Methods and kits 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 DNA fragmentation of sperm in a semen sample, comprising the steps of: embedding the semen sample in a gel having a pore size of 2 nm or more and less than 60 nm to obtain a sperm-embedded gel; performing a lysis treatment on the sperm-embedded gel with a lysis solution to lyse the nuclear proteins of the sperm, wherein the lysis solution comprises urea and sodium dodecyl sulfate; performing a DNA staining on the lysed gel; and observing whether a halo is formed at the periphery of the head of each sperm on the stained gel, wherein the presence of halo formation is indicative of the presence of sperm DNA fragmentation. The present invention also discloses a kit for performing the method.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for detecting sperm DNA fragmentation in a semen sample. The present application also relates to a kit for performing the method. BACKGROUND

[0002] Sperm DNA integrity is one of the major factors affecting embryo quality, embryo implantation and embryo development. When sperm DNA is fragmented (including single- and double-stranded DNA breaks) due to errors in spermiogenesis, sperm apoptosis, oxidative stress or radiation, i.e. sperm DNA fragmentation (SDF), it can cause male infertility, in vitro fertilization (IVF) failure and miscarriage. Therefore, the detection of sperm DNA fragmentation has played an important role in fertility screening and artificial reproduction treatments.

[0003] In the past, comet assay (CA) {also known as single cell gel electrophoresis (SCGE) assay} was considered as one of the standard methods for evaluating DNA fragmentation in sperm, which mainly involves embedding sperm in agarose gel and lysing the nuclear proteins [including protamine] of sperm under high salt conditions using lysis solution, so that the originally intact DNA forms a nucleoid with the nuclear matrix, i.e. in a chromatin dispersed state. Then the agarose gel is subjected to electrophoresis and DNA staining, so that the fragmented DNA forms a comet tail due to the movement in the direction of the anode, and the degree of sperm DNA fragmentation is calculated by using software for analysis. However, the CA assay requires the use of electrophoresis (usually about 20 minutes) and analysis software to determine the degree of sperm DNA fragmentation, which has the disadvantages of more complicated operation and time-consuming, which is not suitable for industrial practical application.

[0004] To improve the shortcomings of the above-mentioned CA assay, relevant researchers in the field developed the sperm chromatin dispersion (SCD) test. The SCD test mainly uses DNA denaturing solution to convert double-stranded DNA in sperm into single-stranded DNA after embedding sperm in agarose gel, and then lysing the nuclear proteins (including protamine) of sperm, at which time the DNA is loosened to form DNA loops due to the lysis of protamine, and the size of the DNA loop is positively correlated with the degree of DNA integrity. Then, after DNA staining, these DNA loops form halos of different sizes, wherein when the halo width is greater than or equal to 1 / 3 of the diameter of the sperm head, it represents no DNA fragmentation, and when there is no halo or the halo width is less than 1 / 3 of the diameter of the sperm head, it represents DNA fragmentation. Although the SCD test eliminates the need for electrophoresis and the use of analysis software required by the CA test, the determination method by observing the halo size is prone to have differences in the interpretation results due to different testers. SUMMARY

[0005] To develop a more convenient and rapid method for detecting DNA fragmentation of sperm in a semen sample, the present application provides a method for detecting DNA fragmentation of sperm in a semen sample, and the method is called sperm DNA fragment release (SDFR) assay.

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

[0007] (a) embedding the semen sample in a gel having a pore size of 2 nm or more and less than 60 nm, thereby obtaining a sperm-embedded gel, wherein the gel comprises an ingredient selected from the group consisting of acrylamide, acrylic acid, methacrylic acid, N-isopropyl acrylamide, alginate, and polyethylene glycol;

[0008] (b) performing a lysis treatment on the sperm-embedded gel with a lysis solution to lyse the nuclear proteins of the sperm, wherein the lysis solution comprises urea having a concentration ranging from 0.5 to 4 M and sodium dodecyl sulfate having a concentration ranging from 0.05 to 0.5% (w / v, g / mL);

[0009] (c) performing a DNA staining on the lysed gel; and

[0010] (d) observing whether a halo is formed at the periphery of the head of each sperm in the stained gel, wherein the presence of halo formation is indicative of the presence of sperm DNA fragmentation.

[0011] Preferably, in step (a), the gel has a pore size ranging from 2.5 to 25 nm.

[0012] More preferably, in step (a), the gel has a pore size ranging from 3 to 10 nm.

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

[0014] (a) embedding the semen sample in a polyacrylamide gel having an acrylamide content ranging from 3 to 24% (w / v, g / mL), thereby obtaining a sperm-embedded polyacrylamide gel;

[0015] (b) subjecting the sperm-embedded polyacrylamide gel to a lysis treatment with a lysis solution to lyse the nuclear proteins of the sperm, wherein the lysis solution comprises urea at a concentration ranging from 0.5 to 4 M and sodium dodecyl sulfate at a concentration ranging from 0.05 to 0.5% (w / v, g / mL);

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

[0017] (d) observing whether a halo is formed at the periphery of the head of each sperm on the stained polyacrylamide gel, wherein the presence of halo formation is indicative of the presence of sperm DNA fragmentation.

