The evaluation kit of human spermatozoa lipid peroxidation

Abstract

This invention is an evaluation kit for lipid peroxidation of human spermatozoa, used to assess male sperm quality in research centers and infertility treatment. The process involves mixing human spermatozoa with PBS at an optimal dose and temperature, then separating them with centrifugal force at a specific speed and time. The separated sperm is diluted with PBS and treated with ferrous sulfate and ascorbic acid at optimal concentrations to promote oxidation. After incubation, trichloroacetic acid is added, and the sample is separated again with a centrifuge to remove peroxides from the spermatozoa. The sample is treated with TBA and NaOH and subjected to thermal stress. Finally, the absorbance is measured with a spectrophotometer at the appropriate wavelength. Through numerical calculations based on MDA, the amount of lipid peroxidation and oxidative damage in the sperm sample is evaluated.

Description

The Evaluation Kit of Human Spermatozoa Lipid Peroxidation

[0001] Male fertility can be affected by oxidative stress due to the presence of reactive oxygen species (ROS). This occurs when free radicals are produced more than the body's antioxidant defenses, leading to damage to biomolecules such as lipids, proteins, and DNA. Sperm cells are particularly susceptible to oxidative stress due to factors such as the presence of NADPH oxidase enzyme, unsaturated fatty acids in the membrane, and loss of cytoplasm during development. ROS are produced intracellularly through various mechanisms, such as enzymatic reactions in mitochondria, plasma membranes, endoplasmic reticulum, and peroxisomes. Externally, sources of ROS include ultraviolet radiation, tobacco, toxins, and pesticides. An imbalance between ROS and antioxidants can lead to conditions such as asthenozoospermia, teratozoospermia, and DNA fragmentation. Lipid peroxidation, where ROS targets lipids, is a major issue in oxidative damage. Hydroxyl and hydroproxyl radicals play a significant role in lipid peroxidation, causing structural damage to cells, tissues, and organs. The process of lipid peroxidation involves initiation, distribution, and termination stages, ultimately resulting in harmful aldehyde products like malondialdehyde. Measuring malondialdehyde levels using thiobarbituric acid (TBA) is common to assess the extent of lipid peroxidation. Due to the importance of this assessment in understanding cell health, an intracellular kit has been developed to evaluate lipid peroxidation in human sperm cells. Researchers emphasize the need for a simple and cost-effective method to measure lipid peroxidation in both sperm cells and other cells beyond reproduction.

[0002] G01N 33 / 50 – C12N 5 / 076 – A61K 35 / 52 – A61K 9 / 00

[0003] CN110846403

[0004] Application of CT47A11 gene methylation in sperm activity detection agent and kit

[0005] The invention relates to application of methylation level of a human CT47A11 gene promoter region in preparing a detection agent for detecting sperm activity. The human CT47A11 gene promoter region ispositioned a region corresponding to space from 120114278th to 120116978th of human X chromosome. Analysis, research and verification on the basis of whole genome methylation sequencing results findsthat the methylation level of the human CT47A11 gene promoter region can serve as a molecular marker to detect the sperm activity, and when methylation rate of the region is 0, it can be thought thatactivity of corresponding sperms is weak. The molecular marker can provide assistant evaluation information for diagnosis of asthenozoospermia and selection of sperms before artificial fertilization.

[0006] The mentioned kit is a sperm analyzer like our claimed kit but our invention focuses on the human Spermatozoa Lipid Peroxidation examination through a process that is different from the mentioned patent’s method.

[0007] WO2010041865

[0008] METHOD AND KIT FOR SEMEN DIAGNOSIS THROUGH COLOUR CHANGES IN METHYLENE BLUE AND SEMEN QUALITY EVALUATION USING SAME

[0009] The present invention relates to a method and a kit for diagnosis of sperm quality (vitality and resilience) through observation of colour changes visible with the naked eye in a solution combining methylene blue and sperm. The invention comprises a method for semen diagnosis using colour coding (with a standard colour table) charting the various colours the semen may change into, and a diagnostic kit for testing semen using said method. When combined with methylene blue, healthier and more active sperm fades the original blue colour more rapidly due to respiration activity by the sperm, which allows for sperm quality evaluation. The above principles are adapted to kit form inside a tube which encases a methylene blue solution into which sample semen is introduced for easy evaluation.

