Culture solution, culture method, thawing solution, thawing method, human embryo, and infertility treatment method

Abstract

[Problem] To reduce the influence of active oxygen to reduce oxidative stress on human embryos and contribute to the recreation of the environment in a fallopian tube, thereby improving the quality of fertilized eggs and the success rate of implantation. [Solution] By adding an appropriate amount of ergothioneine to a culture solution and a thawing solution for human embryos, the influence of active oxygen is reduced to reduce oxidative stress on the human embryos, the environment in a fallopian tube is satisfactorily recreated, and the quality of fertilized eggs and the success rate of implantation are improved. However, when the amount of ergothioneine is too large, a reducing action becomes strong and cell division is suppressed.

Description

Culture medium, culture method, thawing medium, thawing method, human embryo, and infertility treatment method

[0001] The present invention relates to the culture of cells such as human embryos, and more particularly to a culture medium, a culture method, a thawing medium, a thawing method, a human embryo (human fertilized embryo), and a method for infertility treatment using ergothioneine.

[0002] Ergothioneine (C9H 15 N3O2S) is a type of amino acid and an antioxidant with a chemical structure shown in Figure 18. It is taken up into cells by a membrane transporter (OCTN1) or its derivatives and eliminates reactive oxygen species. Reactive oxygen species generated within cells include superoxide, hydrogen peroxide, hydroxyl radicals, and singlet oxygen, and ergothioneine is particularly effective as an antioxidant against hydroxyl radicals.

[0003] In addition to its antioxidant properties, ergothioneine also inhibits various enzymes that cause cell destruction, such as TNF-α (tumor necrosis factor-α), a substance that causes inflammatory reactions, inhibiting the production of lipid peroxides (oxidation of cell membranes), inhibiting elastase activity, and inhibiting MMP-1 (Matrix metalloproteinase-1), a collagen-degrading enzyme, thereby maintaining cellular health.

[0004] Furthermore, ergothioneine has the following excellent properties: a) It does not break down even when heated to 120°C for two hours; b) It is resistant to both acid and alkali and is extremely stable; c) It accumulates in areas of high oxidative stress, such as the kidneys, liver, eye lens, mitochondrial membranes, and hemoglobin membranes, preventing oxidation and improving metabolism and cell differentiation; d) It has greater antioxidant power than vitamin C, CoQ10, glutathione, and vitamin E; e) It reduces oxidized vitamin C and alpha-lipoic acid; f) It chelates heavy metals and excretes them from the body; g) It eliminates intracellular ROS (reactive oxygen species) and protects DNA and enzymes. Ergothioneine has the following excellent properties and has been certified as a medical food by the FDA (U.S. Food and Drug Administration), ensuring its safety.

[0005] Incidentally, background technology for such cell culture media using ergothioneine is the paper described in Non-Patent Document 1 below, which states that "supplementation of ergothioneine during culture improved the quality of bovine embryos." Figure 19 shows the appearance of a normal blastocyst from a bovine fertilized egg, as disclosed in Document 1. Figure 19 (A) shows the appearance of a blastocyst after culturing the fertilized egg for eight days, Figure 19 (B) shows the blastocyst breaking out of the shell surrounding the fertilized egg, and Figure 19 (C) shows the appearance of the blastocyst after breaking out. In all cases, the cell nuclei have been stained with a special dye (Hoechst staining, similar to Figure 20).

[0006] In contrast, Figure 20 shows the state of thawing blastocysts that were flash-frozen with a cryoprotectant. Figures (A) and (B) show the case when the culture medium does not contain ergothioneine (labeled Erg(-)), and Figures (C) and (D) show the case when the culture medium contains ergothioneine (labeled Erg(+)). Figures (A) and (C) show the state of cell number, and Figures (B) and (D) show the state of apoptosis (cell suicide). Figure (E) shows the case when most of the cells have committed suicide, and Figure (F) shows the case when no cells have committed suicide.

