Method and kit for detecting sperm DNA fragmentation

Abstract

The present invention relates to a method and a kit for detecting sperm DNA fragmentation. The present invention discloses a method for detecting sperm DNA fragmentation in a semen sample, comprising the following steps: subjecting the semen sample to gel embedding, DNA denaturation, and lysis, thereby denaturing the sperm DNA and lysing the nucleoprotein, wherein the lysis is performed using a lysis solution containing urea and sodium dodecyl sulfate; subjecting the lysed gel to DNA staining; and observing the stained gel for the formation of a halo around the head of each sperm. The absence of a halo or a halo width less than one-third of the diameter of the sperm head indicates the presence of sperm DNA fragmentation. The present invention also discloses a kit for performing this method.

Description

Technical Field

[0001] The present invention relates to a method for detecting DNA fragmentation in spermatozoa in a semen sample and a kit for performing the method. Background Art

[0002] Sperm DNA integrity is a major factor affecting embryo quality, embryo implantation, and embryo development. Sperm DNA fragmentation (SDF), caused by factors such as spermiogenesis errors, sperm apoptosis, oxidative stress, or radiation, can lead to male infertility, in vitro fertilization (IVF) failure, and miscarriage. Therefore, detecting sperm DNA fragmentation plays a crucial role in fertility screening and artificial reproduction treatments.

[0003] Currently available methods for detecting sperm DNA fragmentation include sperm chromatin structure assay (SCSA), terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay, DNA Breakage Detection-Fluorescence in Situ Hybridization (DBD-FISH) test, comet assay (CA), and sperm chromatin dispersion (SCD) test. Among them, the SCD test is the most widely used.

[0004] The SCD test primarily involves embedding sperm in agarose gel and then using a DNA denaturing solution to selectively convert the double-stranded DNA in the sperm into single-stranded DNA. The sperm's nuclear proteins (including protamine) are then lysed. The protamine cleavage causes the DNA to loosen, forming DNA loops. The size of the DNA loops is positively correlated with the integrity of the DNA. Following DNA staining, these DNA loops form halos of varying sizes. A halo width greater than or equal to 1 / 3 the diameter of the sperm head indicates no DNA fragmentation, while the absence of a halo or a halo width less than 1 / 3 the diameter of the sperm head indicates DNA fragmentation. However, the SCD test has the disadvantages of being complex and time-consuming. In addition, if the test time is too long, the tail of the sperm may become curled, and this will become more obvious as time goes by. Moreover, because some semen samples contain white blood cells, the white blood cells will also form a halo after the SCD test, which can easily be confused with the sperm with curled tails, making it difficult to interpret the results.

[0005] Therefore, there is still a need in the art to develop a detection method that is convenient, fast and reliable to meet the needs of the industry. Summary of the Invention

[0006] Thus, in a first aspect, the present invention provides a method for detecting DNA fragmentation in sperm in a semen sample, comprising the following steps:

[0007] (a) embedding the semen sample in a gel to obtain a gel containing sperm, wherein the gel comprises a component selected from the group consisting of agarose, acrylamide, alginate and vinyl chloride;

[0008] (b) performing a DNA denaturation treatment on the gel containing the sperm using a DNA denaturing solution to denature the DNA of the sperm;

[0009] (c) lysing the DNA-denatured gel with a lysis buffer to lyse the sperm nucleoprotein, wherein the lysis buffer comprises urea having a concentration ranging from 0.5 to 4 M and sodium lauryl sulfate having a concentration ranging from 0.05 to 0.5% (w / v, g / mL);

[0010] (d) subjecting the lysed gel to DNA staining; and

[0011] (e) Observe whether a halo is formed around the head of each sperm on the stained gel. The absence of a halo or a halo width less than 1 / 3 of the diameter of the sperm head indicates the presence of sperm DNA fragmentation.

[0012] Preferably, in step (a), the gel further comprises an acid-base indicator selected from the group consisting of phenol red, methyl violet, methyl orange, methyl red, Congo red, and combinations thereof.

[0013] In a second aspect, the present invention provides a method for detecting DNA fragmentation in sperm in a semen sample, comprising the following steps:

[0014] (a) subjecting an agarose gel to a heat treatment, then mixing a DNA denaturing solution with the semen sample, and then subjecting the resulting mixture to gel polymerization, thereby obtaining a gel containing spermatozoa with denatured DNA embedded therein;

[0015] (b) lysing the gel containing the denatured sperm using a lysis solution to lyse the sperm nucleoprotein, wherein the lysis solution comprises urea having a concentration ranging from 0.5 to 4 M and sodium lauryl sulfate having a concentration ranging from 0.05 to 0.5% (w / v, g / mL);

[0016] (c) subjecting the lysed gel to DNA staining; and

[0017] (d) Observe whether a halo is formed around the head of each sperm on the stained gel. The absence of a halo or a halo width less than 1 / 3 of the diameter of the sperm head indicates the presence of sperm DNA fragmentation.

