Patent Application
20230075204
The present disclosure relates to a method for detecting the presence of sperm DNA fragmentation in a semen sample. The present disclosure also relates to a kit for detecting sperm DNA fragmentation in a semen sample.
Sperm DNA integrity is crucial for embryo quality, embryo implantation, and embryo development. Sperm DNA fragmentation (SDF) can be caused by extrinsic factors, such as radiation, environmental pollutants, and chemotherapeutics, as well as intrinsic factors, such as defective spermatogenesis, sperm apoptosis, and oxidative stress. SDF may cause male infertility, failed in vitro fertilization (IVF), and miscarriage. Therefore, the detection of SDF is important for fertility testing and assisted reproductive techniques (ARTs).
Conventional methods for detecting SDF 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.
Comet assay (CA), also known as single cell gel electrophoresis (SCGE), is a sensitive technique for detecting SDF. The procedures of CA involve embedding sperm cells in an agarose gel on a microscope slide, and then immersing the microscope slide in a lysis solution to break open the cell membrane and lyse the cellular proteins (e.g., protamine). Thereafter, the agarose gel is exposed to an electric field to attract negatively charged fragments of DNA toward the anode and form a comet-like structure. In the comet-like structure, the undamaged DNA nucleoid part is referred to as “head,” and the trailing damaged DNA streak is referred to as “tail.” After DNA staining with a fluorescent dye, the comet-like structure is visualized using a fluorescence microscope. Analysis of the comet tail can be performed by hand or with software, the fluorescence intensity of the comet tail indicating the extent of DNA damage. However, the operation of CA is complicated and time-consuming because of electrophoresis and software analysis processes, and thus CA cannot meet the needs of the industry.
SCD test is a modified halo assay that utilizes chemical methods to detect SDF. The procedures of the SCD test involve embedding sperm cells in an agarose gel, followed by DNA denaturation and deproteinization. Particularly, the double-stranded (DS) DNA of each sperm cell is denatured into a single-stranded (SS) DNA during the DNA denaturation. The nuclear protein (including protamine) of each sperm cell is lysed during the deproteinization. Therefore, DNA loops would be dispersed from the nuclear protein to the periphery of each sperm cell. After DNA staining with 4′,6-diamidino-2-phenylindole (DAPI) or the Diff-Quik reagent, the dispersed DNA loops are monitored by fluorescence or brightfield microscopy. The DNA loops of the sperm cell with DNA fragmentation are smaller than that of the sperm cell without DNA fragmentation, and more difficult to be stained. More specifically, the head of the sperm cell without DNA fragmentation shows as a large halo (i.e., the halo width is at least one-third of the diameter of the core head of the sperm cell). In contrast, the head of the sperm cell with DNA fragmentation shows as a small halo or no halo (i.e., the halo width is smaller than one-third of the diameter of the core head of the sperm cell). However, determining the halo width is difficult.
There is a need to develop a method for rapid and accurate detection of SDF.
In a first aspect, the present disclosure provides a method for detecting sperm DNA fragmentation (SDF) in a semen sample. The method includes:
In a second aspect, the present disclosure provides a method for detecting SDF in a semen sample. The method includes:
In a third aspect, the present disclosure provides a kit for detecting SDF in a semen sample. The kit includes:
a gel-forming formulation comprising acrylamide, acrylic acid, methacrylic acid, N-isopropylacrylamide (NIPAM), alginate, or polyethylene glycol (PEG), the component having a concentration ranging from 10% (w/v, g/mL) to 70% (w/v, g/mL);
a lysis solution; and
a DNA staining reagent.
The present disclosure provides a method for detecting sperm DNA fragmentation in a semen sample, which includes:
In certain embodiments, in step (a), the gel has a pore size from 2 to 60 nm. In other preferred embodiments, the gel has a pore size ranging from 2.5 nm to 25 nm, or from 3 nm to 10 nm, or from 3 nm to 9 nm, or from 2 nm to 8 nm.
In a preferred embodiment, the gel is a polyacrylamide gel.
According to the present disclosure, the polyacrylamide gel may be formed by reacting acrylamide and bis-acrylamide in the presence of an initiator.
