Embryo transfer culture medium, composition for coloring embryo culture medium

Abstract

To provide a culture medium for embryo transplantation for improving visibility of the culture medium for embryo transplantation at a transplantation site and lowering a difficulty level of embryo transplantation.SOLUTION: Provided is a culture medium for embryo transplantation which contains a coloring agent, where a color tone is any cold color-based color of blue-based color, green-based color, purple-based color or grey-based color. It is preferable that the cold color-based color have less than 80 of R component value in an RGB color model, and the coloring agent be at least one selected from the group consisting of Fast green FCF, Evans blue, brilliant blue FCF, Patent blue, Naphthol green B and Indigo carmine, and salts, hydrates, or derivatives thereof.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a culture medium for embryo transfer and a composition for coloring an embryo culture medium. [Background technology]

[0002] Embryo transfer is a reproductive technique in which embryos (fertilized eggs) collected from female animals, embryos produced by in vitro fertilization, genetically engineered embryos, or embryos thawed from cryopreservation are transferred into female individuals. Embryo transfer is widely used as an artificial reproductive method in fields such as livestock farming, as it is useful for utilizing eggs from superior females, preserving specific breeds and lines, improving the efficiency of breeding, and improving productivity. Embryo transfer in humans has also become an important technology applied in the medical field to treat infertility.

[0003] The general steps from in vitro fertilization to embryo transfer are the induced ovulation of donor females, oocyte retrieval, male gamete collection and preparation, oocyte and male gamete fertilization, zygote and embryo culture, selection of cultured embryos, and transfer of the selected embryos into the uterine cavity of a female individual. After embryo transfer, the female individual will undergo key events leading to embryo implantation, pregnancy, and birth.

[0004] As a medium for culturing animal embryos, for example, Patent Document 1 discloses an embryo culture medium supplemented with acetylcarnitine and lipoic acid to improve embryonic development. Patent Document 2 discloses a culture medium that maintains antibacterial activity by combining a neutral aqueous solution containing lactic acid and a pH indicator with an acidic aqueous solution containing pyruvic acid and an antibiotic. Patent Document 3 discloses a culture medium containing a fluorescent dye sensitive to reactive oxygen species that enables sex determination of mammalian embryos and detection of regressed embryos. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2018-525008 [Patent Document 2] Japanese Patent Application Laid-Open No. 2000-253871 [Patent Document 3] Japanese Patent Application Laid-Open No. 2007-068415 Summary of the Invention [Problem to be solved by the invention]

[0006] Conventional culture media are also used during embryo transfer, and their color is pale red or colorless. Furthermore, because the uterine tissue and fallopian tube tissue into which the embryos are transferred are also pale red, the transplantation culture medium containing the embryos and the transplantation site are similar in color, making the embryo transfer culture medium difficult to see. Therefore, when an ultrafine glass capillary that has absorbed the embryos in the transplantation culture medium is inserted into the uterus or fallopian tube and the embryos are discharged together with the transplantation culture medium, it is difficult to accurately visually confirm whether the embryo transfer culture medium has leaked out of the uterus or fallopian tube. Furthermore, because it is difficult to distinguish the transplantation culture medium containing the embryos from the transplantation site, there is a problem in that only experienced individuals who can recognize these subtle color differences can perform embryo transfers. For these reasons, there is a need for the development of an embryo transfer culture medium that has a color different from that of uterine tissue and fallopian tube tissue in order to reduce the technical difficulty of embryo transfer.

[0007] Therefore, an object of the present invention is to provide an embryo transfer culture medium that improves the visibility of the embryo transfer culture medium at the transfer site and reduces the difficulty of the embryo transfer technique. [Means for solving the problem]

[0008] As a result of extensive research into the above-mentioned problems, the inventors discovered that the visibility of the embryo transfer culture medium at the transfer site can be improved by adding a pigment that changes the color tone of the embryo transfer culture medium to a cooler color, and thus completed the present invention.