[0010] The said method and kit is similar to our kit according to their purpose and also the color experiments, but in our process the dye solution is only a part of it and more progress is made in examining the semen.

[0011] United States Patent Application 20120225417

[0012] Methods, Compositions and Kits for the Evaluation and Preservation of Sperm

[0013] The invention concerns methods and kits for evaluating the semen quality. The invention also concerns compounds and compositions for preserving a sperm sample, especially during cryopreservation. The invention is based on a correlation between sperm phosphodiesterase (PDE) activity and sperm fertility and on the use of selective phosphodiesterase inhibitors, more particularly PDE10 inhibitors.

[0014] The process of the mentioned methods is measuring sperm phosphodiesterase (PDE) enzymatic activity in sperm samples to examine the quality and maintain its activity while our kit examines the quality of men's sperm based on the oxidation rate and calculates the lipid peroxidation amount of semen and sperm samples.

[0015] The evaluation kit for lipid peroxidation of human sperm includes chambers with different solutions like ferrous sulfate, ascorbic acid, trichloroacetic acid, and thiobarbituric acid. Solution A is made with a molarity of 0. 5 to 5 mM, solution B from 1 to 20 mM of sodium ascorbate, and solution C with trichloroacetic acid at 5 to 75% concentration. Solution D is prepared using tri-barbituric acid at 0. 1 to 5% concentration and sodium hydroxide. The sperm sample is processed by centrifugation with a force of 50 to 800 g to get a concentration of 10 to 30 million sperm. The kit evaluates sperm quality based on oxidation rate, calculating lipid peroxidation in semen and sperm samples. The aim is to determine the amount of lipid peroxidation in human sperm, providing valuable insights into male fertility.

[0016] About 40 to 50 percent of infertility cases are related to male factors. In infertility centers, for the treatment of infertile couples, the separation of healthy male sperms is done based on more motility and normal morphology, which is one of the most important stages of egg fertilization in the laboratory, and its success rate mainly depends on the selection of healthy sperm. It is used to inject into the egg. However, the methods that are usually used to separate sperm in infertility centers are based on the selection of sperm according to the sperm's motility and appearance, these two parameters are the only predictors of internal lipid oxidative health. And the sperm membrane, which actually constitutes half of the future genome of the fetus, is not because there are sperms with normal morphology that do not have sufficient oxidative health and suffer from oxidative stress and abnormal lipid peroxidation for various reasons. Oxidative stress occurs when the production of oxygen free radicals exceeds the body's natural antioxidant defense mechanisms, which causes damage to biomolecules such as lipids, proteins, and DNA, and as a result, impairs the normal function of sperm. One of the most important reasons for male infertility is the negative effects caused by this oxidative stress. The result of this wrong choice can be fertilized eggs with sperms with oxidative stress damage, which leads to a significant weakening or decrease in the amount of Implantation and as a result the failure of fertilization and the continuation of infertility. Therefore, the ability to correctly diagnose and evaluate healthy sperm based on the health level of the oxidative stress of the sperm sample and its suitability for egg fertilization can be considered as one of the success factors of in vitro fertilization. In this invention, by using lipid peroxidation evaluation technology and other specific techniques, sperm with normal or abnormal levels of peroxidation will be recognized. The main purpose of this invention is to develop and prepare a portable kit to detect and evaluate the state of lipid peroxidation of human sperm samples.Solution of Problem

[0017] In the process of creating the invented kit, to access and evaluate the amount of lipid peroxidation in the sperm membrane and cytoplasm, several step-by-step processes and stages include the preparation of oxidation promoter solution and dialysis and separation of human spermatozoa membrane and optimization of its effectiveness process, preparation of solutions Acidic to make it possible to release peroxide indicators from cellular composite complexes by using an acidic solution with effective parameters and preparing a coloring solution for the indicators. Peroxidation of human spermatozoa sample and observable and calculating the amount of MDA as an indicator of peroxidation of semen sample. To start the operation process, the preparation of the cement sample is also designed based on the operation method of the kit. On this basis, at this point, the conditions of the cement sample should be optimized, which is subjected to a preparation test with a special method using different concentrations, numbers, and forces of centrifugation and different diluting solutions. Different methods have been tested and the method with the best result in lipid peroxidation measurement is selected and applied in the kit.