In certain embodiments, a ratio of acrylamide to bis-acrylamide ranges from 19:1 (w/w) to 199:1 (w/w). In an exemplary embodiment, the ratio of acrylamide to bis-acrylamide ranges from 24:1 (w/w) to 99:1 (w/w). In another exemplary embodiment, the ratio of acrylamide to bis-acrylamide is 29:1 (w/w). In yet another exemplary embodiment, the ratio of acrylamide to bis-acrylamide is 37.5:1 (w/w).
According to the present disclosure, the initiator may be selected from the group consisting of ammonium persulfate (APS), N,N,N′,N′-tetramethylethylenediamine (TEMED), riboflavin-5′-phosphate sodium, 3-(dimethylamino)propionitrile, and any combination thereof. In a preferred embodiment, the initiator is a combination of APS and TEMED.
According to the present disclosure, the semen sample may be collected from a male subject at any time. In one embodiment, the semen sample is collected from a male subject who has experienced sexual abstinence for at least 2 to 3 days but not greater than 10 days.
According to the present disclosure, the semen sample may be fresh or frozen (e.g., may be in a frozen form stored in liquid nitrogen (−196° C.)).
As used herein, the term “subject” refers to any animal of interest, such as primates (e.g., humans, apes, and monkeys), non-primate mammals (e.g., pigs, cows, sheep, horses, goats, dogs, cats, mice, and rats), fish, and amphibians. In certain embodiments, the subject is a human.
According to the present disclosure, the semen sample may be diluted with a diluent to have a sperm concentration ranging from 4×106 cells/mL to 2.8×107 cells/mL.
Examples of the diluent may include, but are not limited to, Earle's medium, human tubal fluid (HTF) medium, tris-buffered saline (TBS), phosphate-buffered saline (PBS), and saline.
In certain embodiments, the semen sample is diluted with HTF medium to have a sperm concentration of 1×107 cells/mL.
As used herein, the term “lysis solution” can be used interchangeably with the terms “cell lysis solution” and “protein lysis solution.”
According to the present disclosure, in the lysis solution, sodium lauryl sulfate is used as an ionic surfactant, and urea is used as a protein denaturant. These two components can improve the lysis of protamine and thus the DNA loops can be easily released from the protamine to the periphery of the head of the sperm cell, and then be monitored as a halo via DNA staining, thereby reducing the time of lysis treatment (e.g., to less than 5 minutes).
According to the present disclosure, the lysis solution may further include an additional ionic or nonionic surfactant.
In certain embodiments, the additional ionic surfactant may be selected from the group consisting of sodium deoxycholate, sodium cholate, sodium lauroyl sarcosinate, and any combination thereof.
In certain embodiments, the additional nonionic surfactant may be selected from the group consisting of Triton X-100, Nonoxynol-40 (NP-40), Pluronic F-127 (F-127), Tween-20, and any combination thereof. In an exemplary embodiment, the additional nonionic surfactant is Triton X-100.
According to the present disclosure, the lysis solution may further include an additional protein denaturant. Examples of the additional protein denaturant may include, but are not limited to, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate, guanidinium chloride, and combinations thereof.
According to the present disclosure, the lysis solution may further include a reducing agent. Examples of the reducing agent may include, but are not limited to, dithiothreitol (DTT), β-m mercaptoethanol, dithioerythritol (DTE), tributylphosphine (TBP), tris(2-carboxyethyl) phosphine (TCEP) hydrochloride, and combinations thereof. In an exemplary embodiment, the reducing agent is DTT or TCEP at a concentration of 0.05-0.2 M.
According to the present disclosure, the lysis solution may further include salts. Examples of the salts may include, but are not limited to, sodium chloride (NaCl), potassium chloride (KCl), and combinations thereof.
According to the present disclosure, the lysis solution may further include a titrant. Examples of the titrant may include, but are not limited to, sodium hydroxide (NaOH), hydrochloric acid (HCl), and a combination thereof.
In certain embodiments, the lysis solution may further include 0.15 M to 3 M of NaCl, 0.05 M to 0.2 M of a reducing agent such as DTT or TECP, 0.1% (v/v) to 5% (v/v) of Triton X-100, and 0.01 M to 0.02 M of NaOH.
