Stable medium and method for prolonging survival time of human semen sample

Abstract

The invention discloses a stable medium for prolonging the survival time of a human semen sample and a method thereof. The medium is composed of a pH buffer agent, an inorganic salt, an organic compound and amino acid with oxidation resistance. When the human semen is mixed with the stable medium, the survival time of the semen can be effectively prolonged to 72 hours at most when the temperature is kept within the range of + / -20% of the human body temperature.

Description

TECHNICAL FIELD

[0001] The present invention relates to the maintenance or preservation of biological material activity in vitro. In particular, the present invention relates to a stabilizing medium for prolonging the survival time of human semen samples and methods thereof. BACKGROUND

[0002] Human semen, a biological complex composed of spermatozoa and seminal plasma, plays a crucial role in male fertility assessment. It not only serves as a core component in various clinical and diagnostic fields such as assisted reproductive technology (ART), sperm bank storage, and medical research, but also holds significant importance for individuals facing genetic disease risks, undergoing chemotherapy, or choosing to delay parenthood. Semen analysis, which encompasses the comprehensive evaluation of sperm count, motility, morphology, and volume, is a critical step in determining male reproductive health and the suitability of the aforementioned applications. Additionally, it is used to verify the presence of sperm in post-vasectomy semen samples.

[0003] Traditional semen analysis methods often require on-site collection, which not only causes psychological burden and performance anxiety in patients but also presents practical operational challenges. Moreover, due to the vulnerability of sperm in vitro, the private collection method provided by clinics is often subject to strict time constraints. Although current solutions such as cryopreservation and mail-order semen analysis kits exist, they still have certain limitations. Specifically, cryopreservation may adversely affect sperm quality, while mail-order kits require ensuring the stability of semen during transportation and have relatively limited storage periods.

[0004] In view of this, there is an urgent need for a more efficient and durable method and medium for preserving human semen. This method should avoid the use of compounds such as glycerol that may cause DNA damage and does not rely on cryopreservation technology. The goal is to ensure that semen samples can maintain activity for a longer period of time in various application scenarios, such as infertility diagnosis, semen donation, artificial insemination, in vitro fertilization, post-vasectomy semen analysis (PVSA), and laboratory research. SUMMARY

[0005] The primary objective of this patent disclosure is to propose an innovative technical solution to address the challenges faced by existing technologies in long-term storage or preservation of semen samples.

[0006] Another objective is to develop a new medium that can effectively preserve and stabilize human semen samples for a long period of time.

[0007] A further goal is to break through the limitations of semen sample survival time, thereby enhancing the flexibility and feasibility of such samples during transportation and analysis.

[0008] The present patent also aims to find the optimal dilution ratio between semen and the preservation medium in order to extend the survival time of sperm from the usual one hour to a maximum of three days.

[0009] Another important goal is to explore a method that can safely and effectively transport a human semen sample from the collection site to the semen analysis test center.

[0010] At the same time, the present patent also aims to simplify the process of semen preservation and stabilization, avoiding the use of complex laboratory operations such as cryopreservation or centrifugation.

[0011] In addition, it is also desirable to achieve a technology that can maintain the stability of a human semen sample during transportation at room temperature.

[0012] To achieve the objectives set out in the present disclosure, we provide a stabilization medium capable of extending the survival time of human semen. This stabilization medium contains one or more pH buffers, inorganic salts, organic compounds, and amino acids with antioxidant properties. When human semen is mixed with this stabilization medium and stored within a predetermined temperature range, the survival time of the semen can be extended to a predetermined period. In certain embodiments, the semen can survive for up to 72 hours, while the storage temperature range is maintained between 36.1 and 37.2 degrees Celsius.

[0013] In certain embodiments, the concentration of amino acids in the stabilization medium is in the range of 0.01 to 0.5 millimoles (mmol).

[0014] Specific amino acid species can include alanine (ala), aspartic acid (asp), asparagine (asn), glutamic acid (glu), alanyl-glutamine (ala-gln), glycine (gly), proline (pro), and serine (ser).

[0015] Organic compounds can include glucose, sodium lactate (L-isomer), sodium pyruvate, and taurine (tau). In these embodiments, the concentration of taurine ranges between 0.01 and 10.0 mmol, the concentration of glucose can range between 0.05 and 5.0 mmol, the concentration of sodium lactate (L-isomer) ranges between 5.0 and 20.0 mmol, and the concentration of sodium pyruvate ranges between 0.1 and 1.0 mmol.