[0018] Preferably, in step (a), the agarose is pre-mixed with an acid-base indicator selected from the group consisting of phenol red, methyl violet, methyl orange, methyl red, Congo red, and combinations thereof before being heat-treated.

[0019] In a third aspect, the present invention provides a method for detecting DNA fragmentation in sperm in a semen sample, comprising the following steps:

[0020] (a) mixing the semen sample, a DNA denaturing solution, and a colloid-forming component, and then subjecting the resulting mixture to gel polymerization to obtain a gel containing denatured spermatozoa, wherein the colloid-forming component is selected from the group consisting of acrylamide, alginate, and vinyl chloride;

[0021] (b) lysing the gel containing the denatured sperm using a lysis solution to lyse the sperm nucleoprotein, wherein the lysis solution comprises urea having a concentration ranging from 0.5 to 4 M and sodium lauryl sulfate having a concentration ranging from 0.05 to 0.5% (w / v, g / mL);

[0022] (c) subjecting the lysed gel to DNA staining; and

[0023] (d) Observe whether a halo is formed around the head of each sperm on the stained gel. The absence of a halo or a halo width less than 1 / 3 of the diameter of the sperm head indicates the presence of sperm DNA fragmentation.

[0024] Preferably, in step (a), the mixture further comprises an acid-base indicator selected from the group consisting of phenol red, methyl violet, methyl orange, methyl red, Congo red, and combinations thereof.

[0025] Preferably, the DNA denaturing solution is an acid selected from the group consisting of hydrochloric acid, acetic acid, nitric acid, sulfuric acid, and combinations thereof.

[0026] Preferably, the DNA denaturing solution is a base selected from the group consisting of sodium hydroxide, potassium hydroxide, calcium hydroxide, and combinations thereof.

[0027] Preferably, the lysate further comprises a protein denaturant selected from the group consisting of 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonic acid hydrate, guanidine hydrochloride, and combinations thereof.

[0028] Preferably, the lysis solution further comprises an ionic surfactant selected from the group consisting of sodium deoxycholate, sodium cholate, sodium lauroyl sarcosinate, and combinations thereof.

[0029] In a fourth aspect, the present invention provides a colloid-forming formula, a DNA denaturing solution, and a lysis solution for preparing a kit for detecting DNA fragmentation of sperm in a semen sample, wherein the steps of using the kit to detect DNA fragmentation of sperm in a semen sample include: steps (a) to (e) described in the first aspect above, steps (a) to (d) described in the second aspect above, or steps (a) to (d) described in the third aspect above.

[0030] Preferably, the kit further comprises a DNA staining reagent.

[0031] In a fifth aspect, the present invention provides a kit for detecting DNA fragmentation of sperm in a semen sample, comprising:

[0032] a colloid-forming formulation comprising an ingredient selected from the group consisting of agarose, acrylamide, alginate, and vinyl chloride;

[0033] a DNA denaturing solution;

[0034] a lysis solution comprising urea having a concentration ranging from 0.5 to 4 M and sodium lauryl sulfate having a concentration ranging from 0.05 to 0.5% (w / v, g / mL); and

[0035] A DNA staining reagent.

[0036] Preferably, the colloid-forming formulation further comprises an acid-base indicator selected from the group consisting of phenol red, methyl violet, methyl orange, methyl red, Congo red, and combinations thereof.

[0037] Preferably, the DNA denaturing solution is an acid selected from the group consisting of hydrochloric acid, acetic acid, nitric acid, sulfuric acid, and combinations thereof.

[0038] Preferably, the DNA denaturing solution is a base selected from the group consisting of sodium hydroxide, potassium hydroxide, calcium hydroxide, and combinations thereof.

[0039] Preferably, the kit further comprises a carrier for carrying the semen sample, wherein the carrier is covered with a film containing 0.25 to 1.5% (w / v, g / L) agarose. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The present invention will be described in detail below with reference to the accompanying drawings and embodiments. The above and other objects and features of the present invention will become more apparent by referring to the following description, the appended claims and the accompanying drawings, in which:

[0041] Figure 1 The results of DNA staining of the AG gels of each group are shown; and

[0042] Figure 2 A correlation diagram is shown between the DFI index measured by the method of the present invention and that measured by the existing SCD test. DETAILED DESCRIPTION

[0043] For the purpose of clearly describing the present invention, it will be understood that the word "comprising" means "including but not limited to," and that the word "comprises" has a corresponding meaning.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used to practice the present invention. Of course, the present invention is in no way limited to the methods and materials described.