In an exemplary embodiment, the lysis solution includes 1 M urea, 0.05% (w/v, g/mL) of SDS, 2.5 M NaCl, 0.1 M DTT or TCEP, 1% (v/v) of Triton X-100, and 0.02 M NaOH. In another exemplary embodiment, the lysis solution includes 4 M urea, 0.05% (w/v, g/mL) of SDS, 0.15 M NaCl, 0.2 M DTT or TCEP, 0.5% (v/v) of Triton X-100, and 0.01 M NaOH. In yet another exemplary embodiment, the lysis solution includes 0.5 M urea, 0.5% (w/v, g/mL) of SDS, 3 M NaCl, 0.05 M DTT or TCEP, 5% (v/v) of Triton X-100, and 0.015 M NaOH.
According to the present disclosure, the lysis solution may be adjusted to have a desired pH value. In certain embodiments, the lysis solution may have a pH value ranging from 7 to 9. In an exemplary embodiment, the lysis solution may have a pH value ranging from 7 to 8.2. In another exemplary embodiment, the lysis solution has a pH value of 7.5.
According to the present disclosure, the DNA staining is conducted using a staining 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.
The present disclosure also provides a method for detecting SDF in a semen sample, which includes:
In certain embodiments, in step (a), the polyacrylamide gel is formed from a polyacrylamide gel forming solution containing acrylamide at a concentration ranging from 3 to 22% (w/v), 4 to 22% (w/v), or 7 to 13% (w/v).
As used herein, a polyacrylamide gel-forming solution refers to a solution comprising a mixture of acrylamide, bis-acrylamide, and one or more initiators, which are mixed together immediately prior to the process of forming the polyacrylamide gel.
According to the present disclosure, in step (a), the gel such as a polyacrylamide gel may have a pore size ranging from 3 nm to 10 nm or 3 nm to 9 nm.
The details of the operating conditions and reagents (i.e., the preparation of the semen sample, the ratio of acrylamide to bis-acrylamide, the initiator, the lysis solution, the DNA staining method, etc.) of this method are generally the same as those described above.
The present methods are easier to operate than the conventional tests, such as SCD and CA tests. Particularly, the person conducting the test only needs to identify the presence of a halo formation in the present methods, rather than to estimate the halo width in conventional tests.
Moreover, the present disclosure provides a kit for detecting sperm DNA fragmentation in a semen sample. The kit includes:
a gel-forming formulation comprising acrylamide, acrylic acid, methacrylic acid, NIPAM, alginate, or PEG, at a concentration ranging from 10% (w/v, g/mL) to 70% (w/v, g/mL);
a lysis solution including urea at a concentration ranging from 0.5 M to 4 M and SDS at a concentration ranging from 0.05% (w/v) to 0.5% (w/v); and
a DNA staining reagent.
Preferably, the lysis solution contains 0.5-1 M urea and 0.05-0.1% SDS (w/v).
Preferably, the lysis solution further comprises a reducing agent such as DTT or TCEP, at 0.05-0.2 M.
In certain embodiments, the gel-forming formulation comprises acrylamide and bis-acrylamide.
According to the present disclosure, the gel-forming formulation may further include an initiator as described above.
In certain embodiments, acrylamide and bis-acrylamide are in one container and the initiator is in another container (e.g., microcentrifuge tubes, glass bottles, or plastic bottles).
According to the present disclosure, the kit may further include a solid support for carrying the semen sample. The solid support includes a support base and an agarose layer disposed on a surface of the support base, and the agarose layer has an agarose concentration ranging from 0.25% (w/v, g/L) to 1.5% (w/v, g/L).
Examples of the support base may include, but are not limited to, a microscope slide and a well-plate.
In an exemplary embodiment, the support base is a microscope slide, and a surface of the microscope slide has been overlaid with a layer of 1% (w/v, g/L) agarose.
The detail of the lysis solution applied in this kit is generally the same as that described above.
According to the present disclosure, the DNA staining reagent may be selected from the group consisting of Diff-Quik solution, Wright-Giemsa solution, PI, SYBR Green, DAPI, and acridine orange.