[0018] Preferably, in step (a), the polyacrylamide gel has an acrylamide content ranging from 4 to 22% (w / v, g / mL).

[0019] Preferably, in step (a), the polyacrylamide gel has a pore size ranging from 3 to 10 nm.

[0020] Preferably, in step (a), the polyacrylamide gel is formed by using acrylamide and bisacrylamide in a ratio ranging from 19: 1 (w / w) to 199: 1 (w / w).

[0021] More preferably, in step (a), the polyacrylamide gel is formed by using acrylamide and bisacrylamide in a ratio ranging from 24: 1 (w / w) to 99: 1 (w / w).

[0022] Preferably, the lysis solution further comprises a protein denaturant selected from the group consisting of 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonic acid hydrate, guanidine hydrochloride, and combinations thereof.

[0023] Preferably, the lysis solution further comprises an ionic interfacial surfactant selected from the group consisting of sodium deoxycholate, sodium cholate, sodium lauryl sarcosinate, and combinations thereof.

[0024] Preferably, the DNA staining is performed using a method selected from the group consisting of Diff-Quik staining, Wright-Giemsa staining, propidium iodide staining, SYBR Green staining, DAPI staining, and acridine orange staining.

[0025] In a third aspect, the present invention provides the use of a colloid forming formulation and a lysis buffer supply for preparing a kit for detecting DNA fragmentation of sperm in a semen sample, wherein the step of using the kit to detect DNA fragmentation of sperm in a semen sample includes: steps (a) to (d) as described in the first aspect above, or steps (a) to (d) as described in the second aspect above.

[0026] Preferably, the kit further includes a DNA staining reagent.

[0027] In a fourth aspect, the present invention provides a kit for detecting DNA fragmentation in sperm in a semen sample, comprising:

[0028] A colloid-forming formulation comprising an ingredient having a concentration range of 10 to 70% (w / v, g / mL), the ingredient being selected from the group consisting of: acrylamide, acrylic acid, methacrylic acid, N-isopropylacrylamide, alginate and polyethylene glycol;

[0029] A lysis buffer comprising urea at a concentration ranging from 0.5 to 4 M and sodium dodecyl sulfate at a concentration ranging from 0.05 to 0.5% (w / v, g / mL); and

[0030] A DNA staining reagent.

[0031] Preferably, the colloid-forming formulation further comprises an initiator selected from the group consisting of: ammonium persulfate, tetramethylethylenediamine, sodium vitamin B2-5'-phosphate, 3-(dimethylamino)propionitrile, and combinations thereof.

[0032] Preferably, 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.

[0033] Preferably, the kit further comprises a carrier for carrying the semen sample, the carrier being covered with a film containing 0.25 to 1.5% (w / v, g / L) agarose. Attached Figure Description

[0034] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. Therefore, the present invention, as well as other objects and features, will become more apparent from the following description, the appended claims, and the accompanying drawings, in which:

[0035] Figure 1is a schematic diagram of a DNA fragmentation detection method using a gel having a pore size of 2 nm or more and less than 60 nm to embed a semen sample;

[0036] Figure 2 is a schematic diagram of a DNA fragmentation detection method using a gel having a pore size of 60 nm or more to embed a semen sample;

[0037] Figure 3 shows the results observed by DNA staining of the PAG gels of each group;

[0038] Figure 4 shows the results observed by DNA staining of the PAG gels of the experimental groups and the AG gels of the SCD and CA groups, where the arrows indicate the presence of halo formation; and

[0039] Figure 5 shows a correlation plot between the DFI index measured by the method of the present application and that measured by the existing comet assay. DETAILED DESCRIPTION

[0040] For the purposes of the present application, it is to be understood that the words "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. A person of ordinary skill in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used in the practice of the present application. Of course, the present application is in no way limited to the methods and materials described.

[0042] In order to simplify the existing method for detecting sperm DNA fragmentation and make it easier to read, the applicant found through research results that after embedding the semen sample in a polyacrylamide gel with an acrylamide concentration of 4 to 22% (w / v, g / mL), only the lysis treatment of nuclear proteins and DNA staining are needed, that is, the presence or absence of halo formation can be observed to determine whether the semen sample has DNA fragmentation simply and accurately. The applicant concluded that under the condition of using a gel with a pore size of 2 nm or more and less than 60 nm to embed the semen sample and then lyse the nuclear proteins, the DNA fragment 1 with high integrity will not diffuse out of the head 3 and form a halo because the volume of its circular structure is larger than the pore size of the gel, only the DNA fragment 2 with low integrity will (see the schematic diagram of Figure 1 Therefore, the method of the present application can directly determine whether there is sperm DNA fragmentation by observing whether a halo is formed around the head 3 of the sperm. Conversely, under the condition of using a gel with a pore size of 60 nm or more to embed the semen sample and then lyse the nuclear proteins, both DNA fragments 1 and 2 with high and low integrity will diffuse out of the head 3 and form a halo (see the schematic diagram of Figure 2 Therefore, the method of the present application can directly determine whether there is sperm DNA fragmentation by observing whether a halo is formed around the head 3 of the sperm. Conversely, under the condition of using a gel with a pore size of 60 nm or more to embed the semen sample and then lyse the nuclear proteins, both DNA fragments 1 and 2 with high and low integrity will diffuse out of the head 3 and form a halo (see the schematic diagram of