[0007] Comparing these figures, in a, (A) Erg(-), the cell nuclei are enlarged due to damage to genes and other organs caused by the cryoprotectant. Also, as in (B), cell damage has occurred and many cells have committed suicide. In b, (C) Erg(+), the cells are not enlarged and are numerous. Also, as in (D), few cells have committed suicide and almost no cell damage has occurred. In this way, the use of ergothioneine removes active oxygen from cells before and after thawing, resulting in little cell damage and almost no cell suicide.

[0008] Figure 21 shows the relationship between the rate of cleavage-stage embryos and blastocysts (vertical axis) and the amount of ergothioneine added to the culture medium (horizontal axis). According to this, a) in the case of a culture medium lacking ergothioneine (see the graph marked "0" on the horizontal axis), approximately 78% of fertilized eggs became cleavage-stage embryos, and approximately 48% became blastocysts. b) At ergothioneine concentrations of 0.05 mM and 0.1 mM, the rates of both cleavage-stage embryos and blastocysts were similar, but the rate of blastocysts increased to approximately 80%. c) However, at ergothioneine concentrations of 0.5 mM, the rate of cleavage-stage embryos decreased to 56% and the rate of blastocysts decreased to 32%. d) At ergothioneine concentrations of 1 mM, the rate of cleavage-stage embryos decreased to 47%, and almost no blastocysts were observed.

[0009] As mentioned above, adding a large amount of ergothioneine to the culture medium will not necessarily produce the desired effect. Furthermore, the optimal amount of ergothioneine to be added varies depending on the species. The example in Figure 21 is from cow's eggs, which have high antioxidant capacity, while the optimal amount for mice and rabbits is said to be around 1 mM.

[0010] Figure 22 shows the relationship between the presence or absence of ergothioneine and cell number during the culture process. Figure (A) shows the cell number, and Figure (B) shows the number of apoptotic cells. Both figures show the cell numbers for early blastocysts (BL), blastocysts (XBL) at the time of evacuation, and evacuation blastocysts (HBL) (corresponding to Figure 19 (A) to (C)). The amount of ergothioneine was set to 0.1 mM as in Figure 21. Referring to the graph in Figure 22 (A), the cell number is slightly higher in the absence of ergothioneine at the early stage, but the number of blastocysts is higher in the presence of ergothioneine at the time of evacuation and after evacuation. On the other hand, as shown in Figure 22 (B), the number of apoptotic cells is lower in the presence of ergothioneine at all stages.

[0011] Figure 22(C) is a graph showing the percentage of apoptotic cells relative to the total cells. In cases where the percentage of apoptotic cells relative to normal cells was 5% or less, the percentage was overwhelmingly higher with ergothioneine than without it. In cases where the percentage was 5-10%, the percentage peaked in the absence of ergothioneine, and in cases where the percentage was 10% or more, the percentage was overwhelmingly lower with ergothioneine.

[0012] Non-Patent Document 2 below presents the results of culturing pig eggs with the addition of ergothioneine. Figure 23 (Figure 1 in the same document) shows the relative changes in intracellular glutathione (GSH) and reactive oxygen species (ROS). Figure 24 (Figure 4A in the same document) shows the cleavage rates of embryos at each stage (1 cell, 2-3 cells, 4-5 cells, 6-8 cells) on day 2 of culture in culture medium containing various concentrations of ergothioneine (10, 50, 100 μM), as well as the combined cleavage rate (CL Rate). Figure 25 (Figure 4B in the same document) shows the cleavage rates of blastocysts at each stage (early blastocyst, expanded blastocyst, budding blastocyst) on day 7 of culture, as well as the combined blastocyst rate (BL Rate). As shown in Figure 24, during cell proliferation, cell fragmentation was less and cell growth was better when cultured in a culture medium with a low concentration of ergothioneine (10 μM). However, as shown in Figure 25, while good results were obtained with a low concentration of ergothioneine (10 μM) up to the blastocyst stage, better results were obtained with a high concentration of ergothioneine (50 μM) during germination (hatching). This indicates that ergothioneine has the opposing effects of suppressing cell proliferation and promoting cell differentiation.