The disclosure will be further illustrated by way of the following examples, which are intended for the purpose of illustration and should not be construed as limiting the disclosure in practice.
The lysis solution used in the following experiments contained 2.5 M NaCl, 0.2 M DTT, 4 M urea, 1% Triton X-100, 0.5% (w/v) SDS, and 0.005 M sodium hydroxide (NaOH), and had a pH value ranging from 7.5 to 8.2.
A semen sample without SDF of male Subject 1 (age between 22-40 years old) was collected, followed by liquefaction at room temperature. 30 μL of the liquefied semen sample was subjected to determination of the number of sperm cells using a semen quality analyzer (X1 PRO, LensHooke) in accordance with the manufacturer's instructions. Afterwards, a suitable amount of HTF medium was added to dilute the semen sample to reach sperm cell concentration from 0.07×105 cells/μL to 0.28×105 cells/μL. Two aliquots (70 μL each) of the diluted semen suspension of Subject 1 were used for the following experiments.
One aliquot served as an experiment sample, and the other aliquot served as a DNase-treated sample. Each aliquot was added with 69.2 μL of an acrylamide/bis-acrylamide solution comprising 30% (w/v) acrylamide (Bio-Rad) and 30.8 μL of 0.01 M phosphate-buffered saline (PBS), followed by mixing with 1.5 μL of 10% ammonium persulfate (APS) and 1.5 μL of N,N,N′,N′-tetramethylethylenediamine (TEMED). 20 μL of the respective resultant mixture containing 12% (w/v) acrylamide was placed on an agarose layer (containing 1% (w/v) agarose) disposed on a surface of a microscope slide, followed by being left standing at room temperature for 3 to 5 minutes, such that the sperm cells were embedded in a polyacrylamide gel and immobilized on the microscope slide. The resultant sperm cells-embedded polyacrylamide gel (PAG) was subjected to the following DNA hydrolysis treatment and is referred to as “sperm cells-PAG” hereinafter.
The sperm cells-PAG of the DNase-treated sample was treated with 0.1% Triton X-100, and was then washed with water twice for about 3 minutes, followed by conducting a DNA hydrolysis treatment using 2 U endonuclease DNase I (Cat. No. E1010, Zymo Research) for 30 minutes, to fragment the sperm DNA thereof. However, the sperm cells-PAG of the experiment sample received no such treatment and the sperm DNA is not fragmented.
Thereafter, about 200-300 μL of a lysis solution as described in General Experimental Material was added to the sperm cells-PAG of each of the experiment sample and the DNase-treated sample at room temperature for about 5-20 minutes. After washing with water two times, the lysed sperm cells-PAG was subjected to Diff-Quik staining for 1 minute using a DNA staining protocol well-known to those skilled in the art. The stained sperm cells-PAG was then observed and photographed under an optical microscope (BX-53, Olympus) at 100× and 200× magnifications.
Referring to
In another experiment, endonuclease Alu I (Cat. No. R0137S, NEB) was used to replace endonuclease DNase I, and similar results were observed (data not shown).
These results show that the present method effectively detected sperm DNA fragmentation in a semen sample and is referred to as “sperm DNA fragment release (SDFR) assay” hereinafter.
A semen sample of male Subject 2, age between 22-40 years old, was collected. Three aliquots were prepared according to Example 1. One aliquot served as an experimental sample detected by the present method, and the other two aliquots served as comparative samples 1 and 2, detected by comparison methods.
The SDF of the experimental sample was detected according to the SDFR method described in Example 1.
The SDF of comparative sample 1 was detected using the SCD test according to the following operating procedures. First, comparative sample 1 was mixed with 0.7% (w/v, g/mL) liquefied low melting agarose gel (Alfa Aesar) (in PBS). The mixture was placed on an agarose layer (containing 1% (w/v, g/L) agarose) disposed on a surface of a microscope slide at 4° C. for 5 minutes, such that the sperm cells were embedded in the low melting agarose gel and were immobilized on the microscope slide. The resultant sperm cells-embedding agarose gel (AG) is referred to as “sperm cells-AG” hereinafter.