[0043] Therefore, the method of the present application can directly determine whether there is sperm DNA fragmentation by observing whether a halo is formed around the head 3 of the sperm. Conversely, under the condition of using a gel with a pore size of 60 nm or more to embed the semen sample and then lyse the nuclear proteins, both DNA fragments 1 and 2 with high and low integrity will diffuse out of the head 3 and form a halo (see the schematic diagram of

[0044] (a) embedding the semen sample in a gel with a pore size of 2 nm or more and less than 60 nm, thereby obtaining a sperm-embedded gel, wherein the gel comprises a component selected from the group consisting of acrylamide, acrylic acid, methacrylic acid, N-isopropyl acrylamide (NIPAM), alginate, and polyethylene glycol (PEG);

[0045] (b) subjecting the sperm-embedded gel to a lysis treatment with a lysis solution, wherein the lysis solution comprises urea at a concentration ranging from 0.5 to 4 M and sodium dodecyl sulfate (SDS) at a concentration ranging from 0.05 to 0.5% (w / v, g / mL), to lyse the nuclear proteins of the sperm;

[0046] (c) subjecting the lysed gel to a DNA staining; and;

[0047] (d) observing whether a halo is formed at the periphery of the head of each sperm on the stained gel, wherein the presence of halo formation is indicative of the presence of sperm DNA fragmentation.

[0048] According to the present application, in step (a), the gel has a pore size of 3 nm or greater and less than 60 nm. Preferably, the gel has a pore size ranging from 2.5 to 25 nm. More preferably, the gel has a pore size ranging from 3 to 10 nm.

[0049] Preferably, the gel is a polyacrylamide gel.

[0050] The present application also provides a method for detecting DNA fragmentation of sperm in a semen sample, comprising the following steps:

[0051] (a) embedding the semen sample in a polyacrylamide gel having an acrylamide content ranging from 3 to 24% (w / v, g / mL), thereby obtaining a sperm-embedded polyacrylamide gel;

[0052] (b) subjecting the sperm-embedded polyacrylamide gel to a lysis treatment with a lysis solution, wherein the lysis solution comprises urea at a concentration ranging from 0.5 to 4 M and sodium dodecyl sulfate (SDS) at a concentration ranging from 0.05 to 0.5% (w / v, g / mL), to lyse the nuclear proteins of the sperm;

[0053] (c) subjecting the lysed polyacrylamide gel to a DNA staining; and

[0054] (d) observing whether a halo is formed at the periphery of the head of each sperm on the stained polyacrylamide gel, wherein the presence of halo formation is indicative of the presence of sperm DNA fragmentation.

[0055] Preferably, in step (a), the polyacrylamide gel has an acrylamide content ranging from 4 to 22% (w / v, g / mL).

[0056] According to the present application, in step (a), the polyacrylamide gel has a range of pore sizes falling within 3 to 10 nm.

[0057] According to the present application, the semen sample can be collected from a subject at any time. Preferably, the semen sample is collected from a subject who has experienced at least 2-3 days but not more than 10 days of abstinence.

[0058] According to the present application, the semen sample can be a fresh or cryopreserved [e.g., frozen in liquid nitrogen (-196°C)] sample.

[0059] As used herein, the term "subject" means any animal of interest, such as primates: humans, apes, and simia; and non-primate mammals: pigs, cows, sheeps, horses, goats, dogs, cats, mice, and rats; fish; and amphibians. In a preferred embodiment of the present application, the subject is a human.

[0060] According to the present application, the semen sample can be diluted with a diluent prior to being subjected to detection, such that the sperm concentration falls within the range of 4 x 10 6 to 2.8 x 10 7 Diluents suitable for use in the present application include, but are not limited to, Earle's medium, Human Tubal Fluid (HTF) medium (also known as Reprocell medium), Tris-buffered saline (TBS), phosphate buffered saline (PBS), and saline.

[0061] In a preferred embodiment of the present application, the semen sample is diluted with HTF medium prior to being subjected to detection, such that the sperm concentration is 1 x 10 7 cells / mL.

[0062] According to the present application, in step (a), the polyacrylamide gel is formed by using acrylamide and bis-acrylamide in the presence of an initiator.