[0016] In certain embodiments, the pH buffer can contain 3-(N-morpholino)propanesulfonic acid (MOPS) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) at a concentration of 10.0 to 25.0 mmol.

[0017] The inorganic salts can include sodium chloride (NaCl), potassium chloride (KCl), magnesium sulfate heptahydrate (MgS04-7H20), disodium hydrogen phosphate dihydrate (Na2P04-2H20), sodium bicarbonate (NaHC03), and calcium chloride dihydrate (CaCl2-2H20). Among them, the concentration of CaCl2-2H20 ranges between 0.8 and 2.8 mmol, the concentration of MgS04-7H20 ranges between 0.2 and 4 mmol, the concentration of NaCl ranges between 75.0 and 105 mmol, the concentration of KCl ranges between 3.5 and 7.5 mmol, the concentration of Na2P04-2H20 ranges between 0.05 and 1.5 mmol, and the concentration of NaHC03ranges between 2.0 and 10.0 mmol.

[0018] In some embodiments, the stabilizing medium can further include antioxidants such as acetyl L-carnitine, alpha-lipoic acid, and N-acetyl-L-cysteine. In addition, the stabilizing medium can further include compounds for adjusting the pH value such as sodium dihydrogen phosphate dihydrate (NaH2P04-2H20), MgS04-7H20, CaCl2-2H20, sodium lactate (L-isomer), and sodium pyruvate. At the same time, antibiotics, chelating agents, and the like can be added to the stabilizing medium.

[0019] In certain embodiments, the pH value of the stabilizing medium is between 7.2 and 8.

[0020] In another aspect, the present application also provides a method for extending the survival time of human semen. The method includes mixing human semen with a stabilizing medium and maintaining the mixture within a temperature range of 36.1 to 37.2 degrees Celsius, thereby enabling the survival time of the semen to be extended to a maximum of 72 hours.

[0021] In another embodiment, the present application also provides a kit including a stabilizing medium. The stabilizing medium includes one or more pH buffers, one or more inorganic salts, one or more organic compounds, and one or more amino acids having antioxidant properties, thereby enabling the survival time of human semen to be extended to a maximum of 72 hours when the semen is mixed with the stabilizing medium and maintained within a temperature range of 36.1 to 37.2 degrees Celsius.

[0022] The stable medium of the present application can effectively protect and stabilize human semen samples for up to 72 hours without the need for refrigeration or freezing. The stable medium extends the effective survival time of sperm from the usual 1 hour to a maximum of 3 days. Therefore, the stable medium provides more flexible logistics options for the transportation of semen samples and ensures that the semen samples maintain persistent activity in various application scenarios, including infertility diagnosis, semen donation, artificial insemination, in vitro fertilization, post vasectomy semen analysis (PVSA), and other laboratory applications and research. In addition, since the stable medium does not contain compounds such as glycerol that can cause damage to DNA, it can avoid damage to the genetic material of sperm, thereby improving sperm survival rate, increasing fertilization rate, reducing the risk of genetic abnormalities in preserved semen cells, and possibly improving reproductive outcomes.

[0023] These and other aspects of the embodiments described herein will become more fully understood when considered in connection with the following description and the accompanying drawings. It will be appreciated that the following description, while indicating preferred embodiments and numerous specific details, is given by way of example only and is not intended to limit the scope of the application. Various modifications and alterations to the embodiments described herein will become apparent to those skilled in the art from the following description, and it is intended that all such modifications and alterations be considered part of this application. BRIEF DESCRIPTION OF DRAWINGS

[0024] By carefully observing the accompanying drawings, we can gain a deeper understanding of the core and complexity of the present application. These drawings are intended to clearly demonstrate the various objectives, features, components, and advantages of the present application to experts in the field. When these drawings are combined with the text of the patent application, we can more fully grasp the objectives, functions, and advantages of the present application. They are extremely important for converting the technical details of the present application into practical and feasible operating steps, so that professionals in this field can easily understand and implement them.

[0025] Figures 1-2 As a table, it lists the specific composition of the stable medium disclosed in the present application.

[0026] Figures 3-4 As a chart, it demonstrates the ability of the stable medium disclosed in the present application to maintain the forward motility parameters of semen samples over a long period of time.

[0027] Figures 5-6 As a chart, it reflects the ability of the stable medium disclosed in the present application to maintain the non-forward motility parameters of semen samples over a long period of time.