[0013] Theriogenology 85 (2016) 688-697 "L-ergothioneine supplementation during culture improves quality of bovine in vitro-produced embryos" G. Zullo, G. Albero, C. Neglia, C. De Canditiis, G. Bifulco, G. Campanile, B. GasparriniJournal of Veterinary Science, J Vet Sci. 2023 Mar;24(2):e24 "Antioxidant effect of ergothioneine on in vitro maturation of porcine oocytes" Ji-Young Jeong et al.

[0014] In the human fallopian tube, reactive oxygen species are generated during the process of fertilization and early division of the egg. Reactive oxygen species are known to adversely affect the structure and function of cells, potentially affecting the quality of human embryos, fertilization rates, miscarriage, and chromosomes. The fallopian tube has antioxidant properties that reduce the effects of reactive oxygen species, making it important to fully replicate the fallopian tube environment in human embryo culture.

[0015] None of the conventional culture media for human embryos adequately reproduces the environment inside the fallopian tube, which results in a low probability of developing early embryos or cleaving embryos into blastocysts. In particular, currently commercially available culture media, although containing the minimum nutrients necessary for blastocyst development, are susceptible to the effects of oxidative stress and are therefore not ideal for embryo growth and differentiation.

[0016] On the other hand, as mentioned above in Non-Patent Document 1, it has been shown that adding ergothioneine to the culture medium and culturing fertilized eggs reduces cell damage such as apoptosis and produces good blastocysts, but this document is based on bovine fertilized eggs, and the results do not directly apply to the culture of human embryos. However, by culturing with the addition of ergothioneine, it is possible that good culture results can also be obtained for human embryos.

[0017] In addition, the fallopian tubes where early human embryos develop are filled with fallopian tube fluid, which provides nutrients to the embryos. Fallopian tube fluid contains primarily lactic acid, pyruvate, and glucose, and is known to contain high concentrations of ergothioneine. This allows cell division to occur without genetic damage caused by reactive oxygen species in the cells. Therefore, adding ergothioneine to the culture medium for human embryos may be able to recreate the environment inside the fallopian tube.

[0018] Furthermore, as described in Non-Patent Document 2 above, it has been found that adding ergothioneine to a culture medium inhibits cell proliferation and promotes cell differentiation, and in this case, it is essential to consider the amount of ergothioneine to be added. In particular, from cell fertilization to blastocyst formation, it is necessary to suppress reactive oxygen species without inhibiting cell proliferation, so a low concentration of ergothioneine is sufficient. However, when cells germinate and implant, it is desirable to slightly increase the concentration of ergothioneine to promote cell differentiation and germination. However, clinically, implantation will not occur unless cells germinate, so the ergothioneine concentration at the time of germination is the most important factor.

[0019] Furthermore, taking into consideration Non-Patent Documents 1 and 2, the optimal ergothioneine concentration differs depending on the type of animal and organ, and from this point of view as well, it is necessary to find the optimal value.

[0020] The present invention focuses on the above points, and its object is to reduce the effects of reactive oxygen species and alleviate oxidative stress on human embryos. Another object is to contribute to reproducing the environment inside the fallopian tube. Yet another object is to determine the amount of ergothioneine to be added that is suitable for culturing human embryos. Yet another object is to improve the quality of fertilized eggs and the success rate of implantation.

[0021] The present invention is characterized by adding ergothioneine to a culture medium for culturing human embryos or thawed human embryos. The reducing action of ergothioneine reduces the effects of reactive oxygen species.

[0022] Another invention is characterized in that ergothioneine is added to a thawing solution used when thawing frozen human embryos. According to one main aspect, the thawing solution comprises a thawing solution 1 that is used first and a thawing solution 2 that is used subsequently, and the concentration of ergothioneine in thawing solution 1 is higher than the concentration of ergothioneine in thawing solution 2. By providing a relatively high concentration of ergothioneine in thawing solution 1, reactive oxygen species released from cryoprotectants such as DMSO can be rapidly reduced or removed immediately after thawing.

[0023] According to yet another invention, the amount of ergothioneine in the thawing solution when thawing frozen human embryos is higher than the amount of ergothioneine in the culture solution for the thawed human embryos. Due to the need to eliminate the effects of cryoprotectants and promote differentiation during thawing, it is advisable to add ergothioneine at a high concentration. The above and other objects, features, and advantages of the present invention will become apparent from the following detailed description and accompanying drawings.