[0063] Preferably, the acrylamide and bisacrylamide are in a ratio falling within a range of 19:1 (w / w) to 199:1 (w / w), more preferably 24:1 (w / w) to 99:1 (w / w). In some embodiments, the acrylamide and bisacrylamide are in a ratio of 29:1 (w / w). In some embodiments, the acrylamide and bisacrylamide are in a ratio of 37.5:1 (w / w).

[0064] According to the present application, the initiator can be selected from the group consisting of ammonium persulfate (APS), tetramethylethylene diamine (TEMED), riboflavin-5'-phosphate sodium, 3-(dimethylamino)propionitrile, and combinations thereof. In a preferred embodiment of the present application, the initiator is a combination of APS and TEMED. In particular, the step of embedding sperm using a polyacrylamide gel and a combination of APS and TEMED can be performed entirely at room temperature without the use of any heating or cooling equipment, making the detection more convenient and rapid.

[0065] As used herein, the terms "lysis solution", "cell lysis solution" and "protein lysis solution" can be used interchangeably.

[0066] According to the present application, the sodium dodecyl sulfate and urea added in the lysis solution are an ionic surfactant and a protein denaturant, respectively, which act to facilitate the lysis of protamine, thereby accelerating the separation of DNA originally intertwined with protamine and further allowing the release of DNA fragments to the periphery of the head of sperm to form a halo, thereby shortening the lysis time (e.g., within 5 minutes) and avoiding the curling of the tail of sperm.

[0067] According to the present application, the lysis solution further comprises other surfactants, which can be ionic surfactants or nonionic surfactants. Preferably, the ionic surfactants are selected from the group consisting of sodium deoxycholate, sodium cholate, sodium lauroyl sarcosinate, and combinations thereof. Preferably, the nonionic surfactants are selected from the group consisting of Triton X-100, Nonoxynol-40 (NP-40), Pluronic F-127, Tween-20, and combinations thereof. In a preferred embodiment of the present application, the nonionic surfactant is Triton X-100.

[0068] According to the present application, the lysis solution further comprises other protein denaturants. Preferably, the protein denaturants are selected from the group consisting of 3-[(3-Cholamidopropyl)dimethylammonio]-l-propanesulfonate hydrate, guanidinium chloride, and combinations thereof.

[0069] According to the present application, the lysis solution further comprises a reducing agent, a salt, and a titrant for adjusting the pH.

[0070] Preferably, the reducing agent is selected from the group consisting of dithiothreitol (DTT), β-mercaptoethanol, dithioerythritol (DTE), tributylphosphine (TBP), Tris(2-carboxyethyl)phosphine hydrochloride, and combinations thereof. In a preferred embodiment of the present application, the reducing agent is DTT.

[0071] Preferably, the salt is selected from the group consisting of sodium chloride (NaCl), potassium chloride (KCl), and combinations thereof.

[0072] Preferably, the titration solution is selected from the group consisting of sodium hydroxide (NaOH), hydrochloric acid (HC1), and combinations thereof.

[0073] More preferably, the lysis solution 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 with high concentration of NaCl facilitates the halo formation of DNA after being separated from protamine.

[0074] In some embodiments, the lysis solution 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 solution 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 solution comprises 0.5 M urea, 0.5% SDS, 3 M NaCl, 0.05 M DTT, 5% Triton X-100, and 0.015 M NaOH.

[0075] According to the present application, the lysis solution can be adjusted to have a desired pH value by the titration solution. Preferably, the lysis solution has a pH value ranging from 7 to 9. More preferably, the lysis solution has a pH value ranging from 7 to 8.2. In one preferred embodiment of the present application, the lysis solution has a pH value of 7.5.

[0076] According to the present application, the DNA staining can be 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.

[0077] The present application also provides a kit for detecting DNA fragmentation of sperm in a semen sample, comprising:

[0078] a gel-forming formulation comprising a component selected from the group consisting of acrylamide, acrylic acid, methacrylic acid, N-isopropyl acrylamide, alginate, and polyethylene glycol, having a concentration ranging from 10 to 70% (w / v, g / mL);

[0079] a lysis solution comprising urea having a concentration ranging from 0.5 to 4 M and SDS having a concentration ranging from 0.05 to 0.5% (w / v, g / mL); and

[0080] a DNA staining reagent.

[0081] As used herein, the term "kit" means a packaged product containing reagents or materials for detecting a disease or a clinical sign thereof. The kit can contain one or more boxes or containers for holding the reagents or materials, as well as labels and / or instructions for indicating the use of the contents of the kit.

[0082] Preferably, the component is acrylamide.

[0083] According to the present application, the gel-forming formulation further comprises a starter as described above. Preferably, acrylamide and the starter are placed in separate containers (e.g., microfuge tubes, glass or plastic bottles).