[0028] Figures 7-8 As a chart, it reveals the ability of the stable medium disclosed in the present application to maintain the stability of the immotility parameters of semen samples over a long period of time.

[0029] Figures 9-10 As a graph, the ability of the stabilizing medium disclosed in the present application to maintain the morphological parameters of the semen sample stable over time is illustrated.

[0030] Figure 11 As a graph, the difference in the forward movement parameters of a pure human semen sample and a semen sample to which the stabilizing medium has been added, according to the present application, at T=0, 24, 48 and 72 hours is compared.

[0031] Figure 12 As a graph, the comparison of the non-forward movement parameters of a pure human semen sample and a semen sample to which the stabilizing medium has been added, according to the present application, at T=0, 24, 48 and 72 hours is illustrated.

[0032] Figure 13 As a graph, the difference in the morphological parameters of a pure human semen sample and a semen sample to which the stabilizing medium has been added, according to the present application, at T=0, 24, 48 and 72 hours is compared.

[0033] Figure 14 As a graph, the comparison of the immotility parameters of a pure human semen sample and a semen sample to which the stabilizing medium has been added, according to the present application, at T=0, 24, 48 and 72 hours is illustrated.

[0034] Figure 15 As a graph, the comparison of the total movement parameters of a pure human semen sample and a semen sample to which the stabilizing medium has been added, according to the present application, at T=0, 24, 48 and 72 hours is illustrated.

[0035] Figure 16 As a table, the results of the DNA fragmentation test of a pure human semen sample and a semen sample to which the stabilizing medium has been added, according to the present application, at T=0 and 72 hours are listed.

[0036] Figure 17 As a block diagram, the composition of the kit containing the stabilizing medium designed according to the present application is described.

[0037] Figure 18 As a flowchart, the specific steps for implementing the stabilization of a human semen sample according to the present application are detailed. DETAILED DESCRIPTION

[0038] The various features and functions of the stabilizing medium disclosed in the present application are fully described below in conjunction with the accompanying drawings. The terms used herein are intended only for the specific embodiments and do not limit the broad applicability of the present application.

[0039] This detailed description is intended to present current preferred embodiments of the application, but is not meant to limit the only form of the application. Unless explicitly stated otherwise, different embodiments can implement the same or similar functions in any order, and all are within the scope of the present application.

[0040] The embodiments are chosen and described in order to best illustrate the principles of the application and its practical application, and to thereby enable one of ordinary skill in the art to best utilize the application in various embodiments and with various modifications as are suited to the particular use contemplated.

[0041] Reference to "an embodiment" or "one embodiment" in this document means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the application. The appearances of "an embodiment" or "one embodiment" in various places in the specification are not necessarily all referring to the same embodiment.

[0042] As previously mentioned, the technology for long-term storage of semen samples still needs improvement. The present application provides a stabilizing medium and its method of use, which can stabilize human semen samples for up to 72 hours without the need for freezing and refrigeration. This stabilizing medium significantly extends the shelf life of sperm from the usual 1 hour to a maximum of 3 days. Therefore, it greatly increases the convenience of semen sample transportation and ensures the long-lasting viability of semen samples in various applications, such as infertility diagnosis, semen donation, artificial insemination, in vitro fertilization, post-vasectomy semen analysis (PVSA), and laboratory research, etc. In addition, the stabilizing medium does not contain compounds such as glycerol that can damage the genetic material of sperm, thereby increasing sperm survival rate, increasing fertilization rate, reducing the risk of genetic abnormalities, and possibly improving reproductive outcomes.

[0043] The following definitions are provided for key terms used in this document:

[0044] "Semen sample" refers to an organic fluid secreted by the male gonads and other sex organs of male or hermaphroditic animals, which can fertilize female eggs.

[0045] "Stabilizing medium" is a substance or mixture of substances designed to maintain the integrity, viability, and stability of biological samples or materials during storage or transportation. It aims to prevent the degradation, damage, or change of biological components, ensuring their suitability for subsequent analysis or processing.

[0046] "Viability time" refers to the ability of biological materials to remain alive and functionally normal within a certain period of time, which is an important indicator of their integrity and functionality.

[0047] "Extended viability time" refers to the ability of biological materials to remain alive and functionally normal for a longer period of time.

[0048] "Preservation" refers to the process of maintaining the quality, state, or existence of a substance, solution, or material.

[0049] "Transport" refers to the act of moving a collected sample, material, or substance from one location to another.

[0050] "Sperm survival rate" is defined as the percentage of live sperm in a semen sample.