[0024] According to the present invention, an appropriate amount of ergothioneine is added to the culture medium for human embryos or the thawing solution used to thaw frozen human embryos, thereby reducing the effects of reactive oxygen and mitigating oxidative stress on human embryos. In addition, the environment inside the fallopian tube is well reproduced, improving the quality of fertilized eggs and the success rate of implantation, making this method suitable for culturing human embryos.

[0025] 1 shows the development of a typical human embryo during culture. 2 shows the development when a human embryo is cultured using a conventional culture medium. 3 shows the development when a human embryo is cultured using a culture medium in an example of the present invention. 4 shows the development when a frozen human embryo is thawed and cultured using a conventional culture medium. 5 shows the development when a frozen human embryo is thawed and cultured using a culture medium in an example of the present invention. 6 shows details of the culture medium in Example 3 in which the present invention is applied to frozen human embryos. 7 shows the amount of ergothioneine added and the state of cell division in Example 3. 8 shows the amount of ergothioneine added and the state of cell division in Example 3. 9 shows the amount of ergothioneine added and the state of cell division in Example 3. 10 shows the amount of ergothioneine added and the state of cell division in Example 3. 11 shows the amount of ergothioneine added and the state of cell division in Example 3. 12 shows the amount of ergothioneine added and the state of cell division in Example 3. 13 shows the amount of ergothioneine added and the state of cell division in Example 3. 14 shows the amount of ergothioneine added and the state of cell division in Example 3. 15 shows the amount of ergothioneine added and the state of cell division in Example 3. 16 shows the amount of ergothioneine added and the state of cell division in Example 3. 17 shows the amount of ergothioneine added and the state of cell division in Example 3. 18 shows details of the culture medium in a reference example in which the above example is applied to mouse embryos. FIG. 1 shows the relationship between the amount of ergothioneine added and the state of cell division in the Reference Example. FIG. 2 shows the relationship between the amount of ergothioneine added and the state of cell division in the Reference Example. FIG. 3 shows the relationship between the amount of ergothioneine added and the state of cell division in the Reference Example. FIG. 4 shows the chemical structure of ergothioneine. FIG. 1 is cited from Non-Patent Document 1. FIG. 2 is cited from Non-Patent Document 1. FIG. 3 is cited from Non-Patent Document 1. FIG. 4 and FIG. 5 are cited from Non-Patent Document 1. FIG. 1 is cited from Non-Patent Document 2. FIG. 4A is cited from Non-Patent Document 2. FIG. 4B is cited from Non-Patent Document 2.

[0026] The best mode for carrying out the present invention will be described in detail below with reference to examples.

[0027] First, to facilitate understanding of the present invention, the development of a typical human embryo during culture will be described with reference to the micrograph in Figure 1. In Figure 1, (A) shows a normal fertilized egg. This divides as shown in Figure 1 (B) to (D), becoming four cells on the second day after fertilization and eight cells on the third day after fertilization. Next, as shown in Figure 1 (E) to (F), the embryo progresses from the precompaction stage, in which cells begin to cluster together, to the late compaction stage, and on the fourth day after fertilization, it becomes a morula stage embryo as shown in Figure 1 (G).

[0028] It then progresses through the early blastocyst stage shown in Figure (H), and becomes a blastocyst on the fifth day of fertilization as shown in Figure (I), and then an expanded blastocyst on the sixth day of fertilization as shown in Figure (J). It then progresses from the emerged blastocyst shown in Figure (K) to the fully emerged blastocyst shown in Figure (L). Figures (A) to (G) represent the early embryo stage, and Figures (H) to (L) represent the blastocyst stage.