[0045] To improve existing methods for detecting sperm DNA fragmentation, the applicants have diligently discovered that mixing a semen sample, a DNA denaturing solution, and heated, liquefied agarose for gel embedding simultaneously denatures the semen sample's DNA during the embedding process. Further lysis treatment with a lysis solution containing urea and sodium dodecylsulfate (SDS) accelerates the cleavage of nuclear proteins, effectively shortening the total detection time. Furthermore, the applicants discovered that premixing the agarose with an acid-base indicator before heating and liquefying allows confirmation of the presence of the DNA denaturing solution by observing a color change, making the operation more convenient for the tester. Furthermore, some acid-base indicators have disinfecting and antibacterial properties, making the gel less susceptible to contamination during storage.

[0046] Therefore, the present invention provides a method for detecting DNA fragmentation of sperm in a semen sample, comprising the following steps:

[0047] (a) embedding the semen sample in a gel to obtain a gel containing sperm, wherein the gel comprises a component selected from the group consisting of agarose, acrylamide, alginate and vinyl chloride;

[0048] (b) performing a DNA denaturation treatment on the gel containing the sperm using a DNA denaturing solution to denature the DNA of the sperm;

[0049] (c) lysing the DNA-denatured gel with a lysis buffer to lyse the sperm nucleoprotein, wherein the lysis buffer comprises urea having a concentration ranging from 0.5 to 4 M and sodium lauryl sulfate having a concentration ranging from 0.05 to 0.5% (w / v, g / mL);

[0050] (d) subjecting the lysed gel to DNA staining; and

[0051] (e) Observe the stained gel to see whether there is a halo formation around the head of each sperm. The absence of a halo or a halo width less than 1 / 3 of the diameter of the sperm head indicates the presence of sperm DNA fragmentation.

[0052] According to the present invention, in step (a), the gel further comprises an acid-base indicator selected from the group consisting of phenol red, methyl violet, methyl orange, methyl red, Congo red, and combinations thereof. In a preferred embodiment of the present invention, the acid-base indicator is phenol red.

[0053] Preferably, the gel is agarose gel.

[0054] According to the present invention, the agarose gel may have an agarose concentration ranging from 1 to 3% (w / v, g / mL). In a preferred embodiment of the present invention, the agarose gel has an agarose concentration of 1.25% (w / v, g / mL).

[0055] According to the present invention, the agarose gel is first placed in a microwave oven or a thermostatic bath and heated and liquefied at a temperature range of 95 to 100° C. before being used to embed the semen sample. In a preferred embodiment of the present invention, the temperature used for the heating and liquefaction is 95° C.

[0056] According to the present invention, the semen sample can be collected from an individual at any time. Preferably, the semen sample is collected from an individual who has experienced at least 2-3 days but no more than 10 days of abstinence.

[0057] According to the present invention, the semen sample can be a fresh sample or a cryopreserved sample [eg, frozen in liquid nitrogen (-196° C.)].

[0058] As used herein, the term "subject" refers to any animal of interest, such as primates (humans, apes, and simians); non-primate mammals (pigs, cows, sheep, horses, goats, dogs, cats, mice, and rats); fish; and amphibians. In a preferred embodiment of the present invention, the subject is a human.

[0059] According to the present invention, the semen sample can be diluted with a diluent before being used for detection, so that the sperm concentration range is within 4×10 6 to 1.5×10 7 cells / mL. Suitable diluents for use in the present invention include, but are not limited to, Earle's medium, human tubal fluid (HTF) medium (also known as germ cell culture medium), Tris-buffered saline (TBS), phosphate-buffered saline (PBS), and saline.

[0060] In a preferred embodiment of the present invention, the semen sample is diluted with HTF culture medium before being used for detection to obtain a 1×10 7 Sperm concentration of cells / mL.

[0061] According to the present invention, the DNA denaturing solution can be an acid or an alkali, and can be present in a normality range of 0.05 to 0.08 N to restrict the DNA denaturation process. In some embodiments, the normality ranges from 0.06 to 0.07 N.

[0062] Preferably, the DNA denaturing solution is an acid selected from the group consisting of hydrochloric acid, acetic acid, nitric acid, sulfuric acid, and combinations thereof. In some embodiments, the DNA denaturing solution is hydrochloric acid.

[0063] Preferably, the DNA denaturing solution is a base selected from the group consisting of sodium hydroxide, potassium hydroxide, calcium hydroxide, and combinations thereof. In some embodiments, the DNA denaturing solution is sodium hydroxide.

[0064] As used herein, the terms "lysis solution," "cell lysis solution," and "protein lysis solution" are used interchangeably.