[0051] "pH buffer" is a substance or mixture of substances that is capable of maintaining the pH of a solution relatively stable, even when acid or base is added.

[0052] "Inorganic salt" is a compound composed of positively charged cations and negatively charged anions, which are electrically neutral as a whole.

[0053] "Organic compound" refers to a chemical compound in which one or more carbon atoms are bonded to other elemental atoms through covalent bonds.

[0054] "Amino acid with antioxidant properties" refers generally herein to an amino acid that neutralizes or mitigates oxidative stress, preventing the harmful effects of free radicals in biological or chemical systems.

[0055] "Antioxidant" refers generally herein to a substance or mixture of substances that is capable of scavenging and neutralizing free radicals or reactive oxygen species. Reactive oxygen species are highly reactive oxygen-containing molecules or ions that cause cellular damage by triggering oxidative stress. Antioxidants play a crucial role in preventing or slowing down oxidative damage caused by free radicals, contributing to cellular health.

[0056] "pH modifying compound" refers generally to a substance or mixture of substances that is capable of changing the acidity or basicity (pH) of a solution.

[0057] "Antibiotic" refers generally to a substance or mixture of substances that is capable of killing or inhibiting the growth of bacterial cells.

[0058] "Chelating agent" refers generally herein to a substance or mixture of substances that is capable of binding to metal ions through multiple coordination sites to form a complex.

[0059] "pH" refers generally to a measure of the acidity or basicity of a solution.

[0060] "Concentration" refers generally herein to the amount or quantity of a substance contained in a specific volume or mass of a mixture. The concentration of a solution is typically expressed in terms of mass, volume, or moles.

[0061] "Predetermined time period" refers generally to a specific known length of time that is set in advance for a particular purpose.

[0062] "Predetermined temperature range" refers generally to a specific known interval of temperatures that is set in advance for a particular purpose.

[0063] The word “one or more” here usually refers to at least one, but can also refer to more than one.

[0064] Figure 1 Table 100 details the composition of the stabilizing medium according to this disclosure. Table 100 shows the compound classes 105 and their concentration ranges 110 in the stabilizing medium. The stabilizing medium, as a buffer medium, is designed to preserve human semen, thereby enabling the storage and transport of human semen to laboratories and other locations for analysis and various applications. These applications include, but are not limited to, semen donation, artificial insemination, and in vitro fertilization. The stabilizing medium contains at least four major classes of compounds 105: amino acids, pH buffers, inorganic salts, and organic compounds.

[0065] In some embodiments, the stabilizing medium contains a variety of amino acids, including but not limited to alanine, aspartic acid, asparagine, glutamic acid, alanyl-glutamine, glycine, proline, serine, and taurine, to utilize their antioxidant properties. These amino acids with antioxidant properties can protect sperm cells from cold shock and help maintain epigenetic homeostasis. Antioxidants are crucial in mitigating oxidative stress, as untreated oxidative stress can lead to sperm pathology, including adenosine triphosphate (ATP) depletion, insufficient axonal phosphorylation, lipid peroxidation, and reduced motility and viability. Furthermore, amino acids and antioxidants play a key role in protecting sperm cells from reactive oxygen species (ROS) damage. Failure to effectively manage ROS can lead to degradation and damage to sperm samples, ultimately undermining the effectiveness of the stabilizing medium. In some embodiments, the amino acids include one or more of alanine, aspartic acid, asparagine, glutamic acid, alanyl-glutamine, glycine, proline, and serine.