[0029] Next, an example of culturing human embryos using a conventional culture medium with informed consent will be described with reference to Figure 2. In this example, ICSI was performed, and the culture medium used was "HiGROW OVIT Plus," a product name manufactured by Fuso Pharmaceutical Industries, Ltd. Its ingredients are Sodium Chloride, Potassium Chloride, Magnesium Sulfate, Calcium Chloride, Potassium Dihydrogen Phosphate, Glucose, Sodium Pyruvate, Sodium L-Lactate, Sodium Bicarbonate, L-Alanine, L-Asparagine, L-Aspartic Acid, L-Glutamic Acid, Glycine, L-Proline, L-Serine, L-Arginine, L-Cystine, L-Histidine, L-Isoleucine, L-Leucine, L-Lysine, L-Methionine, L-Phenylalanine, L-Threonine, L-Tryptophan, L-Tyrosine, L-Valine, Taurine, L-Alanyl-L-Glutamine, EDTA, Gentamicin Sulfate, HAS, pH (after equilibration) 7.3-7.5 (37℃, 6% The conditions were: CO2, osmolality 255-275 mOsm / kg; sterility test: no microbial growth; endotoxin test: less than 0.1 EU / mL; mouse embryo culture test: blastocyst development rate 80% or higher (1-cell embryos, cultured for 4 days). b. Culture was performed using a Vitrolife EmbryoScope+ Time-Lapse System incubator. Culture conditions were: temperature: 37.0°C, humidity: room humidity, 6% oxygen, 5% carbon dioxide, and 90% nitrogen. These are typical culture conditions. c. Microscopic photographs were taken using an Olympus IX71 inverted microscope and an Olympus CS230B camera.

[0030] Figures 2(A)-(F) show micrographs of the embryos as they progress from 2 cells to 4 cells to 8 cells to a morula and then to a blastocyst. Focusing on Figure 2(A) and comparing it with Figure 1(B), fragmentation occurs during cell division into two. As shown in Figures 2(B) and 2(C), fragmentation also progresses during cell division into four and eight. Therefore, when comparing the morula in Figure 2(D) and the blastocyst in Figure 2(E) with Figures 1(G)-(J), they cannot be considered to be good embryos.

[0031] In contrast, Figure 3 shows the culture state of embryos collected at the same time from the same woman, with informed consent, when 2 mg (milligrams) of ergothioneine was added to 100 ml (milliliters) of culture medium. The conditions were the same except for the addition of ergothioneine to the culture medium. a) The culture medium and incubator used were those shown in Figure 2 above. b) The ergothioneine used was "L-(+)-Ergothioneine," a product name manufactured by Nagara Science Co., Ltd.

[0032] The blood concentration of ergothioneine in humans is approximately 1 mg to 4 mg ("to" indicates "1 mg to 4 mg"; the same applies below), and adding too much can cause excessive reduction, so we chose about half that amount. Too much ergothioneine, on the other hand, can cause a stronger reduction, disrupting the balance of the redox system within cells and inhibiting cell division. However, when cultured in a culture medium containing added ergothioneine, as shown in Figure 3, most cells did not fragment and cell division proceeded smoothly in all stages. Therefore, implanting the blastocysts of this example into the uterus is likely to improve pregnancy rates. Compared to Figure 1, this result is favorable for the development of human embryos.

[0033] In contrast, similar cultures were performed on mouse embryos. Ergothioneine was added to the culture medium at varying concentrations (0 mg, 0.1 mg, 0.5 mg, 1.0 mg, 2.0 mg, 3.0 mg, 5.0 mg, 10.0 mg, 25.0 mg, 50.0 mg, 75.0 mg, and 100.0 mg per 100 ml of culture medium) and mixed. Mouse embryos were cultured in each concentration of culture medium and observed for cell proliferation over several days. Good results were obtained with ergothioneine additions ranging from 2.0 mg to 10.0 mg. Comparing these results with those for human embryos described above, it appears that the results for mouse embryos can generally be applied to human embryos as well.