[0018] In the following, to prepare the oxidation and dialysis promoter solution and to separate the membrane and release the oxidized and peroxide substances of the sperm in the evaluation kit, different materials, temperature, dose, and time were tested, among which ferrous sulfate and sodium ascorbate with Optimum concentration and dose have been obtained and selected as suitable materials with appropriate response and are used as promoters in the kit. The resulting mixture is tested for optimal effectiveness at different times and temperatures, which is kept at the optimal time and temperature and is ready for the next steps. It should be noted that by conducting research and using different samples and materials, one of the innovative points in this kit is the exclusive use of the aforementioned promoter materials in the evaluation process.

[0019] To prepare a solution with the possibility of releasing peroxide indicators from cellular composite complexes, various acid solutions with different percentages and times and at different temperatures were tested and the solution with the best result included TCA with a concentration of about 40%, as well as temperature and Optimum time is used as the desired acid solution in the kit. Next, to measure the level of lipid peroxidation, the sperm sample was tested under different centrifugal forces for different periods.

[0020] To prepare the coloring solution color the visible solution and calculate the amount of MDA as an indicator of peroxidation of the spermatozoa sample, various materials were tested, in the invention kit, a special step with coloring solutions based on TBA and NaOH is tested with different concentration, dose and normality and the materials with selected parameters are included in the kit. It should be noted that to see the results, after adding special dyeing solutions, the sample is kept at different temperatures and times so that the dyeing process is created optimally. Optimum time and duration are obtained by testing and indicated and applied in the desired process by the kit.

[0021] After preparing and using the required solutions of the evaluation kit to determine the conditions for calculating and evaluating the peroxide state of the semen sample, in the last step, the absorption reading of Kot containing lipid oxidation indicator substances of the human spermatozoa sample is examined using a spectrophotometer, which is Note that the amount of absorption and color intensity can be calculated and separated. The method of calculating the amount of peroxidation for the human sperm sample is explained in the instructions in the invention kit. Therefore, an invented kit based on a native and optimized scientific method and based on available materials and today's knowledge is used to diagnose male infertility problems.Advantage Effects of the Invention

[0022] 1- It is a completely exclusive kit for calculating the peroxidation of human spermatozoa, while most of the existing and common kits are used for non-specific calculations such as blood, serum, and cells, and for this reason, there is a significant difference in the preparation method and used materials.

[0023] 2- Reducing the number of stages of the evaluation process compared to the previous samples, so that in the previous inventions, about 7 different types of solutions are added to the prototype, and in this kit, this number has been changed and reduced to 4 solutions.

[0024] 3- Reducing the evaluation time and the evaluation process is faster than the previous examples so that in the previous inventions it was 3 hours and in the claimed invention this time has been significantly reduced and has reached about 2 hours.

[0025] 4- The kit is cheaper than the previous samples due to the use of fewer raw materials needed for the evaluation and measurement process.

[0026] Shows a flowchart of the stages of the evaluation process of lipid peroxidation of human spermatozoa by the claimed kit.

[0027] Shows the phases of the evaluation process of lipid peroxidation of human spermatozoa which is performed by the kit: In the first place, after preparing the sperm suspension containing the sperm suspension sample along with the diluent, it is placed in the microtube after centrifugation, that is, about 20 million sperm samples and placed in the microtube. Then solution A is poured on the sample and placed in a water bath at 37 degrees Celsius for 60 minutes. In the continuation of the process, solution B was poured into the microtube containing the sample and placed in a cold temperature below 5 degrees for 5 minutes, and then after centrifugation at 800 g for 12 minutes, one milliliter of the supernatant solution was separated and exposed to the dye solution. Donor C contains 500 microliters of 2% TBA in 0.2 normal NaOH and is exposed to a 100°C water bath for 10 minutes. It stays At the last stage of the process, the same coat sample containing sperm peroxidation markers prepared is examined using a spectrophotometer and the resulting evaluation is calculated and recorded using a special method.Examples

[0028] Evaluation of peroxidation of human spermatozoa is used by an inventive kit, and how to use the kit is as follows: after preparing a sample of human semen, and washing it with a centrifuge, to evaluate the quality of sperm based on the level of lipid peroxidation, a suspension of about 20 Millions of sperm are prepared. Then solution A is poured on the sample and placed in a water bath at 37 degrees Celsius for 60 minutes. In the continuation of the process, solution B was poured into the microtube containing the sample and placed in a cold temperature below 5 degrees for 5 minutes, and then after centrifugation at 800 g for 12 minutes, one milliliter of the supernatant solution was separated and exposed to the dye solution. The donor C is placed and exposed to a 100°C water bath for 10 minutes. At the last stage of the process, the same coat sample containing sperm peroxidation indicators prepared using a spectrophotometer is examined and the resulting evaluation is calculated and recorded using a special method, and the quality of the sperm sample is measured and determined based on this.