Thereafter, about 200-300 μL of a denaturing solution containing 0.1 N HCl was added to the sperm cells-AG of the comparative sample 1 at room temperature for about 7 minutes. The denatured sperm cells-AG was then treated with about 200-300 μL of a lysis solution as described in General Experimental Material at room temperature for about 5-20 minutes.
After washing with water two times, the lysed sperm cells-AG was subjected to Diff-Quik staining for 1 minute using a staining protocol well-known to those skilled in the art. The stained sperm cells-AG was then observed and photographed under an optical microscope (BX-53, Olympus) at 100× and 200× magnifications.
In addition, comparative sample 2 was subjected to detection of SDF using CA which was performed similar to the operating procedures of the SCD test described above, except that: the sperm cells-AG was not subjected to DNA denaturation treatment; and before DNA staining, the lysed sperm cells-AG was subjected to electrophoresis for about 20 minutes, followed by washing with 0.01 M PBS (pH 7.4) for about 5 minutes.
The procedures of the above-mentioned 3 different processes are summarized in Table 1.
The DFI (%) of each sample was calculated using technology known to those skilled in the art.
Referring to
These results show that the present method (i.e., the SDFR assay) requires fewer operating steps (SDFR dispenses with DNA denaturation treatment and electrophoresis), detects SDF quickly, and has a similar detection effect.
In addition, another aliquot of the semen sample of Subject 2 was placed in a cryoprotectant solution, and was then stored in liquid nitrogen for 72 hours in accordance with the fifth edition of the WHO laboratory manual for the examination and processing of human semen. Thereafter, the frozen semen sample was thawed at room temperature, and then subjected to determination of SDF according to the method described in Example 1. The DFI determined in the frozen semen sample was similar to that determined in the experimental sample (data not shown). The results indicate that the present method can effectively detect SDF in a frozen semen sample.
Fourteen semen samples from male Subjects (age between 22-40 years old) were collected and diluted to semen suspensions according to Example 1. The diluted semen suspensions were subjected to detection of SDF according to the present method similar to that performed for the experimental sample described in Example 1, and the DFI of the respective semen sample was calculated using technology known to those skilled in the art.
In addition, the 14 semen samples were also subjected to CA, and the operating procedures of the CA were similar to those described in Example 2, except that: a polyacrylamide gel was used to embed the semen sample instead, and ImageJ software was used to quantify the number of sperm cells with comet tails, so as to calculate DFI.
The DFIs, respectively determined based on the present method and the CA method, were then analyzed using linear regression and Pearson's correlation analysis to determine the correlation therebetween, and a coefficient of determination (R2 value) was calculated.
Referring to
The result indicates that the accuracy of the method of the present disclosure is similar to that of the CA.
A semen sample of male Subject 4 (age between 22-40 years old) was collected and was treated with 0.1% Triton X-100. The semen sample was then washed with water twice for about 3 minutes, followed by conducting a DNA hydrolysis treatment using 2 U endonuclease DNase I (Cat. No. E1010, Zymo Research) for 30 minutes to fragment the sperm DNA thereof. Portions of the hydrolyzed semen sample were used as 8 test samples (i.e., test samples 1 to 8).
The respective test sample was sequentially subjected to an embedding process, a lysis treatment, and DNA staining generally according to the procedures described in Example 1, except that the conditions shown in Table 2 were used for the embedding process.
The sperm cell concentration of each test semen sample is calculated by its dilution fold. The total sperm cell number in each test needs to be constant. The DFI of each test sample was calculated using technology known to those skilled in the art.
As shown in Table 3 below, the DFI's determined in test samples 0 to 7 were similar. The result indicates that the polyacrylamide gel formed from a polyacrylamide gel-forming solution containing acrylamide at a concentration ranging from 3 to 22% (w/v) or 4 to 22% (w/v) can be used in the present method.
The polyacrylamide gel formed from a polyacrylamide gel-forming solution containing acrylamide at a concentration ranging from 4% to 22% (w/v) is calculated to have a pore size ranging from 8.8 nm to 3.2 nm (see B. M. A. Carvalho, et al. (2014), Sep. Purif. Rev., 43:241-262). In contrast, the AG under the SCD or CA test is calculated to have a pore size ranging from 70 nm to 600 nm (see Janaky Narayanan, et al. (2006), J. Phys. Conf. Ser., 28:83-86).