[0009] That is, the present invention provides the following [1] to [8]. [1] A culture medium for embryo transfer, characterized by containing a pigment and having a cool color tone of any of blue, green, purple, or gray. [2] The culture medium for embryo transfer according to [1] above, wherein the cool colors have an R component value of less than 80 in the RGB color model. [3] The culture medium for embryo transfer described in [2] above, characterized in that the cool colors have an R component value of less than 80 in the RGB color model, a sum of the G and B component values ​​of 100 or more and 360 or less, and the G component value or the B component value is greater than the R component value. [4] The culture medium for embryo transfer described in [1] above, characterized in that the cool colors have an R component value of 80 or more and 150 or less, a sum of G and B component values ​​of 100 or more and 360 or less in the RGB color model, and the G component value or the B component value is greater than the R component value. [5] The culture medium for embryo transfer described in [1] above, characterized in that the cool colors have a ratio of the G component value or the B component value to the R component value of 0.9 or more in the RGB color model, and the G component value or the B component value is 50 or more and 200 or less. [6] The culture medium for embryo transfer described in [5] above, characterized in that the cool colors have a sum of the G and B component values ​​of 100 or more and 360 or less in the RGB color model. [7] The culture medium for embryo transfer according to any one of [1] to [6] above, characterized in that the dye is at least one selected from the group consisting of Fast Green FCF, Evans Blue, Brilliant Blue FCF, Patent Blue, Naphthol Green B, and indigo carmine, as well as salts, hydrates, or derivatives thereof. [8] A culture medium for embryo transfer, characterized by containing at least one dye selected from the group consisting of Fast Green FCF, Evans Blue, Brilliant Blue FCF, Patent Blue, Naphthol Green B, and indigo carmine, and derivatives thereof. [9] A composition for coloring an embryo culture medium, comprising at least one dye selected from the group consisting of Fast Green FCF, Evans Blue, Brilliant Blue FCF, Patent Blue, Naphthol Green B, and indigo carmine, and derivatives thereof. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a culture medium for embryo transfer that improves the visibility of the embryo transfer site. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 shows the cytotoxicity of 23 types of dyes to embryos in Example 2. The horizontal axis shows the abbreviations of the dyes, which correspond to the dyes in Table 1 below. PBS is phosphate-buffered saline, and DMSO is dimethyl sulfoxide. [Figure 2] FIG. 2 shows a photograph of six types of dyes selected in Example 2 dropped onto a petri dish. [Figure 3] FIG. 3 is a diagram showing the RGB values ​​of the six pigments selected in Example 2 and the color tone of the uterus. [Figure 4] FIG. 4 shows the cytotoxicity when two types of dyes are combined in Example 2. [Figure 5] FIG. 5 shows the cytotoxicity at different dye concentrations in Example 2. [Figure 6] FIG. 6 shows the genotoxicity of the embryo transfer culture medium of the present invention to embryos. [Figure 7] FIG. 7 shows the results of gene expression analysis to examine the influence of the culture medium for embryo transfer of the present invention on embryo development. [Figure 8A] FIG. 8A shows the results of examining the effect of the embryo transfer culture medium of the present invention (containing Brilliant Blue FCF as a dye) on the implantation of a fertilized egg into the uterus after embryo transfer. [Figure 8B]FIG. 8B shows the results of examining the effect of the embryo transfer culture medium of the present invention (containing Fast Green FCF as a dye) on the implantation of a fertilized egg into the uterus after embryo transfer. [Figure 8C] FIG. 8C shows the results of examining the effect of the embryo transfer culture medium of the present invention (containing Evans blue as a dye) on the implantation of a fertilized egg into the uterus after embryo transfer. [Figure 8D] FIG. 8D shows the results of examining the effect of the embryo transfer culture medium of the present invention (containing naphthol green B as a dye) on the implantation of a fertilized egg into the uterus after embryo transfer. [Figure 8E] FIG. 8E shows the results of examining the effect of the embryo transfer culture medium of the present invention (containing patent blue as a dye) on the implantation of a fertilized egg into the uterus after embryo transfer. [Figure 9] FIG. 9 shows the effect of the culture medium for embryo transfer of the present invention on the growth of mice born by embryo transfer. [Figure 10] FIG. 10 shows the effect of the culture medium for embryo transfer of the present invention on the fertility of mice born by embryo transfer. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, embodiments of the embryo transfer culture medium according to the present invention (hereinafter also referred to as the "culture medium of the present invention") and the composition for coloring the embryo culture medium according to the present invention (hereinafter also referred to as the "composition of the present invention") will be described in detail. The culture medium of the present invention and the composition of the present invention described in the embodiments are merely examples for the purpose of explaining the present invention, and the present invention is not limited thereto.

[0013] [Definition] As used herein, the term "embryo" refers to an individual mammal in the early stages of development. The term "embryo" also encompasses embryos (fertilized eggs) collected from female animals, embryos produced by in vitro fertilization, genetically engineered embryos, and embryos thawed from cryopreservation. Examples of mammals include humans, mice, rats, guinea pigs, monkeys, dogs, cows, horses, pigs, sheep, goats, rabbits, cats, and hamsters. As used herein, "culture medium" refers to a medium used for handling, collecting, and manipulating gametes and embryos, but is not limited to a medium for culture. In this specification, the terms "RGB color model" and "RGB" refer to a method of expressing color in which the three elements of red component (R component), green component (G component), and blue component (B component) are numerically represented in 256 levels.

[0014] As used herein, "cool colors" refers to blue, green, purple, or gray colors. Examples of blue colors include blue, cyan, ultramarine blue, navy blue, and indigo. Examples of green colors include green, viridian, verdigris, hunter green, and moss green. Examples of purple colors include purple, wistaria, heliotrope, violet, and pansy. Examples of gray colors include gray, fog blue, ash gray, dove gray, and steel gray.