[0066] The pH buffers in the stabilizing medium may include 3-(N-morpholino)propanesulfonic acid (MOPS) and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES). These pH buffers help maintain the pH value, which is crucial for sperm motility. The inorganic salts in the stabilizing medium may be selected from the group including sodium chloride (NaCl), potassium chloride (KCl), magnesium sulfate heptahydrate (MgSO4·7H2O), disodium hydrogen phosphate dihydrate (Na2PO4·2H2O), sodium bicarbonate (NaHCO3), and calcium chloride dihydrate (CaCl2·2H2O). Inorganic salts are essential for maintaining sperm cell integrity and viability during storage. For example, the main functions of inorganic salts in the stabilizing medium may include osmotic pressure regulation, ion balance, pH buffering, prevention of ice crystal formation, and energy metabolism. The organic compounds in the stabilizing medium may be selected from the group including glucose, sodium lactate (L-isomer), sodium pyruvate, and taurine (tau). These organic compounds play a crucial role in enhancing cell viability and protecting the sample from damage during storage. For example, the primary functions of organic compounds in stabilizing media may include membrane stabilization, osmotic pressure regulation, energy supply, mimicking physiological environments, viscosity adjustment, and nutrient supply. In some embodiments, the stabilizing media may contain antibiotics, such as gentamicin or penicillin, to prevent microbial contamination of the semen sample, maintain its sterility, and ensure it is protected from harmful microorganisms. A balance must be maintained when using antibiotics; excessive use may be harmful to sperm cells, while insufficient use may make the sample susceptible to microbial contamination. In some embodiments, the stabilizing media contains chelating agents, such as EDTA, which prolong the survival time of the semen sample by stimulating sperm motility. However, excessive EDTA may damage sperm cells, while insufficient EDTA may fail to effectively stimulate sperm motility, thus affecting sperm viability. In some embodiments, the stabilizing medium comprises one or more pH-modifying compounds, possibly selected from the group consisting of sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), magnesium sulfate heptahydrate (MgSO4·7H2O), calcium chloride dihydrate (CaCl2·2H2O), sodium lactate (L-isomer), and sodium pyruvate. In some embodiments, the stabilizing medium comprises one or more antioxidants, possibly selected from the group consisting of acetyl-L-carnitine, α-lipoic acid, and N-acetyl-L-cysteine.

[0067] In some embodiments, the stabilizing medium is prepared by dissolving components such as amino acids, pH buffers, inorganic salts, and organic compounds in water.

[0068] The combination of compounds in category 105 listed in Table 100 enables stable preservation of human semen for up to 72 hours, far exceeding the typically one-hour preservation time provided by conventional or existing media. In some embodiments, the stabilizing medium may be refrigerated with ice packs to maintain the temperature between 2 and 12 degrees Celsius. Before adding the human semen sample to the stabilizing medium, ensure that the medium has reached room temperature to fully exert its stabilizing effect. During mixing, the mixture containing the human semen sample and the stabilizing medium should be thoroughly stirred until homogeneous. After mixing, the temperature of the mixture should be maintained close to body temperature. Specifically, in one embodiment, the mixture temperature should be maintained between 36.1 and 37.2 degrees Celsius; in another embodiment, the mixture temperature should be maintained within ±20% of body temperature. Furthermore, the pH of the stabilizing medium should be controlled between 7.2 and 8.

[0069] Figure 2 Table 200 details the composition of the stabilizing medium in this disclosure. Table 200 includes compound categories 205, specific compounds 210, and their concentration ranges 220 in the stabilizing medium. Compound category 205 primarily includes amino acids, pH buffers, inorganic salts, and organic compounds. The types of amino acids include, but are not limited to, alanine (ALA), aspartic acid (ASP), asparagine (ASN), glutamic acid (GLU), alanyl-glutamine (ALA-GLN), glycine (GLY), proline (Pro), and serine (SER). The concentration of each amino acid in the stabilizing medium must be strictly controlled between 0.01 and 0.5 mmol. Maintaining an appropriate concentration of amino acids is crucial; excessive concentrations may lead to antioxidant overload, damaging sperm cells, while insufficient concentrations may weaken the protective effect of the stabilizing medium on sperm cells. In some embodiments, the stabilizing medium may also contain antioxidants such as acetyl-L-carnitine, α-lipoic acid, and N-acetyl-L-cysteine ​​to further protect the sample from oxidative stress.

[0070] Stabilizing media also contain pH buffers, such as MOPS or HEPES, at concentrations ranging from 10.0 to 25.0 mmol. These buffers are designed to maintain the optimal pH range (7.2 to 8) required by sperm. Excessive MOPS can make the stabilizing media too acidic, while insufficient MOPS / HEPES content can reduce its buffering capacity, affecting sperm motility and function. In addition, stabilizing media may also contain pH-modifying compounds such as NaH2PO4·2H2O (which can act as an acid or base), MgSO4·7H2O and CaCl2·2H2O (which have a neutral to slightly acidic effect on pH), as well as sodium lactate (L-isomer) and sodium pyruvate (which can act as an acid or base under certain conditions).