[0034] Next, Example 2 of the present invention will be described with reference to Figures 4 and 5. This example illustrates the thawing and culturing of discarded frozen embryos from humans for which informed consent has been obtained. Human embryos are frozen by dehydration using DMSO, ethylene glycol, or the like, followed by rapid freezing. For example, they are cryopreserved using a cryoprotectant manufactured by ReproLife. The frozen embryos were thawed using the following thawing solutions (reconstitution solution or melting solution) 1 and thawing solution 2. The amounts of ergothioneine added per 100 ml of thawing solution were as follows: a. Thawing solution 1: 100 mg to 600 mg of ergothioneine (4.37 mMol to 26.20 mMol); b. Thawing solution 2: 10 mg to 300 mg of ergothioneine (0.437 mMol to 13.10 mMol). The culture medium and ergothioneine were the same as those used in the above-mentioned examples. As described above, by using a relatively high concentration of ergothioneine in Thawing Solution 1, it is possible to rapidly reduce or eliminate the reactive oxygen species released from cryoprotectants such as DMSO immediately after thawing, thereby reducing or eliminating the side effects of the cryoprotectant, which is thought to promote cell differentiation. In other words, cell damage can be reduced more effectively than when a high concentration of ergothioneine is added to the culture medium.

[0035] Figure 4 shows the results of thawing and culturing in a thawing solution without ergothioneine. Embryo (A-1) cultured for 5-6 hours results in (A-2), where the cells enlarge and proteins and genes are repairing themselves. If the repair is successful, pregnancy will occur. In contrast, embryo (B-1) cultured for 5-6 hours after thawing results in (B-2), which has darkened and become oxidized. In this example, pregnancy did not occur. Embryo (C-1) cultured for 5-6 hours after thawing results in (C-2), where cell breakdown has occurred and pregnancy did not occur.

[0036] On the other hand, when thawed and cultured in a thawing solution containing ergothioneine, the results are as shown in Figure 5 (A) to (F). All of these are examples of different embryos, with (A), (B), and (F) showing embryos that erupted in 6 hours, (C) showing embryos that erupted in 15 hours, (D) and (E) showing embryos that erupted in 18 hours, and (G) showing embryos before eruption. As shown in these examples, the cells enlarged and erupted without repairing proteins or genes, demonstrating the antioxidant effects and differentiation-promoting effectiveness of ergothioneine, even in the thawing solution.

[0037] Cryoprotectants for fertilized eggs include those manufactured by Kitazato Corporation (https: / / www.kitazato.co.jp / ) and ReproLife Co., Ltd. (https: / / reprolife.jp / ). Both cryoprotectants are widely used worldwide, and their core ingredient is DMSO (dimethyl sulfoxide). DMSO is an organic solvent, and while its toxicity is considered low among organic solvents, it is not completely negligible. Even the extremely low concentrations (e.g., 0.1 wt%) commonly used DMSO can adversely affect cell function. When used as a cryoprotectant, it is usually in the form of a 10% (v / v) DMSO solution, which is said to be almost completely gone upon thawing. However, its toxicity remains. While its cytotoxicity is counteracted by antioxidants such as trehalose, it still needs to be actively eliminated by entering the cells. Furthermore, for blastocyst germination, ergothioneine must be added at a relatively high concentration. Optimization is required to achieve germination while mitigating DMSO toxicity and cell proliferation. This embodiment focuses on this point.

[0038] Next, Example 3 of the present invention will be described with reference to Figures 6 to 13. In this example, discarded frozen embryos from humans who had given informed consent were thawed and cultured in a culture medium supplemented with ergothioneine. Figure 6 shows the ergothioneine concentration in the culture medium used in the experiment. Figure 6 (A) shows the components of the stock solution; 10 mg of ergothioneine was added to 1 ml of purified water was used for the study. Figure 6 (B) shows the relationship between the ergothioneine concentration, the volume of stock solution, and the volume of culture medium in thawed human embryo samples 1 to 14. The corresponding relationship between the ergothioneine concentration in samples 1 to 14 and the wells of the embryoscope is also shown. The culture medium and ergothioneine were the same as those used in the above-mentioned examples.

[0039] The results of culturing human embryos at various ergothioneine concentrations are shown in Figures 7 to 13. From these, it can be seen that, per 100 ml of culture medium, a. 1 mg to 100 mg of ergothioneine: Cell division occurs normally (see Figures 7 to 10). b. 200 mg to 300 mg of ergothioneine: Ergothioneine counteracts the side effects of DMSO and promotes differentiation, resulting in very active cell division (see Figure 11). c. 500 mg of ergothioneine: Cell proliferation is inhibited (see Figure 12). d. 700 mg of ergothioneine: Cells gradually shrink and degenerate to a black color (see Figure 13). e. 1000 mg of ergothioneine: Cells shrink from the beginning of cell division, degenerate to a black color, and die (see Figure 13).