The results show that when a polyacrylamide gel having a pore size of 3.3 nm to 8.2 nm was used to embed the semen sample, followed by lysing the nuclear protein, the DNA loops without fragmentation (see the symbol “1” in
On the contrary, when a gel having a pore size of ≥70 nm (e.g., agarose gel) is used to embed the semen sample, followed by lysing the nuclear protein, the DNA loops without fragmentation (see the symbol “1” in
The lysis solution used in this experiment contained 2.5 M NaCl, 1 M urea, 1% Triton X-100, 0.05% SDS, a reducing agent (0.05 M DTT, 0.2 M DTT, 0.05 M TCEP, or 0.2 M TCEP), 0.4 M Tris (Tris(hydroxymethyl)aminomethane), 0.05 M Na2EDTA (Ethylenediaminetetraacetic Acid, Disodium Salt) and 0.005 M NaOH, and had a pH value ranging from 7.5 to 8.2.
A portion of a semen sample of a male subject A was collected and diluted according to Example 1.
One aliquot (70 μL each) of the diluted semen suspension was used for the experiments. The aliquot was added with 70 μL of a 30% (w/v, g/mL) acrylamide/bis-acrylamide solution (Bio-Rad), followed by mixing with 15 μL of 1% APS and 15 μL of 10% TEMED to initiate gel polymerization (gel forming). 15 μL of the respective resultant mixture containing 12.35% (w/v) acrylamide was immediately placed on an agarose layer (containing 1% (w/v, g/L) agarose) disposed on a surface of a microscope slide, followed by being left standing at room temperature for 3 to 5 minutes to continue gel polymerization (gel forming), such that the sperm cells were embedded in a polyacrylamide gel and were immobilized on the microscope slide. The resultant sperm cells-embedded polyacrylamide gel (PAG) is referred to as “sperm cells-PAG” hereinafter, which was treated with DNase.
The sperm cells-PAG of the sample was treated with 0.1% Triton X-100, and was then washed with water twice for about 3 minutes, followed by conducting a DNA hydrolysis treatment using 2 U endonuclease DNase I (Cat. No. E1010, Zymo Research) for 30 minutes, to fragment the sperm DNA thereof.
Thereafter, about 200-300 μL of a lysis solution as described above in this Example was added to the sperm cells-PAG of each of the experiment sample and the sample at room temperature for about 10 minutes. After washing with water two times, the lysed sperm cells-PAG was subjected to Diff-Quik staining for 1 minute using a DNA staining protocol and then destained with 75% ethanol. The stained sperm cells-PAG was then observed and photographed under an optical microscope (BX-53, Olympus) at 100× and 200× magnifications.
The other portions of the semen sample of Subject A were used as test samples. The respective test sample was sequentially subjected to a diluting process, an embedding process, a DNase-treatment, a lysis treatment, and DNA staining generally according to the procedures of the above portion, except that the conditions shown in Table 4 were used for the diluting and embedding processes.
The sperm cell concentration of each test semen sample is calculated by its dilution fold. The total numbers of sperm cell number used in each test are constant. The DFI of each test sample was calculated using technology known to those skilled in the art.
Tables 5-8 shows the results of DFI (%) vs. different acrylamide concentrations in the gel forming solution; the lysis solution contained 1 M urea, 0.05% SDS (w/v), and different concentrations of DTT or TCEP.
The invention, and the manner and process of making and using it, are now described in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, to make and use the same. It is to be understood that the foregoing describes preferred embodiments of the present invention and that modifications may be made therein without departing from the scope of the present invention as set forth in the claims. To particularly point out and distinctly claim the subject matter regarded as invention, the following claims conclude the specification.
| Number | Date | Country | Kind |
|---|---|---|---|
| 109145792 | Dec 2020 | TW | national |
This application is a continuation-in-part of U.S. application Ser. No. 17/643,161, filed on Dec. 7, 2021, which claims the priority to Taiwanese Application NO:109145792, filed Dec. 23, 2020. The contents of which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17643161 | Dec 2021 | US |
| Child | 18047242 | US |