[0015] As used herein, "warm colors" refers to red, yellow, or orange colors. Examples of red colors include red, pink, cyclamen pink, vermilion, and cherry pink. Examples of yellow colors include yellow, cadmium yellow, cream yellow, lemon yellow, and sunflower. Examples of orange colors include orange, mandarin orange, topaz, Indian red, and salmon pink.

[0016] [Embryotransplantation culture medium] The culture medium of the present invention is intended for use in embryo transfer, and is characterized by containing a pigment and having a cool color tone of any of blue, green, purple, and gray. This makes it possible to provide an embryo transfer culture medium that improves the visibility of the embryo transfer culture medium at the embryo transfer site. The culture medium of the present invention may also be used to culture embryos for use in embryo transfer. Therefore, embryos may be cultured in the culture medium of the present invention, and the culture medium of the present invention containing the cultured embryos may be used directly for embryo transfer. First, each component constituting the culture medium of the present invention will be described in detail. Note that the content of each component refers to the content in the culture medium unless otherwise specified.

[0017] (dye) The pigment is a substance used to color the culture medium primarily in cool colors. There are no particular limitations on the pigment, as long as it colors the culture medium in colors other than warm colors. Examples of pigments include natural pigments such as mineral pigments, plant pigments, and animal pigments, and synthetic pigments such as organic pigments and organic pigments.

[0018] Specific examples of dyes include Fast Green FCF (Green No. 3), Brilliant Blue FCF (Blue No. 1), Evans Blue (Direct Blue 53), Patent Blue, Naphthol Green B, Indigo Carmine (Blue No. 2), and salts, hydrates, or derivatives thereof. Preferred dyes are Fast Green FCF and Brilliant Blue FCF from the viewpoints of visibility and safety at the embryo transfer site. The salts may be any pharmacologically acceptable salt, such as inorganic salts of sodium, potassium, calcium, magnesium, etc.; organic bases such as trimethylamine and ethanolamine; and inorganic acids such as hydrochloric acid and phosphoric acid. Examples of the derivatives include alcohol derivatives and ether derivatives. These dyes may be used alone or in combination of two or more. Specific examples of combinations of two dyes include Fast Green FCF and Brilliant Blue FCF, Fast Green FCF and Evans Blue, Fast Green FCF and Patent Blue, Fast Green FCF and Naphthol Green B, Fast Green FCF and Indigo Carmine, Brilliant Blue FCF and Evans Blue, Brilliant Blue FCF and Patent Blue, Brilliant Blue FCF and Naphthol Green B, Brilliant Blue FCF and Indigo Carmine, Evans Blue and Patent Blue, Evans Blue and Naphthol Green B, Evans Blue and Indigo Carmine, Patent Blue and Naphthol Green B, Patent Blue and Indigo Carmine, and Naphthol Green B and Indigo Carmine.

[0019] The content of the dye in the embryo transfer culture medium is not particularly limited as long as it provides the desired visibility of the embryo transfer culture medium. The content of the dye can be adjusted appropriately depending on the dye used, and for example, the content of the dye relative to the total embryo transfer culture medium can be 0.0003 to 0.05% by mass. When Fast Green FCF is used as the dye, the content can be 0.00125 to 0.05% by mass, preferably 0.0025 to 0.04% by mass, and more preferably 0.005 to 0.02% by mass. When Brilliant Blue FCF is used as the dye, the content can be 0.00125 to 0.05% by mass, preferably 0.025 to 0.04% by mass, and more preferably 0.005 to 0.02% by mass. When Evans Blue is used as the dye, the content is 0.00125 to 0.0075% by mass, preferably 0.0025 to 0.006% by mass, and more preferably 0.003 to 0.0055% by mass. When Patent Blue is used as the dye, the content is 0.0003 to 0.002% by mass, preferably 0.0006 to 0.0015% by mass, and more preferably 0.0008 to 0.0013% by mass. When Naphthol Green B is used as the dye, the content is 0.01 to 0.12% by mass, preferably 0.02 to 0.08% by mass, and more preferably 0.03 to 0.06% by mass. When Indigo Carmine is used as the dye, the content is 0.0025 to 0.03% by mass, preferably 0.005 to 0.02% by mass, and more preferably 0.007 to 0.015% by mass. By setting the content of the pigment within the above range, it is possible to improve the visibility of the embryo transfer culture medium at the transfer site.

[0020] (color tone) The color tone of the embryo transfer culture medium is not particularly limited as long as it is a color other than the warm colors of the uterine tissue and fallopian tube tissue into which the embryo is transferred. Examples of the color tone of the embryo transfer culture medium include cool colors such as blue, green, purple, and gray. Methods for confirming the color tone include, for example, image analysis of the embryo transfer culture medium, visual inspection by a person skilled in the art, and absorbance. From the viewpoint of accuracy, image analysis is a preferred method for confirming the color tone.