[0065] According to the present invention, sodium lauryl sulfate and urea added to the lysis solution serve as an ionic surfactant and a protein denaturant, respectively. These agents promote the cleavage of protamine, thereby accelerating the separation of DNA fragments originally entangled with protamine and releasing them to the periphery of the sperm head, forming a halo. This shortens the lysis process (e.g., to within 5 minutes) and prevents the sperm tail from curling.

[0066] According to the present invention, the lysis solution further comprises another surfactant, which can be an ionic surfactant or a nonionic surfactant. Preferably, the ionic surfactant is selected from the group consisting of sodium deoxycholate, sodium cholate, sodium lauroyl sarcosinate, and combinations thereof. Preferably, the nonionic surfactant is selected from the group consisting of Triton X-100, Nonoxynol-40 (NP-40), Pluronic F-127 (F-127), Polysorbate 20 (Tween-20), and combinations thereof. In a preferred embodiment of the present invention, the nonionic surfactant is Triton X-100.

[0067] According to the present invention, the lysate further comprises another protein denaturant. Preferably, the protein denaturant is selected from the group consisting of 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate, guanidinium chloride, and combinations thereof.

[0068] According to the present invention, the lysis solution further comprises a reducing agent, salts, and a titrant for adjusting the pH value.

[0069] Preferably, the reducing agent is selected from the group consisting of dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), dithioerythritol (DTE), β-mercaptoethanol (β-ME), glutathione (GSH), dimercaprol, heparin, and combinations thereof. In a preferred embodiment of the present invention, the reducing agent is DTT.

[0070] Preferably, the salt is selected from the group consisting of sodium chloride (NaCl), potassium chloride (KCl), and combinations thereof.

[0071] Preferably, the titrant is selected from the group consisting of sodium hydroxide (NaOH), hydrochloric acid (HCl), and combinations thereof.

[0072] More preferably, the lysis buffer further comprises NaCl having a concentration ranging from 0.15 to 3 M, DTT having a concentration ranging from 0.05 to 0.2 M, Triton X-100 having a concentration ranging from 0.1 to 5%, and NaOH having a concentration ranging from 0.01 to 0.02 M. In particular, the use of DTT and a high concentration of NaCl facilitates the formation of a halo after DNA is separated from protamine.

[0073] In some embodiments, the lysis buffer comprises 1 M urea, 0.05% SDS, 2.5 M NaCl, 0.1 M DTT, 1% Triton X-100, and 0.02 M NaOH. In some embodiments, the lysis buffer comprises 4 M urea, 0.05% SDS, 0.15 M NaCl, 0.2 M DTT, 0.5% Triton X-100, and 0.01 M NaOH. In some embodiments, the lysis buffer comprises 0.5 M urea, 0.5% SDS, 3 M NaCl, 0.05 M DTT, 5% Triton X-100, and 0.015 M NaOH.

[0074] According to the present invention, the lysate can be adjusted to a desired pH using the titrant. Preferably, when the DNA denaturing solution is an acid, the lysate is adjusted to a pH within a range of 7.5 to 9.0. Preferably, when the DNA denaturing solution is an alkali, the lysate is adjusted to a pH within a range of 5.5 to 7.0. In a preferred embodiment of the present invention, the lysate has a pH within a range of 8.2 to 8.5.

[0075] According to the present invention, the DNA staining is performed using a method selected from the group consisting of: Diff-Quik staining, Wright-Giemsa staining, propidium iodide (PI) staining, SYBR Green staining, DAPI staining, and acridine orange staining.

[0076] The present invention also provides a method for detecting DNA fragmentation in sperm in a semen sample, comprising the following steps:

[0077] (a) heating an agarose gel, mixing the a DNA denaturing solution and the semen sample, and then subjecting the resulting mixture to gel polymerization to obtain a gel containing spermatozoa with denatured DNA;

[0078] (b) lysing the gel containing the denatured sperm using a lysis solution to lyse the sperm nucleoprotein, wherein the lysis solution comprises urea having a concentration ranging from 0.5 to 4 M and sodium lauryl sulfate having a concentration ranging from 0.05 to 0.5% (w / v, g / mL);

[0079] (c) subjecting the lysed gel to DNA staining; and

[0080] (d) Observe whether a halo is formed around the head of each sperm on the stained gel. The absence of a halo or a halo width less than 1 / 3 of the diameter of the sperm head indicates the presence of sperm DNA fragmentation.

[0081] According to the present invention, in step (a), the agarose is pre-mixed with a pH indicator as described above before being heat treated.

[0082] According to the present invention, the heat treatment may be performed at a temperature ranging from 95 to 100° C. Preferably, the heat treatment is performed at 95° C.

[0083] According to the present invention, the semen sample, the DNA denaturing solution, the lysis solution, and the DNA stain are as described above.