[0071] The stabilizing medium contains a rich variety of inorganic compounds, including NaCl, KCl, MgSO4·7H2O, Na2PO4·2H2O, NaHCO3, and CaCl2·2H2O, with concentrations controlled within the ranges of 75.0–105 mmol, 3.5–7.5 mmol, 0.2–4.0 mmol, 0.05–1.5 mmol, 2.0–10.0 mmol, and 0.8–2.8 mmol, respectively. These inorganic compounds are crucial for maintaining osmotic balance in the semen sample, thereby ensuring the integrity and function of sperm cells. NaCl and KCl provide essential cations to the semen sample to maintain osmotic balance; Na+, K+, and NaHCO3 help maintain the intracellular pH of the semen sample. The concentrations of these inorganic salts must be strictly controlled to avoid osmotic imbalance that could damage sperm. Furthermore, magnesium ions (Mg2+) provided by MgSO4·7H2O participate in various cellular functions, including nucleic acid stabilization, thereby enhancing the overall stability of sperm cells; sodium ions (Na+) and phosphate ions (PO43-) provided by Na2PO4·2H2O help maintain osmotic pressure balance and regulate pH; and calcium ions (Ca2+) provided by CaCl2·2H2O are crucial for sperm motility. The combined effects of inorganic salts in the stabilizing medium provide an ideal environment for sperm survival, osmotic pressure balance, and basic cellular functions.

[0072] In addition, the stabilizing medium contains organic compounds such as glucose, sodium lactate (L-isomer), sodium pyruvate, and taurine, at concentrations ranging from 0.05 to 5.0 mmol, 5.0 to 20.0 mmol, 0.1 to 1.0 mmol, and 0.01 to 10.0 mmol, respectively. The stabilizing medium containing sodium pyruvate performs well in maintaining the DNA integrity of sperm samples. The addition of glucose is crucial for maintaining sperm cell motility. Sodium lactate helps maintain pH and serves as an energy source for sperm cells. Taurine, as an antioxidant, protects sperm cells from oxidative stress damage and may help maintain osmotic balance within sperm cells. The organic compounds in the stabilizing medium work together to provide strong support for the health and function of sperm cells during storage. The concentrations of these organic compounds also need to be strictly controlled to ensure their beneficial effects on sperm viability while avoiding any adverse effects. The combination of inorganic and organic compounds in the stabilizing medium is designed to create an environment optimal for sperm cell survival and function during storage and handling.

[0073] Figures 3 to 10These are graphs illustrating experimental data from clinical trial validation studies conducted according to this disclosure. These graphs show the results of tests conducted at T=0, T=24 hours, T=48 hours, and T=72 hours after pure human semen samples were mixed with a stabilizing medium at 37°C, aiming to collect data on the performance of the stabilizing medium. Specifically, Figure 3 and Figure 4 (Figures 300 and 400) demonstrate the ability of the stabilizing medium to maintain the forward motility parameters of semen samples over extended periods. As shown, the forward motility parameters of the semen samples can be maintained for up to 72 hours, highlighting the significant effect of the stabilizing medium in maintaining the functional integrity of sperm cells during this extended period. Forward motility is a key indicator for assessing sperm quality and its ability to successfully fertilize an egg. This extended preservation period is particularly important for various reproductive procedures, such as artificial insemination and in vitro fertilization, as these procedures require flexible scheduling. Figure 5 and Figure 6 (Figures 500 and 600) reveal the ability of the stabilizing medium to maintain non-progressive motility parameters in semen samples over extended periods. Similarly, the stability of non-progressive motility parameters over 72 hours underscores the effectiveness of the stabilizing medium in maintaining sperm cell viability and quality. Non-progressive motility, as an important aspect of sperm assessment, represents the proportion of sperm that exhibit motility but lack typical progressive movement. Figure 5 and Figure 6 The data showed dynamic changes in non-progressive motility at specific time points (e.g., T=0, T=24 hours, T=48 hours, and T=72 hours). Understanding these changes is crucial for a comprehensive assessment of sperm health. Long-term preservation results indicate that stabilizing media can effectively mitigate environmental stresses or natural physiological changes that may impair non-progressive motility.

[0074] Figure 7 and Figure 8 (Figures 700 and 800) demonstrate the ability of the stabilizing medium to maintain immobility parameters in semen samples over extended periods. As shown, the stabilizing medium can retain viable sperm cells for up to 72 hours, thus maintaining relatively stable immobility parameters in the semen sample over this period. This stability demonstrates the stabilizing medium's ability to mitigate factors that may impair sperm motility. Therefore, Figure 7 and Figure 8 The stability of the long-term preservation and immobility parameters of viable sperm cells shown in the study highlights the great potential of stable media for long-term storage and preservation of human semen without the need for cryopreservation and / or refrigeration. Figure 9 and Figure 10Figures 900 and 1000 visually demonstrate the ability of stabilizing media to maintain semen sample morphological parameters over extended periods. The figures emphasize the sustained maintenance of normal sperm morphology, further demonstrating the stability and effectiveness of the stabilizing media within a specific timeframe. In assisted reproductive technology, the ability to maintain a stable level of normal sperm morphology over a long period is of paramount value.