[0021] Image analysis of the embryo transfer culture medium is not particularly limited. A specific example of image analysis is the RGB color model. The RGB color model can express color by quantifying three elements: red component (R component), green component (G component), and blue component (B component). The R component value, G component value, and B component value are not particularly limited as long as they improve the visibility of the embryo transfer culture medium. RGB values ​​can be obtained using general image processing software such as Photoshop (registered trademark).

[0022] As for RGB values, for example, the R component value can be less than 80, more preferably less than 70, even more preferably less than 40, and particularly preferably less than 20. When the R component value is less than 80, the total value of the G and B component values ​​is 100 to 360, and the G or B component value is greater than the R component value. The upper limit of the total value of the G and B component values ​​is more preferably 300 or less, even more preferably 250 or less, and particularly preferably 200 or less. On the other hand, the lower limit of the total value of the G and B component values ​​is more preferably 100 or more, even more preferably 130 or more, and particularly preferably 140 or more. By setting the RGB values ​​within the above ranges, the visibility of the embryo transfer culture medium at the transfer site can be improved.

[0023] Furthermore, as for the RGB values, for example, when the R component value is 80 or more and 150 or less, the sum of the G component value and the B component value is 150 or more and 360 or less, and the G component value or the B component value is a value greater than the R component value. In this case, the upper limit of the R component value is more preferably 140 or less, even more preferably 120 or less, and particularly preferably 110 or less. The upper limit of the sum of the G component value and the B component value is more preferably 350 or less, even more preferably 330 or less, and particularly preferably 300 or less. On the other hand, the lower limit of the sum of the G component value and the B component value is more preferably 180 or more, even more preferably 200 or more, and particularly preferably 230 or more.

[0024] Furthermore, as for the RGB values, for example, the G or B component value is 0.9 times or more relative to the R component value, and the G or B component value is 50 to 200. In this case, the G or B component value is preferably 1.2 times or more relative to the R component value, more preferably 1.5 times or more, and even more preferably 2 times or more. In this case, the upper limit of the G or B component value is more preferably 180 or less, even more preferably 160 or less, and particularly preferably 150 or less. Meanwhile, the lower limit of the G or B component value is more preferably 70 or more, even more preferably 80 or more, and particularly preferably 100 or more.

[0025] Additionally, when the G or B component value is 0.9 times or more the R component value and the G or B component value is 50 to 200, the sum of the G and B component values ​​is preferably 100 to 360. In this case, the upper limit of the sum of the G and B component values ​​is more preferably 350 or less, even more preferably 330 or less, and particularly preferably 300 or less. On the other hand, the lower limit of the sum of the G and B component values ​​is more preferably 120 or more, even more preferably 150 or more, and particularly preferably 180 or more.

[0026] By setting the RGB values ​​within the above ranges, the visibility of the embryo transfer culture medium at the transfer site can be improved.

[0027] The quantification of color tones through image analysis may use color space models other than the RGB color space. Examples of color spaces include the HLS color space, the YCC color space, the HSV color space, the L*a*b color space, and the CMYK color space. The number of color components may be two, three, four, or more.

[0028] (basal medium) The basal medium in the culture medium for embryo transfer of the present invention is not particularly limited as long as it is suitable for the embryo or fertilized egg to be cultured. Specific examples of basal media include M16 medium, BMOC2 medium, MTF medium, CZB medium, KSMO medium, mSOF medium, G1 medium, G2 medium, M2 medium, P1 medium, TYH medium, HTF medium, Earle medium, Whitten medium, Peter & Hoppe medium, Ham's F-10 medium, Ham's F-12 medium, Ham's F-12K medium, Global medium, G-MOPS medium, EmbryoGlue medium, Continuous Single Culture medium, AIM V medium, HFDM-1 medium, M-199 medium, L-15 medium, McCoy's 5A medium, MCDB105 medium, MCDB107 medium, MCDB131 medium, MCDB153 medium, MCDB201 medium, NCTC109 medium, NCTC135 medium, Waymouth's Examples of basal media include MB752 / 1 medium, CMRL-1066 medium, Williams' medium E, Brinster's BMOC-3 medium, E8 medium, RPMI medium 1640, Dulbecco's phosphate-buffered saline (D-PBS), Hank's balanced salt solution (HBSS), DMEM (Dulbecco's Modified Eagle Medium), EMEM (Eagle's Minimum Essential Medium), α-MEM (Minimum Essential Medium alpha Modification), IMDM (Iscove's Modified Dulbecco's Medium), and GMEM (Glasgow's MEM). These basal media may be used alone or in combination of two or more. Furthermore, the basal media may contain additional, removed, increased, or decreased medium components depending on the type and condition of the embryo or fertilized egg. These basal media may be selected from known media as appropriate for the embryo or fertilized egg to be cultured.