[0040] These results suggest that: a) generally good results were obtained when the amount of ergothioneine added was less than 700 mg per 100 ml of culture medium. b) When focusing on the occurrence of active cell division, an optimal concentration of 100 mg to 600 mg per 100 ml of culture medium is used. c) The ergothioneine concentration that resulted in the development of released blastocysts was 200 mg to 300 mg per 100 ml of culture medium. Because cryoprotectants generate large amounts of reactive oxygen, it is thought that adding such a large amount of ergothioneine produced better results. d) When focusing on promoting cell differentiation, an ergothioneine concentration of 100 mg to 200 mg / 100 ml (4.36 mMol to 8.74 mMol) prevents cell damage caused by DMSO and other agents during thawing and results in a good pregnancy rate.

[0041] <Reference Example> Next, a reference example in which the present invention was applied to the culture of mouse embryos will be described with reference to Figures 14 to 17. Figure 14 shows the amount of ergothioneine added to the culture medium used in the experiment. Because mouse embryos are young fertilized embryos, it is thought that changes in ergothioneine concentration are unlikely to result in differences in cell growth. Therefore, in this example, 25 μM hydrogen peroxide (H2O2) was added to the culture medium, and similar culture was performed. The ergothioneine, hydrogen peroxide, and culture medium in this example are as shown in Figure 14.

[0042] As a result, as shown in Figures 15 to 17, per 100 ml of culture medium: a. Ergothioneine 5 mg to 10 mg: healthy blastocysts with good germination; b. Ergothioneine 25 mg to 200 mg: impaired cell proliferation; c. Ergothioneine 300 mg to 700 mg: cell division is not possible, and as the ergothioneine concentration increases, cells die.

[0043] Based on the above, we conclude that: a) when thawing frozen embryos, adding 200 mg to 300 mg of ergothioneine per 100 ml of culture medium (≒ 8 mM to 15 mM) to the culture medium after thawing can improve the development of frozen embryos in order to prevent the effects of oxidizing substances such as DMSO. b) The optimum concentration of ergothioneine in the culture medium is thought to be 1 mg to 20 mg per 100 ml of culture medium (≒ 0.04 mM to 0.9 mM). Thus, the results of mouse embryo culture also indicate that there is an optimum amount of ergothioneine to be added to the culture medium. Furthermore, the favorable range of ergothioneine concentrations in mouse embryos in this example largely overlaps with that in human embryos, confirming the validity of the results of the ergothioneine concentrations in human embryos described above.

[0044] <Other Examples> The present invention includes numerous examples, and various modifications can be made based on the above disclosure. Examples include the following: (1) Various methods for producing ergothioneine are known, but the method disclosed in JP 2022-28808 A is preferred. (2) The above examples illustrate the addition of ergothioneine to the embryo culture medium and cryoprotectant. However, this does not preclude the addition of ergothioneine to any of the processes involved in artificial insemination, oocyte collection, sperm collection, insemination (intracytoplasmic sperm injection), culture, embryo transfer, implantation, and frozen embryo transfer. (3) For elderly women, the concentration of ergothioneine in the culture medium may be slightly increased due to the possibility of decreased antioxidant capacity. Similarly, the concentration of ergothioneine in the thawing solution for thawing frozen embryos may also be slightly increased. Furthermore, since mushrooms and fermented foods are rich in natural ergothioneine, increasing the ergothioneine concentration is an option for those who do not consume these foods. (4) As mentioned above, the addition of ergothioneine reduces reactive oxygen species in embryos, enabling normal cell division and reducing miscarriage and chromosomal abnormalities. Furthermore, it significantly reduces cell damage caused by cryoprotectants, preventing compensatory nuclear and cytoplasmic enlargement, allowing cells to maintain their original size and promoting cell differentiation, thereby preventing a decline in the rate of cell proliferation. Therefore, low concentrations of ergothioneine are applicable to difficult-to-freeze cells such as germ cells, embryonic stem cells (ES cells), and induced pluripotent stem cells (iPS cells). (5) In the above examples, the culture medium used was "HiGROW OVIT Plus," a product of Fuso Pharmaceutical Industries, Ltd., and the ergothioneine used was "L-(+)-Ergothioneine," a product of Nagara Science Co., Ltd., but these are merely examples and are not intended to be limiting. The same applies to cryoprotectants, etc. (6) This does not preclude the use of the ergothioneine-added culture medium of the present invention in the culture of embryos from animals other than humans.