[0075] from Figures 3 to 10 It is clear that, compared to existing media, the formulation of the stable medium can effectively preserve human semen for up to 72 hours, while existing media typically only preserve it for 1 hour. Furthermore, the stable medium does not contain DNA-depleting compounds such as glycerol and does not require cryopreservation.

[0076] Currently, we tested purified human semen samples and semen samples mixed with a stabilizing medium at T=0, 24 hours, 48 ​​hours, and 72 hours to evaluate their forward motility, non-forward motility, morphology, immobility, and total motility parameters. A total of 50 purified human semen samples were collected as controls. These samples were mixed with the stabilizing medium at a 1:3 ratio to form stable mixtures (i.e., 1 part semen sample mixed with 3 parts stabilizing medium). Subsequently, 1 ml of the stable mixture was tested at specified time points, and the results were compared with those of the purified semen samples. All tests were performed at room temperature. Figures 11 to 15 (Figures 1100 to 1500) compare the kinematic parameters of pure human semen samples and semen samples treated with a mixed stable medium at T=0, 24 hours, 48 ​​hours, and 72 hours. Data in each figure are based on the average of 50 samples, reflecting the collective average measurements from multiple independent samples. As shown, the semen samples treated with the mixed stable medium exhibit significant advantages and stability across all parameters compared to untreated semen samples. In particular, Figure 16 (Table 1600) shows the DNA fragmentation results of pure human semen samples and semen samples mixed with a stable medium at T=0 and 72 hours. The results show that the percentage of DNA fragmentation in untreated semen samples was significantly increased at 72 hours, while the DNA integrity of semen samples in the stable mixture was maintained for a longer period of time.

[0077] Figure 17A block diagram of a kit 1700 comprising the stabilizing medium described herein is shown. The kit 1700 contains stabilizing media as shown in Table 100 or Table 200. To maintain the temperature of the stabilizing medium between 2 and 12 degrees Celsius until it mixes with human semen to form a mixture, it may be refrigerated with ice packs or other suitable elements. It is particularly important that the stabilizing medium be brought to room temperature before adding the semen sample to ensure optimal stabilization. In some embodiments, the stabilizing medium is stored in a container or vial and housed within the outer casing of the kit 1700. Furthermore, the kit 1700 may also include, but is not limited to, instructions, other containers, transfer tools, temperature regulating or control elements, etc. The outer packaging of the kit 1700 is designed to accommodate these containers, instructions, transfer tools, and temperature regulating or control elements, etc.

[0078] Figure 18 A flowchart illustrating a method for stabilizing human semen samples according to this disclosure is provided. In step 1802, the human semen sample is mixed with a stabilizing medium as shown in Table 100 or Table 200 to form a homogeneous mixture. In some embodiments, the ratio of stabilizing medium to semen sample is 3:1. Stirring may be necessary during mixing to ensure homogeneity. In some cases, the stabilizing medium 120 is pre-cooled and maintained between 2°C and 12°C using a refrigerator, gel ice pack, or other cooling device before use. However, the stabilizing medium must be brought to room temperature before mixing with the human semen sample to ensure stabilization effectiveness. In step 1804, the mixed semen and stabilizing medium are maintained at a temperature range of 36.1 to 37.2°C to extend the semen's survival time to a maximum of 72 hours. In some embodiments, this temperature may be maintained within ±20% of body temperature.

[0079] It should be clarified that although we have described certain forms of the invention in detail, the invention is not limited to these specific forms or component configurations. It will be apparent to those skilled in the art that various modifications can be made without departing from the spirit of the invention. Therefore, the invention should not be considered limited to what is described in the drawings and specifications. The various features described below can be used individually or in combination with other features. However, a single feature may only provide a solution to one problem discussed above, and all problems may not be fully solved by a single feature.

Claims

1. A stable medium for prolonging the survival time of human semen samples, characterized in that, The stable medium contains: One or more pH buffers; One or more inorganic salts; One or more organic compounds; and One or more amino acids with antioxidant properties, which enable the semen to survive for a predetermined period of time when human semen is mixed with a stable medium and maintained within a predetermined temperature range.