[0029] (Culture components) The embryo transfer culture medium of the present invention may contain medium components necessary for culturing embryos or fertilized eggs in the above-mentioned basal medium. The culture components are not particularly limited as long as they are suitable for the embryos or fertilized eggs to be cultured. Examples of medium components include sugars, amino acids, proteins, growth factors, serum, vitamins, antioxidants, antibiotics, buffer solutions, inorganic salts, trace metals, and other additives. Furthermore, the medium may or may not contain a pH indicator such as phenol red. These medium components may be blended alone or in combination of two or more. These medium components may be appropriately selected from known components depending on the embryo or fertilized egg to be cultured.

[0030] Specific examples of sugars include monosaccharides such as glucose, fructose, mannose, and galactose; disaccharides such as sucrose, sucralose, trehalose, maltose, and lactose; trisaccharides such as glucosylsucrose, lactosucrose, and raffinose; tetrasaccharides such as acarbose and maltotetraose; and polysaccharides such as dextran and cyclodextrin.

[0031] Specific examples of amino acids include L-glutamic acid, L-glutamine, L-arginine, L-cystine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-alanine, L-asparagine, L-aspartic acid, L-cysteine, L-hydroxyproline, and taurine.

[0032] Specific examples of proteins include albumin, human serum albumin (HSA), recombinant human albumin, bovine serum albumin (BSA), α-globulin, β-globulin, and proteoglycan.

[0033] Specific examples of growth factors include insulin, insulin-like growth factor (IGF), epidermal growth factor (EGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), leukemia inhibitory factor (LIF), platelet-activating factor (PAF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), transforming growth factor (TGF), and basic fibroblast growth factor (bFGF).

[0034] Specific examples of serum include human serum, human umbilical cord serum (HCS), bovine serum, fetal calf serum (FCS), and horse serum.

[0035] Specific examples of vitamins include vitamin A group such as retinol, retinal, and retinoic acid, vitamin B group such as thiamine (vitamin B1), riboflavin (vitamin B2), vitamin B6, pyridoxine, vitamin B12, niacin, pantothenic acid, folic acid, and biotin, vitamin C group such as ascorbic acid, vitamin E group such as tocopherol, and choline.

[0036] Specific examples of antioxidants include glutathione, N-acetylcysteine, cysteine, ascorbic acid, ascorbic acid derivatives, sodium citrate, α-tocopherol, uric acid, pyruvic acid, glucose, albumin, and ferritin.

[0037] Specific examples of antibiotics include penicillin, streptomycin, and gentamicin.

[0038] Specific examples of the buffer solution include HEPES buffer, MOPS buffer, phosphate buffered saline (PBS), bicarbonate buffer, and the like.

[0039] Specific examples of inorganic salts include sodium chloride, sodium hydroxide, sodium sulfate, sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium carbonate, sodium bicarbonate, potassium chloride, potassium hydroxide, potassium sulfate, potassium phosphate, dipotassium hydrogen phosphate, potassium carbonate, potassium bicarbonate, calcium chloride, calcium sulfate, calcium nitrate, calcium phosphate, calcium carbonate, magnesium chloride, magnesium sulfate, magnesium nitrate, magnesium phosphate, and magnesium carbonate.

[0040] Specific examples of trace metals include iron sulfate, iron nitrate, copper sulfate, copper nitrate, and zinc sulfate.

[0041] Specific examples of other additives include organic acids such as lactic acid, propionic acid, citric acid, and pulvinic acid, lipids such as cholesterol, fatty acids such as linolenic acid, amines such as ethanolamine and putrescine, reducing agents such as mercaptoethanol and 3-mercapto-1,2-propanediol, thickeners such as sodium alginate, polyvinylpyrrolidone, carboxymethylcellulose, and pullulan, chelating agents such as EDTA, and pH indicators such as phenol red.

[0042] Due to the above-mentioned features, the culture medium of the present invention is colored in cool colors by incorporating a pigment, thereby improving the visibility of the culture medium for embryo transfer at the transfer site, thereby enabling not only experienced practitioners but also less experienced practitioners to perform embryo transfer appropriately.

[0043] [Composition for coloring embryo culture medium] The composition of the present invention is for coloring an embryo culture medium, which contains a pigment, thereby improving the visibility of the embryo transfer culture medium at the transfer site. First, each component constituting the composition of the present invention will be described in detail. Note that the content of each component refers to the content in the composition unless otherwise specified.

[0044] (dye) The dye is not particularly limited as long as it colors the embryo culture medium a cool color such as blue, green, purple, or gray. Specific examples of dyes include Fast Green FCF (Green No. 3), Brilliant Blue FCF (Blue No. 1), Evans Blue (Direct Blue 53), Patent Blue, Naphthol Green B, Indigo Carmine (Blue No. 2), and salts, hydrates, or derivatives thereof. Preferred dyes are Fast Green FCF and Brilliant Blue FCF from the viewpoints of safety and visibility of the embryo transfer culture medium at the embryo transfer site. These dyes may be used alone or in combination of two or more or three or more.