[0084] According to the present application, the kit further comprises a carrier for holding the semen sample, which is adapted to allow the gel formed by the gel-forming formulation to be immobilized on its surface for facilitating the various processing steps of the method described above (including lysis and DNA staining). Suitable carriers for use in the present application include, but are not limited to, slides and well-plates.

[0085] According to the present application, the carrier can be coated with a film containing 0.25 to 1.5% (w / v, g / L) agarose to facilitate the immobilization of the gel on the carrier. In a preferred embodiment of the present application, the carrier is a slide coated with a film containing 1% (w / v, g / L) agarose.

[0086] According to the present application, the lysis solution has a pH value and a composition as described above.

[0087] According to the present application, the DNA staining reagent is selected from the group consisting of Diff-Quik stain, Wright-Giemsa stain, propidium iodide, SYBR Green, DAPI, and acridine orange.

[0088] The present application will be further described with reference to the following examples. It is to be understood that the examples are only by way of illustration and should not be construed to limit the scope of the present application.

[0089] <EXAMPLE>

[0090] Experimental subjects and materials:

[0091] 1. Experimental subjects and their semen samples:

[0092] A total of 36 male subjects aged between 22 and 40 years old participated in the clinical experiment of the present application, among which 18 subjects were diagnosed with infertility.

[0093] After the collected semen from each subject was fully liquefied, 30 μL of the semen was taken and the number of sperm contained therein was determined using a sperm quality analyzer (Model X1 PRO, LensHooke). The semen sample was then diluted with Human Tubal Fluid (HTF) medium to obtain a semen sample with a desired concentration.

[0094] 2. The lysis solution used in the following examples has 2.5 M sodium chloride (NaCl), 0.2 M dithiothreitol (DTT), 4 M urea, 1% Triton X-100, 0.5% sodium dodecylsulfate (SDS), and 0.005 M sodium hydroxide (NaOH), wherein NaOH is used to adjust the pH value of the lysis solution to 7.5-8.2.

[0095] Example 1. Detection of sperm DNA fragmentation using polyacrylamide gel (PAG)-embedded semen samples

[0096] Experimental Methods:

[0097] First, a semen sample of a healthy subject was randomly selected from the first item of the above "Experimental Subjects and Materials", and the semen sample was divided into one control group and one experimental group (5-10 x 10 5 To each group of the semen sample, 69.2 μL of a 30% (w / v, g / mL) acrylamide / bis-acrylamide solution (Bio-Rad) and 30.8 μL of 0.01 M PBS were added and mixed, followed by the addition of 1.5 μL of 10% ammonimum persulfate (APS) and 1.5 μL of tetramethylethylenediamine (TEMED). Next, 20 μL of each group of the mixture was taken and placed on a glass slide covered with a film containing 1% (w / v, g / L) agarose so that the mixture could be firmly fixed on the glass slide, and then left to stand at room temperature for 3-5 minutes, so that each group of the semen sample was embedded in a polyacrylamide gel containing 12% (w / v, g / mL) acrylamide and fixed on the glass slide, thereby obtaining each group of the sperm-embedded polyacrylamide gel (hereinafter referred to as PAG gel).

[0098] After that, the experimental group was subjected to DNA cleavage treatment [i.e., DNA hydrolysis] according to the following procedure, while the control group was not treated: The PAG gel of the experimental group was treated with 0.1% Triton X-100 so that the plasma membrane of the sperm was permeabilized, then washed with H2O for about 3 minutes for 2 times, followed by DNA hydrolysis using 2 U of endonuclease DNase I (catalog number E1010, brand Zymo Research) for 30 minutes, so that DNA double-strand breaks (DSBs) and single-strand breaks (SSBs) occurred, thereby causing DNA fragmentation.

[0099] Then, the groups were subjected to lysis treatment in accordance with the following procedure: 200-300 μL of the lysis solution of item 2 of the "Experimental individuals and materials" above was added to the PAG gel of each group and allowed to act at room temperature for 5-20 minutes, so that the sperm were lysed and the nuclear proteins [including protamine] in the sperm were lysed, so that the fragmented DNA of the sperm was released from the sperm chromatin complexed with the nuclear proteins.

[0100] After washing with H2O twice, DNA staining was performed using the sperm morphology rapid staining solution (Diff-Quik method) and according to the techniques well known and customary to those skilled in the art for about 1 minute, and then the stained PAG gel was observed and photographed using an optical microscope (model BX-53, brand Olympus) at a magnification of 100x and 200x.

[0101] Results:

[0102] Figure 3 The results observed by DNA staining of the PAG gels of the groups are shown. From Figure 3 It can be seen that most of the lysed sperm of the experimental groups have halo formation (i.e., appear pink), while the lysed sperm of the control group have almost no halo formation.

[0103] To verify the above results again, the applicant additionally selected another endonuclease Alu I (catalog number R0137S, brand NEB) that causes double-stranded breaks in DNA to replace DNase I to perform the same test, and similar results were also observed (data not shown).

