moiety, a phosphorescent moiety or a radioactive moiety.

7. The method of any one of claim 1-6, further comprising contacting the DNA molecules with an intercalating agent prior to said detecting.

8. The method of claim 7, wherein said intercalating agent is selected from the group consisting of SYBR Green I, SYBR Gold, YoYo, EvaGreen and LCGreen.

9. The method of any one of claims 1-6, further comprising immobilizing said DNA molecules on a solid surface following step (a) and prior to step (b).

10. The method of claim 9, wherein said solid surface is positively charged.

11. The method of claim 10, wherein said solid surface is coated with poly-L-lysine.

12. The method of any one of claims 1-9, wherein the DNA molecules are longer than 20,000 base pairs.

13. The method of any one of claims 1-12, further comprising isolating DNA from sperm cells prior to the labeling under conditions that prevents damage to the DNA molecules.

14. The method of claim 13, wherein said isolating comprises contacting the sperm cells with a buffer comprising an anionic detergent capable of lysing said sperm cells and denaturing protamines of said sperm cells.

15. The method of claim 14, wherein said anionic detergent comprises SDS.

16. The method of claim 15, wherein a concentration of said SDS in said composition is between 2-5 %.

17. The method of claim 14, wherein said buffer further comprises a reducing agent.

18. The method of claim 17, wherein said reducing agent comprises DTT or P- mercaptoethanol.

19. The method of claim 18, wherein said reducing agent is DTT.

20. The method of claim 19, wherein a concentration of said DTT in said composition is between 60-100 mM.

21. The method of any one of claims 1-20, further comprising quantifying a number of damage sites per DNA molecule.

22. A method of assessing whether sperm cells are a candidate for in-vitro fertilization comprising detecting damage to the sperm cells DNA molecules according to any one of claims 1-21, wherein an extent of said damage is indicative whether sperm cells are a candidate for in- vitro fertilization.

23. A method of diagnosing a disease associated with sperm cell damage comprising detecting damage to the sperm cell DNA molecules according to any one of claims 1-21, wherein an extent of said damage above a predetermined threshold is indicative of a disease associated with sperm cell damage.

24. A method of detecting the effect of an agent on sperm cells comprising:(a) contacting sperm cells with the agent; and(b) detecting damage to the sperm cell DNA molecules according to any one of claims 1- 21, wherein a level of damage above a predetermined amount is indicative that the agent is toxic to the sperm cells.

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