[0045] The content of the pigment in the composition of the present invention is not particularly limited as long as it provides the embryo culture medium with the desired visibility of the embryo transfer culture medium, and the content of the pigment can be adjusted to the same content as the pigment in the above-mentioned embryo transfer culture medium.

[0046] (composition) The composition of the composition of the present invention is not particularly limited as long as it does not interfere with the culture of embryos or fertilized eggs. For example, it may be purified water, physiological saline, or any other composition that satisfies the description of the above section [Embryotransplantation Culture Medium] (Basal Medium) or (Culture Medium Components)].

[0047] (How to use) The compositions of the present invention are intended to be added to embryo culture media. The amount of the composition of the present invention to be added to an embryo culture medium is not particularly limited as long as it provides the desired visibility of the embryo transfer culture medium. The amount of the composition of the present invention to be added can be such that the pigment content in the embryo transfer culture medium is as described above. By adding the composition of the present invention in the above amount, the visibility of the embryo transfer culture medium at the transfer site can be improved.

[0048] The color tone of the embryo culture medium to which the composition of the present invention has been added is not particularly limited as long as it exhibits the desired visibility of the embryo transfer culture medium, and may, for example, satisfy the description in the above section [Embryo transfer culture medium] (Color tone).

[0049] Due to the above-mentioned characteristics, the composition of the present invention can color the embryo culture medium containing the embryo in a cool color, thereby improving the visibility of the embryo transfer culture medium at the transfer site, thereby enabling not only experienced practitioners but also less experienced practitioners to properly perform embryo transfer. [Example]

[0050] [Example 1] Selection of dyes It is important that the color of the culture medium for embryo transfer be easily distinguishable from the color of the organ, so we first selected a dye.

[0051] Sixty commercially available dyes were dissolved in phosphate-buffered saline (PBS) to prepare 100x solutions. These solutions were then added to M16 culture medium (Sigma-Aldrich) to achieve three concentrations: 1x, 1 / 2x, and 1 / 4x. M16 medium alone without dye was used as a control. Next, 20 μL drops of each M16 medium were placed on a Petri dish and overlaid with liquid paraffin (Nacalai Tesque). Hereinafter, the concentration (% by mass) listed in the "In Use (1x) Concentration" column in Table 1 below will be referred to as the "1x concentration." Concentrations obtained by diluting the 1x concentration by 2x and 4x will be referred to as the "1 / 2x concentration" and "1 / 4x concentration," respectively. Concentrations obtained by diluting the 1x concentration by 2x and 4x will be referred to as the "2x concentration" and "4x concentration," respectively.

[0052] The color of each drop was checked with the naked eye, and 23 types of pigments that were thought to be easily distinguishable from the color of the organs were selected.

[0053] [Example 2] Narrowing down of dyes It is important that the components of the embryo transfer culture medium do not affect embryo development. Therefore, the cytotoxicity of the 23 dyes selected in Example 1 was evaluated.

[0054] The 23 dyes selected in Example 1 were added to 10% FBS-supplemented EMEM (Fujifilm Wako Pure Chemical Industries, Ltd.) at the concentrations listed in the "In Use (1x) Concentration" column in Table 1 below, and cytotoxicity was evaluated according to the colony formation inhibition test (International Standard ISO 10993-5). As a control, PBS alone or DMSO alone was added to 10% FBS-supplemented EMEM, and a similar evaluation was performed. Briefly, the colony formation inhibition test was performed on a 24-well plate seeded with approximately 50 Chinese hamster V79 cells per well and pre-incubated for 24 hours. Each of the 23 samples was then substituted into each well and cultured at 37°C under 5% CO2 for 6 days. After methanol fixation, Giemsa staining was performed, and the number of colonies was counted visually. The number of colonies in the control was set to 1, and compared with each of the 23 groups. The results are shown in Figure 1. In Figure 1, the vertical axis represents the relative value when the control is set to 1.

[0055] [Table 1]

[0056] As shown in Figure 1, Fast Green FCF, Evans Blue, Brilliant Blue FCF, Patent Blue, Naphthol Green B, and Indigo Carmine were determined to have low cytotoxicity, while the other dyes were determined to be cytotoxic. Therefore, Fast Green FCF, Evans Blue, Brilliant Blue FCF, Patent Blue, Naphthol Green B, and Indigo Carmine were selected as dyes for use in the embryo transfer culture medium. Acid Green 3 was excluded due to its color stability.

[0057] In addition, culture media containing the six selected dyes—Fast Green FCF, Brilliant Blue FCF, Evans Blue, Patent Blue, Naphthol Green B, or indigo carmine—was dissolved in phosphate-buffered saline (PBS), and each dye solution was added to M16 culture medium (Sigma-Aldrich) at three concentrations: 1x, 1 / 2x, and 1 / 4x. Next, 20 μL drops of each M16 medium were prepared on Petri dishes and overlaid with liquid paraffin (Nacalai Tesque). Photographs of the solutions dropped onto the Petri dishes are shown in Figure 2. The (-) on the right in Figure 2 represents a control without added dye.