[0084] The present invention also provides a method for detecting DNA fragmentation in sperm in a semen sample, comprising the following steps:

[0085] (a) mixing the semen sample, a DNA denaturing solution, and a colloid-forming component, and then subjecting the resulting mixture to gel polymerization to obtain a gel containing denatured spermatozoa, wherein the colloid-forming component is selected from the group consisting of acrylamide, alginate, and vinyl chloride;

[0086] (b) lysing the gel containing the denatured sperm using a lysis solution to lyse the sperm nucleoprotein, wherein the lysis solution comprises urea having a concentration ranging from 0.5 to 4 M and sodium lauryl sulfate having a concentration ranging from 0.05 to 0.5% (w / v, g / mL);

[0087] (c) subjecting the lysed gel to DNA staining; and

[0088] (d) Observe whether a halo is formed around the head of each sperm on the stained gel. The absence of a halo or a halo width less than 1 / 3 of the diameter of the sperm head indicates the presence of sperm DNA fragmentation.

[0089] According to the present invention, in step (a), the mixture further comprises an acid-base indicator as described above.

[0090] According to the present invention, the semen sample, the DNA denaturing solution, the lysis solution, and the DNA stain are as described above.

[0091] The present invention also provides a kit for detecting DNA fragmentation of sperm in a semen sample, comprising:

[0092] a gel-forming formulation comprising an ingredient selected from the group consisting of agarose, acrylamide, alginate, and vinyl chloride;

[0093] a DNA denaturing solution;

[0094] a lysis solution comprising urea having a concentration ranging from 0.5 to 4 M and sodium lauryl sulfate having a concentration ranging from 0.05 to 0.5% (w / v, g / mL); and

[0095] - DNA staining reagent.

[0096] As used herein, the term "kit" refers to a packaged product containing reagents or materials for detecting a disease or its clinical signs. The kit may include one or more boxes or containers for containing the reagents or materials, as well as a label and / or instructions for indicating the use of the contents of the kit.

[0097] Preferably, the component is agarose.

[0098] Preferably, the component is acrylamide. For example, the colloid-forming formulation comprises an acrylamide / bis-acrylamide solution and an initiator, and they are placed in separate containers (e.g., microcentrifuge tubes, glass bottles, or plastic bottles).

[0099] Initiators suitable for use in the present invention include, but are not limited to, ammonium persulfate (APS), tetramethylethylenediamine (TEMED), riboflavin-5'-phosphate sodium, and 3-(dimethylamino)propionitrile.

[0100] According to the present invention, the colloid-forming formulation may further comprise an acid-base indicator as described above.

[0101] According to the present invention, the DNA denaturing solution can be an acid or a base as described above.

[0102] According to the present invention, the DNA denaturing solution and the colloid-forming formulation are placed in separate containers (eg, microcentrifuge tubes, glass bottles, or plastic bottles).

[0103] According to the present invention, the kit further includes a carrier for holding the semen sample, which serves to immobilize the gel formed by the colloid-forming formula on its surface, thereby facilitating the various processing steps of the above-described method (including lysis and DNA staining). Suitable carriers for the present invention include, but are not limited to, slides and well plates.

[0104] According to the present invention, the carrier may be covered with a film containing 0.25 to 1.5% (w / v, g / L) agarose to facilitate easier fixation of the gel on the carrier. In a preferred embodiment of the present invention, the carrier is a glass slide covered with a film containing 1% (w / v, g / L) agarose.

[0105] According to the present invention, the formulation and pH value of the lysis solution are as described above.

[0106] According to the present invention, the DNA staining reagent is selected from the group consisting of: Diff-Quik dye, Wright-Giemsa dye, propidium iodide, SYBR Green, DAPI and acridine orange.

[0107] The present invention will be further described with reference to the following examples. However, it should be understood that the examples are for illustration only and should not be construed as limitations on the implementation of the present invention.

[0108] <Example>

[0109] Experimental subjects and materials:

[0110] 1. Experimental subjects and their semen samples:

[0111] A total of 36 male subjects aged between 22 and 40 participated in the experiment of the present invention.

[0112] 100 μL of semen was collected from each subject and the sperm count was measured using a sperm quality analyzer (model X1 PRO, brand Lenshooke). The semen was then diluted with phosphate-buffered saline (PBS) to obtain a semen sample of the desired concentration.

[0113] 2. The lysis solution used in the following examples contained 2.5 M sodium chloride (NaCl), 0.2 M dithiothreitol (DTT), 4 M urea, 1% Triton X-100, 0.05% sodium dodecyl sulfate (SDS), and 0.01 M sodium hydroxide (NaOH). NaOH was used to adjust the pH of the lysis solution to 8.2 to 8.5.