[0104] These experimental results show that the method for detecting sperm DNA fragmentation using a polyacrylamide gel to embed a semen sample according to the present application can effectively detect semen samples with sperm DNA fragmentation caused by endonuclease treatment, and this detection method is named by the applicant as "Sperm DNA fragment release (SDFR) assay".

[0105] Example 2. Comparison of the sperm DNA fragmentation detection method using a polyacrylamide gel according to the present application with existing methods

[0106] To understand the difference in effectiveness between the SDFR test of the present application using a polyacrylamide gel to detect sperm DNA fragmentation and the existing detection method using an agarose gel, the applicant used two existing detection methods, the sperm chromatin dispersion (SCD) test and the comet assay (CA), for comparison.

[0107] Experimental Methods:

[0108] First, a semen sample from a healthy subject randomly selected from the first item of the above "Experimental Subjects and Materials" was divided into an experimental group, an SCD group, and a CA group, and DNA fragmentation was detected according to the different procedures shown in Table 1 below.

[0109] Table 1. Procedures for detecting DNA fragmentation and results interpretation method used in each group

[0110]

[0111] The detection of sperm DNA fragmentation in the experimental group was performed sequentially with reference to the steps of polyacrylamide gel embedding, nuclear protein lysis treatment, and DNA staining described in Example 1 above.

[0112] Detection of sperm DNA fragmentation in the SCD group was performed according to the following steps: first, the semen sample of the SCD group was mixed with 0.7% (w / v, g / mL) low melting agarose gel [i.e., agarose (purchased from Alfa Aesar) dissolved in 100 mL PBS] that was heated to liquefy, then the mixture formed was placed on a glass slide [which was covered with a thin film containing 1% (w / v, g / L) agarose], and then left to stand at 4°C for 5 minutes, so that the semen sample was embedded in the agarose gel and fixed on the glass slide, thereby obtaining an agarose gel embedded with sperm (hereinafter referred to as AG gel). Next, 200-300 μL of a denaturing solution containing 0.1 N hydrochloric acid (HCl) was added to the AG gel and DNA denaturation treatment was performed at room temperature, so that the double-stranded DNA in the sperm was restricted to convert into single-stranded DNA, then 200-300 μL of the lysis solution of item 2 of the above "Experimental subjects and materials" was added to the denatured AG gel and nucleoprotein lysis treatment was performed at room temperature for 5-20 minutes. After washing with H2O twice, DNA staining was performed using the sperm morphology rapid staining solution (Diff-Quik method) and according to the techniques well known and customary to those skilled in the art, then the stained AG gel was observed and photographed using an optical microscope at a magnification of 100x and 200x.

[0113] Detection of sperm DNA fragmentation in the CA group was generally performed by referring to the steps of the SCD group, with the difference that DNA denaturation treatment was not performed, and before DNA staining, the agarose gel after lysis treatment was taken to perform electrophoresis (about 20 minutes) and washed with 0.01 M PBS (pH 7.4) (about 5 minutes).

[0114] Finally, the experimental group, the SCD group, and the CA group respectively calculated the ratio of sperm with DNA fragmentation in all sperm of the semen sample, i.e., the DNA fragmentation index (DFI) (%) according to different result interpretation methods of each group.

[0115] Results:

[0116] Figure 4 The results of the PAG gel of the experimental group and the AG gel of the SCD group and the CA group observed by DNA staining are shown. From the results, it can be seen that the sperm of the experimental group and the SCD group have DNA fragmentation, while the sperm of the CA group do not have DNA fragmentation. Figure 4As can be seen, the experimental group can easily determine whether the DNA is fragmented by observing whether there is a halo. The SCD group needs to compare the size of the halo, which is not easy to determine, and the CA group needs to carefully observe whether there is a comet tail, which is also relatively difficult. Therefore, the SDFR test of the present application needs to be further combined with software analysis. Furthermore, from the DNA staining results of the three groups, they have similar DFI indexes. Therefore, compared with the existing detection method using agarose gel, the SDFR test of the present application has fewer operation steps (no DNA denaturation treatment and electrophoresis), is easier to determine, and can obtain similar detection performance.

[0117] In addition, the applicant further refers to the fifth edition of the World Health Organization Laboratory Manual of Human Semen Analysis to store the semen sample in a cryoprotective solution in liquid nitrogen for 72 hours, and then refers to the above experimental group to detect sperm DNA fragmentation. The experimental results show that there is no significant difference between the measured DFI index and that measured in the above experimental group (data not shown), which indicates that the SDFR test of the present application does not reduce its detection performance due to the freezing preservation of the semen sample, and thus can be used for repeated detection of semen samples.