[0045] According to the present invention, an appropriate amount of ergothioneine is added to the culture medium or thawing solution for human embryos, thereby reducing the effects of reactive oxygen and mitigating oxidative stress on human embryos. In addition, the environment inside the fallopian tube is well reproduced, improving the quality of fertilized eggs and the success rate of implantation, making the present invention suitable for human embryo culture and even infertility treatment.

Claims

1. A culture medium for human embryos, characterized in that ergothioneine is added to the culture medium for culturing human embryos or frozen human embryos.

2. A culture medium for human embryos according to claim 1, characterized in that the concentration of ergothioneine in the culture medium is set to a concentration equivalent to the concentration of ergothioneine in the human fallopian tube.

3. A thawing solution for frozen human embryos, characterized in that ergothioneine is added to the thawing solution used when thawing frozen human embryos.

4. A thawing solution for frozen human embryos as described in claim 3, characterized in that the thawing solution contains thawing solution 1 to be used first and thawing solution 2 to be used thereafter, and the concentration of ergothioneine in thawing solution 1 is higher than the concentration of ergothioneine in thawing solution 2.

5. A solution for thawing frozen human embryos according to claim 4, characterized in that the amount of ergothioneine added per 100 ml of the solution is 100 mg to 600 mg (4.37 mMol to 26.20 mMol) for the solution 1 and 10 mg to 300 mg (0.437 mMol to 13.10 mMol) for the solution 2.

6. A culture medium for thawed human embryos for culturing after thawing, characterized in that less than 700 mg of ergothioneine is added per 100 ml of culture medium.

7. The culture medium for thawed human embryos according to claim 6, characterized in that the amount of ergothioneine added is 100 mg to 600 mg per 100 ml of culture medium.

8. The culture medium for thawed human embryos according to claim 6, characterized in that the amount of ergothioneine added is 200 mg to 300 mg per 100 ml of culture medium.

9. The culture medium for thawed human embryos according to claim 6, characterized in that the amount of ergothioneine added is 100 mg to 200 mg per 100 ml of culture medium.

10. A method for culturing human embryos, characterized by using the culture medium according to claim 1 or 2.

11. A method for thawing a frozen human embryo, characterized by using the thawing solution of any one of claims 3 to 5.

12. A method for culturing thawed human embryos, characterized by using the culture medium of any one of claims 6 to 9.

13. A method for culturing frozen human embryos, comprising thawing frozen human embryos using a thawing solution according to any one of claims 3 to 5, and culturing the thawed human embryos in a culture medium according to any one of claims 6 to 9.

14. A method for culturing frozen human embryos according to claim 13, characterized in that the amount of ergothioneine added to the thawing solution for thawing the frozen human embryos is greater than the amount of ergothioneine added to the culture solution for the thawed human embryos.

15. A human embryo cultured in the culture medium according to claim 1 or 2.

16. A human embryo thawed using the thawing solution of any one of claims 3 to 5.

17. A human embryo cultured in the culture medium of any one of claims 6 to 9.

18. A human embryo thawed using a thawing solution according to any one of claims 3 to 5, and then cultured in a culture medium according to any one of claims 6 to 9.

19. The human embryo according to claim 18, characterized in that the amount of ergothioneine added to the thawing solution for thawing the frozen human embryo is greater than the amount of ergothioneine added to the culture solution for the thawed human embryo.

20. A method for treating infertility, comprising transplanting the human embryo according to any one of claims 15 to 19 into the uterus.

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