[0058] As is clear from FIG. 2, all of the samples were distinguishable from the control even at 1 / 4x the concentration, and in particular, the difference from the control was clear at 1 / 4x the concentration for Fast Green FCF and Brilliant Blue FCF.

[0059] Furthermore, the prepared drops were photographed against a white background, and the R, G, and B component values ​​were measured using ImageJ. For comparison, the R, G, and B component values ​​of the uterus were also measured in the same way. The measurement results for the R, G, and B component values ​​(RGB values) of each culture medium are shown in Figure 3. In Figure 3, the horizontal axis for all samples except the uterus is, from left to right, 1x concentration, 1 / 2x concentration, 1 / 4x concentration, and control (-). The uterus is for each of four individuals.

[0060] In the uterus, the R component value was clearly high, ranging from 130 to 170, the G component value from 70 to 100, and the B component value from 60 to 100. On the other hand, in the case of Fast Green FCF (FG), Brilliant Blue FCF (BB), Evans Blue (EB), Patent Blue (PB), Naphthol Green B (NGB), and Indigo Carmine (IC), the R component value was equal to or lower than the G or B component values. Furthermore, as the concentration increased, the R component value decreased and the ratio of the G and B component values ​​to the R component value increased.

[0061] Next, two combinations of dyes—Fast Green FCF, Brilliant Blue FCF, Evans Blue, Patent Blue, Naphthol Green B, or Indigo Carmine—were added to 10% FBS-supplemented EMEM (Fujifilm Wako Pure Chemical Industries, Ltd.) at a concentration of 1x. Cytotoxicity was evaluated in accordance with the colony formation inhibition test (International Standard ISO 10993-5) as described above. The results are shown in Figure 4. In Figure 4, the vertical axis represents the relative value when the control is set to 1. As is clear from Figure 4, all relative values ​​to the control were 0.6 or higher, confirming low cytotoxicity even when two dyes were combined. For embryo transfer, which requires lower toxicity, combinations other than Evans Blue and Brilliant Blue FCF, Evans Blue and Fast Green FCF, and Evans Blue and Indigo Carmine are preferred, as are combinations that do not include Evans Blue and Indigo Carmine, i.e., combinations of Brilliant Blue FCF, Fast Green FCF, Naphthol Green B, and Patent Blue.

[0062] Furthermore, cytotoxicity was evaluated at various concentrations. Brilliant Blue FCF, Evans Blue, Fast Green FCF, indigo carmine, or naphthol green B was added to 10% FBS-supplemented EMEM (Fujifilm Wako Pure Chemical Industries, Ltd.) at 1x, 2x, or 4x concentrations, and cytotoxicity was evaluated in accordance with the colony formation inhibition test (international standard ISO 10993-5) as described above. The results are shown in Figure 5. In Figure 5, the vertical axis represents the relative value when the control is set to 1. As is clear from Figure 5, all dyes had low cytotoxicity up to a concentration of 1x, Brilliant Blue FCF and Fast Green FCF had low cytotoxicity even at a concentration of 4x, and indigo carmine and naphthol green B had low cytotoxicity even at a concentration of 2x.

[0063] [Example 3] Cytotoxicity evaluation Using the six dyes selected in Example 2, genotoxicity was evaluated by alkaline single-cell gel electrophoresis (comet assay).

[0064] Each dye was added to EMEM supplemented with 10% FBS to achieve the same concentration as in Example 2, and V79 cells were cultured at 37°C for approximately 3 days. The collected cells were then embedded in agarose and subjected to electrophoresis. PBS without dye was used as a negative control, and H2O2 was used as a positive control. Subsequently, the nucleic acids were fluorescently stained with PBS supplemented with GelGreen and observed under a fluorescent microscope. The results are shown in Figure 6.

[0065] As shown in Figure 6, no shooting star-like comets were observed with any of the six dyes, confirming that none of the dyes had any toxicity to genes.

[0066] [Example 4] Gene expression analysis It is important that the components of embryo transfer culture medium do not affect embryonic development. Therefore, we performed comprehensive gene expression analysis using SAGE sequencing to examine their effects on embryonic development. Embryos were cultured for 3 days in M16 medium supplemented with the dyes Acid Green 3 (AG3), Brilliant Blue FCF (BB), Evans Blue (EB), Fast Green FCF (FG), Indigo Carmine (IC), Naphthol Green B (NGB), and Patent Blue (PB). Alternatively, M16 medium, KSOM medium, or WM medium without dyes was used as a control. RNA was extracted using a commercially available RNA extraction kit (ReliaPrep; Promega) and sequenced using an Ion S5 (Life Technologies). After sequencing, data were obtained as FASTQ files. This FASTQ file was uploaded to the iDEP web analysis page (URL: bioinformatics.sdstate.edu / idep93 / ) to generate a correlation diagram. The resulting correlation diagram is shown in Figure 7. In the figure, CTL is M16 medium, which is a control and is frequently used as an embryo culture medium, KSOM is KSOM (Ark Resources), which is one of the embryo culture media, and WM is mWM (Ark Resources), which is one of the embryo culture media.