[0118] Example 3. Accuracy analysis of the detection method of the present application

[0119] Experimental method:

[0120] First, 14 semen samples of subjects (including healthy and infertile subjects) were randomly selected from the above “experimental subjects and materials” item 1, and the semen samples were taken to detect sperm DNA fragmentation using the method of the present application, that is, the steps of polyacrylamide gel embedding, nuclear protein lysis treatment and DNA staining described in the above embodiment 1 were sequentially performed, and the DFI index was calculated according to the above embodiment 2.

[0121] In addition, the 14 semen samples were also taken to perform a comet test, that is, the steps of the CA group of the above embodiment 2 were basically performed, except that polyacrylamide gel embedding was used instead of agarose gel embedding. In order to solve the problem that the comet tail is not easy to observe, the ImageJ analysis software was used to calculate the ratio of sperm showing a comet tail in the semen sample, thereby obtaining the DFI index.

[0122] Then, linear regression and Pearson’s correlation analysis were used to analyze the correlation of the DFI indexes obtained by the detection method of the present application and the existing comet test.

[0123] Results:

[0124] Figure 5 This diagram shows the correlation between the DFI index measured by the method of this invention and that measured by existing comet experiments. Figure 5 As can be seen, the method of this invention and the comet assay have a high coefficient of determination (R²) of 0.9479 in detecting sperm DNA fragmentation. 2 This experimental result shows that the detection performance of the SDFR test of this invention is the same as that of the comet test.

[0125] Example 4. Evaluation of the effectiveness of the detection method of the present invention using polyacrylamide gels with different acrylamide concentrations.

[0126] Experimental methods:

[0127] First, a semen sample from one subject was randomly selected from item 1 of "Experimental Individuals and Materials" above. The semen sample was then subjected to DNA hydrolysis using DNase I as described in Example 1 above. The DNA-hydrolyzed semen sample was then divided into 8 groups. Next, the semen samples from each group were used to detect sperm DNA fragmentation using the method of the present invention.

[0128] The detection of sperm DNA fragmentation in each group was generally performed in sequence according to the steps of polyacrylamide gel embedding, nucleoprotein lysis, and DNA staining described in Example 1 above, and the DFI index was calculated according to the steps described in Example 2 above. The difference was that the sperm samples of each group were embedded in polyacrylamide gels containing 4 to 25% (w / v, g / mL) acrylamide according to the formulations shown in Table 2 below, thereby obtaining PAG gels with different acrylamide concentrations.

[0129] Table 2. Formulations of PAG gels containing different acrylamide concentrations

[0130]

[0131]

[0132] result:

[0133] The results obtained in this experiment are shown in Table 3 below. As can be seen from Table 3, similar DFI indices were obtained using 4 to 22% (w / v, g / mL) acrylamide, while the DFI indices obtained using 25% (w / v, g / mL) acrylamide were significantly deviated. This experimental result shows that the polyacrylamide gel used in the detection method of the present application needs to have acrylamide in a concentration range of 4 to 22% (w / v, g / mL).

[0134] Table 3. DFI indices obtained using PAG gels containing different acrylamide concentrations

[0135] Acrylamide concentration (% w / v, g / mL) DFI (%) 4 81 7 84 10 84 13 82 16 82 19 85 22 85 25 55

[0136] In addition, according to B. M. A. Carvalho, et al. (2014), Sep. Purif. Rev., 43: 241-262, the pore size of the polyacrylamide gel having an acrylamide concentration of 4 to 22% (w / v, g / mL) is expected to fall in a range of about 3.2 to 8.8 nm. In contrast, according to Janaky Narayanan, et al. (2006), J. Phys. Conf. Ser., 28: 83-86, the pore size of the agarose gel used in the existing SCD test and CA test falls in a range of about 70 to 600 nm.

[0137] Based on the above, the applicant has thus inferred that, after embedding the semen sample in a gel having a pore size of 2 nm or more and less than 60 nm [particularly, a polyacrylamide gel having an acrylamide concentration of 3 to 24% (w / v, g / mL)], only the lysis treatment of the nuclear protein and the DNA staining are required, and it is possible to clearly determine whether the semen sample has DNA fragmentation by observing the presence or absence of halo formation. Therefore, the method of the present application is expected to be applied to rapidly detect sperm DNA fragmentation in a human individual, and thus can be used for the evaluation of male infertility.

[0138] All patents and literature cited in this specification are hereby incorporated by reference in their entirety. In the case of conflict, the present detailed description, including definitions will control.

[0139] While the application has been described with reference to the above specific embodiment, it is manifestly intended that modifications and variations thereof will occur to those skilled in the art upon reading this description. It is intended to include all such modifications and variations as fall within the scope of the application. Accordingly, the application is only to be limited by the appended claims.