2. The stable medium according to claim 1, characterized in that, The concentration of one or more amino acids in the stabilizing medium ranges from 0.01 to 0.5 mmol.

3. The stable medium according to claim 1 or 2, characterized in that, The one or more amino acids are selected from the group consisting of alanine (Ala), aspartic acid (ASP), asparagine (ASN), glutamic acid (GLU), alanyl-glutamine (Ala-GLN), glycine (GLY), proline (Pro), and serine (Ser).

4. The stable medium according to any one of claims 1 to 3, characterized in that, The one or more pH buffers comprise 3-(N-morpholino)propanesulfonic acid (MOPS) or 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) in a concentration range of 10.0 to 25.0 mmol.

5. The stable medium according to any one of claims 1 to 4, characterized in that, The one or more inorganic salts are selected from the group consisting of sodium chloride (NaCl), potassium chloride (KCl), magnesium sulfate heptahydrate (MgSO4·7H2O), disodium hydrogen phosphate dihydrate (Na2HPO4·2H2O), sodium bicarbonate (NaHCO3), and calcium chloride dihydrate (CaCl2·2H2O).

6. The stable medium according to any one of claims 1 to 5, characterized in that, The concentration of calcium chloride dihydrate (CaCl2·2H2O) contained in the one or more inorganic salts is between 0.8 and 2.8 mmol.

7. The stable medium according to any one of claims 1 to 6, characterized in that, The concentration of magnesium sulfate heptahydrate (MgSO4·7H2O) contained in the one or more inorganic salts is between 0.2 and 4 mmol.

8. The stable medium according to any one of claims 1 to 7, characterized in that, The concentration of sodium chloride (NaCl) contained in the one or more inorganic salts is between 75.0 and 105 mmol.

9. The stable medium according to any one of claims 1 to 8, characterized in that, The concentration of potassium chloride (KCl) contained in the one or more inorganic salts is between 3.5 and 7.5 mmol.

10. The stable medium according to any one of claims 1 to 9, characterized in that, The concentration of disodium hydrogen phosphate dihydrate (Na2HPO4·2H2O) contained in the one or more inorganic salts is between 0.05 and 1.5 mmol.

11. The stable medium according to any one of claims 1 to 10, characterized in that, The concentration of sodium bicarbonate (NaHCO3) contained in the one or more inorganic salts is between 2.0 and 10.0 mmol.

12. The stable medium according to any one of claims 1 to 11, characterized in that, The one or more organic compounds are selected from the group consisting of glucose, sodium lactate (L-isomer), sodium pyruvate and taurine.

13. The stable medium according to any one of claims 1 to 12, characterized in that, The concentration of taurine (tau) contained in the one or more organic compounds ranges from 0.01 to 10.0 mmol.

14. The stable medium according to any one of claims 1 to 13, characterized in that, The concentration of glucose contained in one or more organic compounds is between 0.05 and 5.0 mmol.

15. The stable medium according to any one of claims 1 to 14, characterized in that, The one or more organic compounds contain sodium lactate (L-isomer) at a concentration between 5.0 and 20.0 mmol.

16. The stable medium according to any one of claims 1 to 15, characterized in that, The one or more organic compounds contain sodium pyruvate in a concentration ranging from 0.1 to 1.0 mmol.

17. The stable medium according to any one of claims 1 to 16, characterized in that, The stabilizing medium also comprises at least one of the following components: one or more antioxidants, one or more pH adjusters, one or more antibiotics, and chelating agents. The one or more antioxidants are selected from acetyl-L-carnitine, α-lipoic acid, and N-acetyl-L-cysteine; the one or more pH adjusters are selected from sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), magnesium sulfate heptahydrate (MgSO4·7H2O), calcium chloride dihydrate (CaCl2·2H2O), sodium lactate (L-isomer), and sodium pyruvate.

18. The stable medium according to any one of claims 1 to 17, characterized in that, The pH of this stable medium is between 7.2 and 8.

19. The stable medium according to any one of claims 1 to 18, characterized in that, The scheduled time period is up to 72 hours, and the scheduled temperature range is set to 36.1 to 37.2 degrees Celsius.

20. A method for prolonging the survival time of a human semen sample, the method comprising the following steps: Human semen is mixed with the stable medium of claim 1 to form a mixture; The mixture of human semen and a stabilizing medium was maintained at a temperature range of 36.1 to 37.2 degrees Celsius, thereby extending the survival time of the semen to a maximum of 72 hours.

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