[0114] Example 1. Effect of lysis treatment of sperm using a lysis solution containing urea and SDS on the detection of sperm DNA fragmentation

[0115] Experimental methods:

[0116] First, a semen sample of a subject was randomly selected from the first item of “Experimental subjects and materials” above, and the semen sample was divided into a control group and four experimental groups (i.e., experimental groups 1 to 4) (each group had 2.5×10 5 Sperm / 25 μL). 100 μL of heated, liquefied 1.25% (w / v, g / L) agarose (Uniregion BioTech Inc.) (premixed with 0.02 mg / mL phenol red) was added to the semen samples of each group and mixed. Then, 25 μL of the resulting mixture was taken and placed on a glass slide (covered with a film containing 1% (w / v, g / L) agarose to firmly fix the mixture on the slide). The mixture was then allowed to stand at 4°C for 5 minutes, thereby embedding the semen samples of each group in the agarose gel and fixing them on the slide. This yielded sperm-embedded agarose gels (hereinafter referred to as AG gels) in each group.

[0117] Next, 200-300 μL of a DNA denaturing solution containing 0.1N hydrochloric acid (HCl) was added to each AG gel and denatured at room temperature for 7 minutes, thereby converting the double-stranded DNA in the sperm into single-stranded DNA. Subsequently, 200-300 μL of the lysis buffer described in item 2 of "Experimental Subjects and Materials" was added to the DNA-denatured AG gels of Experimental Groups 1 to 4 and treated for nuclear protein lysis at room temperature for 2, 5, 10, and 20 minutes, respectively. This lysed the sperm and cleaved the sperm nuclear proteins (including protamine). The cleavage of protamine loosened the DNA, forming DNA loops. The DNA-denatured AG gel in the control group was lysed for 20 minutes in the same manner as in Experimental Group 4 above, with the difference being that a lysis buffer without urea and SDS (i.e., containing 2.5 M NaCl, 0.2 M DTT, 0.2 M Tris, and 1% Triton X-100) was used, equivalent to the lysis buffer used in existing SCD experiments.

[0118] After washing twice with H2O, the gel was dehydrated with 70% ethanol. DNA staining was then performed using Wright-Giemsa according to techniques well known and commonly used by those skilled in the art. The stained AG gel was then observed and photographed using an optical microscope (BX-53, Olympus) at 100x and 200x magnifications.

[0119] Afterwards, the number of sperm without halos or with a halo width less than 1 / 3 of the sperm head diameter (i.e., the number of sperm with DNA fragmentation) was counted, and the ratio of sperm with DNA fragmentation in all sperm in the semen sample, i.e., the DNA fragmentation index (DFI) (%), was further calculated.

[0120] result:

[0121] Figure 1 The results of DNA staining of AG gels of each group are shown. Figure 1As can be seen, there was no significant difference in the DFI index measured between experimental groups 1 to 4 and the control group. Based on this experimental result, the applicant believes that using a lysis solution containing urea and SDS to lyse sperm nucleoprotein can effectively accelerate nucleoprotein lysis, thereby shortening the experimental time required, while also achieving detection efficiency similar to existing methods.

[0122] Example 2. Effect of Mixing Heated Liquefied Agarose and DNA Denaturing Solution to Simultaneously Perform Gel Embedding and DNA Denaturation on the Detection of Sperm DNA Fragmentation

[0123] Experimental methods:

[0124] First, a semen sample of a subject was randomly selected from the first item of “Experimental subjects and materials” above, and the semen sample was divided into a control group and two experimental groups (i.e., experimental groups 1 and 2) (each group had 2.5×10 5 Sperm / 25 μL). Next, sperm DNA fragmentation detection was performed on semen samples of each group as described below.

[0125] The detection of sperm DNA fragmentation in the control group was carried out in the same manner as described in the control group of Example 1 above, including agarose gel embedding, DNA denaturation, nuclear protein lysis, and DNA staining.

[0126] The detection of sperm DNA fragmentation in Experimental Group 1 was carried out in accordance with the steps of agarose gel embedding, DNA denaturation, nuclear protein lysis and DNA staining described in Experimental Group 2 of Example 1 above.

[0127] The detection of sperm DNA fragmentation in Experimental Group 2 was carried out according to the following steps: first, 50 μL of heated and liquefied 1.25% (w / v, g / L) agarose (premixed with 0.02 mg / mL phenol red) was mixed with 25 μL of a DNA denaturing solution containing 0.28N hydrochloric acid, and then added to the semen sample of Experimental Group 2 and mixed. The resulting mixture was then placed on a glass slide [which was covered with a thin film containing 1% (w / v, g / L) agarose] and allowed to stand at 4°C for 6 minutes, so that the semen sample was embedded in the agarose gel and fixed on the glass slide while undergoing DNA denaturation treatment, thereby obtaining an agarose gel embedded with DNA-denatured sperm (hereinafter referred to as DNA-denatured AG gel). Next, add 200-300 μL of the lysis buffer (described in item 2 of "Experimental Subjects and Materials") to the DNA-denatured AG gel and perform a nuclear protein lysis treatment at room temperature for 5 minutes. After washing twice with H₂O, dehydrate with 70% ethanol. Next, stain the DNA using Wright-Giemsa staining techniques well known and commonly used by those skilled in the art. The stained AG gel is then observed and photographed using an optical microscope at 100x and 200x magnifications.