[0029] In the industrial phase, the lipid peroxidation evaluation kit of human spermatozoa is produced in several stages and sections, and the final product is prepared by establishing direct and indirect communication between each section.

[0030] In the supply section of the primary materials of the kit, in this section, all the solutions, reagents, boxes, catalogs, and materials required for the kit are listed and supplied.

[0031] The preparation and packaging section of the promoter solution required for the kit is prepared in this section and is prepared and packed in the required volume for each package of the kit.

[0032] The preparation and packaging department of the acid solution is prepared and packaged in the required volume for each package of the kit.

[0033] The coloring solution preparation and packaging section, where the coloring solution is prepared and packaged in the required volume for each kit package.

[0034] The final packaging part of the kit, where all the solutions and materials needed for a kit are boxed and packed in the required number or according to the customer's order.

[0035] The final visit and confirmation section, where the package ready for the kit is checked based on the customer's order or the market and is closed with a confirmation stamp, and after labeling, the package door is sealed.

[0036] The quality control department, where all chemical, microbial, or physical tests are performed on the consumables required for the kit, and after the materials are approved, they enter the production line.

[0037] The research and development department improves and develops the product by monitoring, investigating, and researching all lines and consumables of the kit.

[0038] The application of kits produced in andrology laboratories around the world in examining the quality of sperm and evaluating the quality of men's sperm in terms of the amount of lipid peroxidation, infertility clinics of the country and the world, and in the treatment of infertile couples in investigating the cause of male infertility and evaluating the quality of men's sperm from the point of view The opinion of oxidative stress, and educational and research centers of reproductive sciences are used to train students, researchers and even in research projects to evaluate the quality of men's sperm.

Claims

An evaluation kit for lipid peroxidation of human spermatozoa including:- A box with holes for placing materials and solutions including:- A chamber containing solution A based on ferrous sulfate and- A chamber containing solution B based on ascorbic acid and- A chamber containing solution C based on trichloroacetic acid and- A container containing solution D based on thiobarbituric acid and sodium hydroxideAccording to claim 1, the evaluation kit for lipid peroxidation of human spermatozoa includes preparation of oxidation promoter solution A based on ferrous sulfate, preparation of oxidation promoter solution B based on ascorbic acid, preparation of acidifying solution C based on trichloroacetic acid, preparation of solution D dyes are based on tri-barbituric acid and sodium hydroxide.According to claim 2, the step of preparing the oxidation promoter solution A based on ferrous sulfate is stated, in which solution A is prepared with a molarity between 0.5 and 5 mM.According to claim 2, the step of preparing the oxidation promoter B solution, which is prepared from sodium ascorbate with a molarity between 1 and 20 mM,According to claim 2, the step of preparing the acidifying solution C is based on trichloroacetic acid, where the acidifying solution C is prepared in a concentration between 5 and 75% and stored at optimal ambient temperature.According to claim 2, the step of preparing solution D is based on tri-barbituric acid, where tri-barbituric acid with concentrations between 0.1 and 5% and sodium hydroxide with normality between 0.01 and 5.00 0 normal is prepared and becomes a uniform solution.According to claim 1, the prototype human spermatozoa is specifically prepared for use in the kit.According to claim 7, in the prototype of human spermatozoa, the semen sample with solutions such as PBS is subjected to a force of g between 50 and 800 using a centrifuge, and the sedimented sperms caused by the force of the centrifuge with a concentration between 10 It is diluted up to 30 million with diluting solutions, including PBS.According to claim 1, the human spermatozoan lipid peroxidation evaluation kit examines the quality of men's sperm based on the oxidation rate and calculates the lipid peroxidation amount of semen and sperm samples.

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