Claims

1. A method for detecting DNA fragmentation in sperm in a human semen sample, characterized in that: The method includes the following steps: (a) The semen sample is embedded in a gel having a pore size of more than 2 nm and less than 60 nm to obtain a sperm-embedded gel, wherein the gel contains a component selected from the group consisting of: acrylamide, acrylic acid, methacrylic acid, N-isopropylacrylamide, alginate and polyethylene glycol. (b) The sperm-embedded gel is subjected to a lysis treatment with a lysis buffer to lyse the sperm nucleoproteins, wherein the lysis buffer contains urea in a concentration range of 0.5 to 4 M and sodium dodecyl sulfate in a concentration range of 0.05% g / mL to 0.5% g / mL. (c) Stain the lysed gel with DNA; as well as (d) Observe whether there is a halo forming around the head of each sperm on the stained gel. The presence of halo formation is a characteristic of sperm DNA fragmentation. This method does not include a DNA denaturation step.

2. The method for detecting DNA fragmentation in sperm in a human semen sample according to claim 1, characterized in that: In step (a), the gel has a pore size ranging from 2.5 to 25 nm.

3. The method for detecting DNA fragmentation in sperm in a human semen sample according to claim 2, characterized in that: In step (a), the gel has a pore size ranging from 3 to 10 nm.

4. A method for detecting DNA fragmentation in sperm in a human semen sample, characterized in that: The method includes the following steps: (a) The semen sample was embedded in a polyacrylamide gel with an acrylamide content ranging from 3% g / mL to 24% g / mL, thereby obtaining a polyacrylamide gel containing sperm. (b) The polyacrylamide gel containing sperm is subjected to a lysis buffer to lyse the nucleoproteins of the sperm, wherein the lysis buffer contains urea in a concentration range of 0.5 to 4 M and sodium dodecyl sulfate in a concentration range of 0.05% g / mL to 0.5% g / mL. (c) Stain the lysed polyacrylamide gel with DNA; as well as (d) Observe whether there is a halo forming around the head of each sperm on the stained polyacrylamide gel. The presence of halo formation is a characteristic of sperm DNA fragmentation. This method does not include a DNA denaturation step.

5. The method for detecting DNA fragmentation in sperm in a human semen sample according to claim 4, characterized in that: In step (a), the polyacrylamide gel has an acrylamide content ranging from 4% g / mL to 22% g / mL.

6. The method for detecting DNA fragmentation in sperm in a human semen sample according to claim 4, characterized in that: In step (a), the polyacrylamide gel has a pore size ranging from 3 to 10 nm.

7. The method for detecting DNA fragmentation in sperm in a human semen sample according to claim 4, characterized in that: In step (a), the polyacrylamide gel is formed by using acrylamide and bisacrylamide in a ratio ranging from 19:1 w / w to 199:1 w / w.

8. The method for detecting DNA fragmentation in sperm in a human semen sample according to claim 7, characterized in that: In step (a), the polyacrylamide gel is formed by using acrylamide and bisacrylamide in a ratio ranging from 24:1 w / w to 99:1 w / w.

9. The method for detecting DNA fragmentation in sperm in a human semen sample according to claim 1 or 4, characterized in that: The lysis buffer further contains a protein denaturing agent selected from the group consisting of: 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonic acid hydrate, guanidine hydrochloride, and combinations thereof.

10. The method for detecting DNA fragmentation in sperm in a human semen sample according to claim 1 or 4, characterized in that: The lysis buffer further contains an ionic surfactant selected from the group consisting of sodium deoxycholate, sodium cholate, sodium lauroyl sarcosinate, and combinations thereof.

11. The method for detecting DNA fragmentation in sperm in a human semen sample according to claim 1 or 4, characterized in that: The DNA staining was performed using a method selected from the group consisting of: Diff-Quik staining, Wright-Giemsa staining, propidium iodide staining, SYBR Green staining, DAPI staining, and acridine orange staining.

12. A kit for detecting DNA fragmentation in sperm in a human semen sample, characterized in that: This kit contains: A colloid-forming formulation comprising an ingredient having a concentration range of 10% g / mL to 70% g / mL, the ingredient being selected from the group consisting of: acrylamide, acrylic acid, methacrylic acid, N-isopropylacrylamide, alginate and polyethylene glycol; A lysis buffer comprising urea at a concentration ranging from 0.5 to 4 M and sodium dodecyl sulfate at a concentration ranging from 0.05% g / mL to 0.5% g / mL; and A DNA staining reagent, This kit does not contain a DNA denaturing solution.

13. The kit for detecting DNA fragmentation in sperm in a human semen sample according to claim 12, characterized in that: The colloid-forming formulation further comprises an initiator selected from the group consisting of: ammonium persulfate, tetramethylethylenediamine, sodium vitamin B2-5'-phosphate, 3-(dimethylamino)propionitrile, and combinations thereof.

14. The kit for detecting DNA fragmentation in sperm in a human semen sample according to claim 12, characterized in that: 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.

15. The kit for detecting DNA fragmentation in sperm in a human semen sample according to claim 12, characterized in that: The kit further includes a carrier for holding the semen sample, which is covered with a film containing 0.25% g / L to 1.5% g / L agarose.

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