[0128] Finally, the DFI index of each group was calculated according to the above Example 1.

[0129] result:

[0130] The results of this experiment are shown in Table 1 below. As can be seen from Table 1, there was no significant difference in the DFI index between experimental groups 1 and 2 and the control group. This experimental result demonstrates that, in addition to using a lysis buffer containing urea and SDS to lyse sperm, combining the agarose gel embedding and DNA denaturation steps can further shorten the experimental time required and achieve detection performance comparable to existing methods.

[0131] Table 1. DFI index of AG gel in each group

[0132] Group DFI (%) control group 19 Experimental Group 1 18 Experimental Group 2 19

[0133] In addition, the applicant attempted to conduct an experiment using the same method as Experimental Group 2, but without the addition of urea and SDS. The experimental results showed that the halo formed by sperm lysis using a lysis buffer without urea and SDS became smaller and less easily discernible (data not shown). Based on this, the applicant believes that the addition of urea and SDS can help loosen DNA structure and diffusion, making it easier to observe.

[0134] Example 3. Accuracy analysis of the detection method of the present invention

[0135] Experimental methods:

[0136] First, semen samples from 22 subjects were randomly selected from item 1 of "Experimental Subjects and Materials" above, and the semen samples were used to detect sperm DNA fragmentation using the method of the present invention. That is, the steps of combined agarose gel embedding and DNA denaturation treatment, nucleoprotein lysis treatment, and DNA staining were followed in sequence with reference to the steps described in Experimental Group 2 of Example 2 above, and the DFI index was calculated as described in Example 1 above.

[0137] In addition, the 22 semen samples were also used to perform the SCD test, that is, the test was performed according to the steps of the control group in Example 1 above.

[0138] Then, linear regression and Pearson's correlation analysis were used to analyze the correlation between the DFI index obtained by the detection method of the present invention and the existing SCD test.

[0139] result:

[0140] Figure 2 The correlation diagram between the DFI index measured by the method of the present invention and that measured by the existing SCD test is shown. Figure 2 It can be seen that the method of the present invention and the SCD test have a coefficient of determination (R) of up to 0.9523 in detecting sperm DNA fragmentation. 2 ). This experimental result shows that the detection performance of the detection method of the present invention is equivalent to that of the SCD test.

[0141] Based on the above experimental results, the applicant believes that mixing a semen sample, a DNA denaturing solution, and heated, liquefied agarose for simultaneous gel embedding and DNA denaturation of the semen sample, followed by nuclear protein lysis using a lysis buffer supplemented with urea and SDS, can effectively shorten the detection time of sperm DNA fragmentation while achieving detection performance similar to that of existing methods. Therefore, the method of the present invention is expected to be applicable to the rapid detection of sperm DNA fragmentation in a human individual and, therefore, to the assessment of male infertility.

[0142] All patents and publications cited in this specification are hereby incorporated by reference in their entirety. In the event of any conflict, the detailed description of this specification (including definitions) will prevail.

[0143] Although the present invention has been described with reference to the specific embodiments above, it is apparent that many modifications and variations can be made without departing from the scope and spirit of the invention. It is therefore intended that the present invention be limited only as indicated by the appended claims.

Claims

1. A method for simultaneously embedding and denaturing sperm DNA in a semen sample and detecting DNA fragmentation, characterized in that: The method includes the following steps: (a) heating an agarose gel, mixing the a DNA denaturing solution with the semen sample, and subjecting the resulting mixture to gel polymerization to obtain a gel containing spermatozoa with denatured DNA, wherein The DNA denaturing solution is an acid or a base; (b) lysing the gel containing the denatured sperm using a lysis solution to lyse the sperm nucleoprotein and obtain a lysed gel, wherein the lysis solution comprises urea having a concentration ranging from 0.5 to 4 M and sodium lauryl sulfate having a concentration ranging from 0.05% g / mL to 0.5% g / mL; (c) subjecting the lysed gel to DNA staining to obtain a stained gel; and (d) Observe whether a halo is formed around the head of each sperm on the stained gel. The absence of a halo or a halo width less than 1 / 3 of the diameter of the sperm head indicates the presence of sperm DNA fragmentation.

2. The method for simultaneously embedding and denaturing sperm DNA in a semen sample and detecting DNA fragmentation according to claim 1, wherein: In step (a), the agarose is pre-mixed with an acid-base indicator selected from the group consisting of phenol red, methyl violet, methyl orange, methyl red, Congo red, and combinations thereof before being heat-treated.

3. A method for simultaneously embedding and denaturing sperm DNA in a semen sample and detecting DNA fragmentation, characterized in that: The method includes the following steps: (a) mixing the semen sample, a DNA denaturing solution, and a colloid-forming component, and then subjecting the resulting mixture to gel polymerization to obtain a gel containing denatured spermatozoa, wherein the colloid-forming component is selected from the group consisting of acrylamide, alginate, and vinyl chloride, and the DNA denaturing solution is an acid or an alkali; (b) lysing the gel containing the denatured sperm using a lysis solution to lyse the sperm nucleoprotein and obtain a lysed gel, wherein the lysis solution comprises urea having a concentration ranging from 0.5 to 4 M and sodium lauryl sulfate having a concentration ranging from 0.05% g / mL to 0.5% g / mL; (c) subjecting the lysed gel to DNA staining to obtain a stained gel; and (d) Observe whether a halo is formed around the head of each sperm on the stained gel. The absence of a halo or a halo width less than 1 / 3 of the diameter of the sperm head indicates the presence of sperm DNA fragmentation.

4. The method for simultaneously embedding and denaturing sperm DNA in a semen sample and detecting DNA fragmentation according to claim 3, wherein: In step (a), the mixture further comprises an acid-base indicator selected from the group consisting of phenol red, methyl violet, methyl orange, methyl red, Congo red, and combinations thereof.

5. The method for simultaneously embedding and denaturing sperm DNA in a semen sample and detecting DNA fragmentation according to any one of claims 1 to 4, wherein: The DNA denaturing solution is an acid selected from the group consisting of hydrochloric acid, acetic acid, nitric acid, sulfuric acid, and combinations thereof.

6. The method for simultaneously embedding and denaturing sperm DNA in a semen sample and detecting DNA fragmentation according to any one of claims 1 to 4, wherein: The DNA denaturing solution is a base selected from the group consisting of sodium hydroxide, potassium hydroxide, calcium hydroxide, and combinations thereof.

7. The method for simultaneously embedding and denaturing sperm DNA in a semen sample and detecting DNA fragmentation according to any one of claims 1 to 4, wherein: The lysate further comprises a protein denaturant selected from the group consisting of 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonic acid hydrate, guanidine hydrochloride, and combinations thereof.

8. The method for simultaneously embedding and denaturing sperm DNA in a semen sample and detecting DNA fragmentation according to any one of claims 1 to 4, wherein: The lysis solution further comprises an ionic surfactant selected from the group consisting of sodium deoxycholate, sodium cholate, sodium lauroyl sarcosinate, and combinations thereof.

9. A kit for simultaneously embedding and denaturing sperm DNA in a semen sample and detecting DNA fragmentation, characterized in that: The kit contains: A colloid-forming formulation and a DNA denaturing solution for simultaneous embedding and denaturation, wherein the colloid-forming formulation comprises an ingredient selected from the group consisting of agarose, acrylamide, alginate, and vinyl chloride, and the DNA denaturing solution is an acid or a base; a lysis solution comprising urea having a concentration ranging from 0.5 to 4 M and sodium lauryl sulfate having a concentration ranging from 0.05% g / mL to 0.5% g / mL; and A DNA staining reagent.

10. The kit for simultaneously embedding and denaturing sperm DNA in a semen sample and detecting DNA fragmentation according to claim 9, wherein: The colloid-forming formulation further comprises an acid-base indicator selected from the group consisting of phenol red, methyl violet, methyl orange, methyl red, Congo red, and combinations thereof.

11. The kit for simultaneously embedding and denaturing sperm DNA in a semen sample and detecting DNA fragmentation according to claim 9, wherein: The DNA denaturing solution is an acid selected from the group consisting of hydrochloric acid, acetic acid, nitric acid, sulfuric acid, and combinations thereof.

12. The kit for simultaneously embedding and denaturing sperm DNA in a semen sample and detecting DNA fragmentation according to claim 9, wherein: The DNA denaturing solution is a base selected from the group consisting of sodium hydroxide, potassium hydroxide, calcium hydroxide, and combinations thereof.

13. The kit for simultaneously embedding and denaturing sperm DNA in a semen sample and detecting DNA fragmentation according to claim 9, wherein: The kit further comprises a carrier for carrying the semen sample, wherein the carrier is covered with a film containing 0.25% g / L to 1.5% g / L agarose.

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