[0067] As is clear from FIG. 7, when any dye was added, gene expression was almost the same as compared to the control, and the overall gene expression pattern was 0.96 or more, similar to the control.

[0068] [Example 5] Embryonic development test It is important that the components of the embryo transfer culture medium do not affect the implantation of the fertilized egg into the uterus after embryo transfer. Therefore, we first performed embryo transfer using the embryo transfer culture medium of the present invention and investigated the implantation rate.

[0069] The embryo transfer procedure is as follows: <From mouse anesthesia to dissection> 1. Prepared and disinfected the equipment. 2. Pseudopregnant mice were anesthetized and placed in a prone position. 3. The dorsal side was shaved with clippers and disinfected with cotton soaked in 70% ethanol. 4. The skin on the dorsal midline was incised approximately 0.5-1.0 cm (skin only). 5. Forceps were inserted into the incision and the left lower abdominal wall was grasped. 6. The grasped abdominal wall was incised approximately 0.5 cm, and the fatty tissue around the ovaries was pulled out from the abdominal cavity, pulling the ovaries, fallopian tubes, and upper part of the uterus out of the body. 7. The removed ovaries, fallopian tubes, upper uterus and adipose tissue were covered with gauze moistened with saline to prevent drying.

[0070] <Embryo preparation> 8. Followed steps 1 to 7, then operated the stereo microscope. 9. Approximately 5-10 embryos were aspirated into a glass capillary under a stereomicroscope. At this time, M16 medium containing Acid Green 3 (AG3), Brilliant Blue FCF (BB), Evans Blue (EB), Fast Green FCF (FG), Naphthol Green B (NGB), and Patent Blue (PB) (all at 1x concentration) was also prepared.

[0071] <Embryo transfer> 10. An injection needle (26-27G) was inserted into the oviduct-uterine junction of the mouse. 11. The syringe needle was removed and a glass capillary was inserted into the site. 12. It was confirmed that the inserted glass capillary was securely inside the uterus. 13. After confirming the insertion into the uterus, the embryos in the capillary were injected into the uterus. 14. Under a stereomicroscope, the M16 medium containing each dye was repeatedly aspirated and discharged from the capillary several times to confirm that no embryos remained in the capillary. 15. The ovaries and other organs pulled out in steps 6 and 7 were returned to the abdominal cavity. 16. The abdominal wall incision made in step 6 was sutured. 17. Embryo transfer was performed in the opposite uterus in the same manner, following steps 6-16. 18. The skin was sutured. 19. Postoperative care was provided, including administration of antagonists and analgesics.

[0072] Implantation was confirmed 5 days after transfer (corresponding to 8.5 days of gestation). Among the implanted uteri, photographs of the uterus using the dye Brilliant Blue FCF (BB) are shown in Figure 8A, those using the dye Fast Green FCF (FG) are shown in Figure 8B, those using the dye Evans Blue (EB) are shown in Figure 8C, those using the dye Naphthol Green B (NGB) are shown in Figure 8D, and those using the dye Patent Blue (PB) are shown in Figure 8E. The implantation rates were not significantly different from the control, suggesting that none of the five dyes affected the implantation rate. Furthermore, during the embryo transfer process, the uterine tissue and the embryo transfer culture medium were easily visually distinguishable, and the embryo transfer culture medium did not leak out of the uterus or fallopian tubes.

[0073] [Example 6] Analysis of next generation individuals Embryos were transferred using an embryo transfer culture medium containing Brilliant Blue FCF (BB), Evans Blue (EB), and Fast Green FCF (FG), and the body weights of the mice (Slc:BDF1 mice) born from these transfers were measured at 4, 6, and 8 weeks of age. The results are shown in Figure 9. In the figure, the horizontal axis represents age in weeks, and the vertical axis represents body weight (kg).

[0074] As is clear from Figure 9, the weight of the offspring after birth was similar to that of the control (negative control), and it was considered that there was no effect on the growth of the offspring of Brilliant Blue FCF (BB), Evans Blue (EB), and Fast Green FCF (FG).

[0075] [Example 7] Fertility of next generation individuals To confirm whether the next generation individuals could produce further offspring, sibling mating was carried out. The next generation of individuals born in Example 6 were kept until they reached sexual maturity (up to 8 weeks after birth). Thereafter, the mature individuals were kept in the same cage for 8 days to allow for natural mating. Since the sexual cycle of mice is approximately 4 days, it was thought that there would be at least two estrus cycles during the cohabitation period. After 8 days of cohabitation, the mice were kept for several days and observed several times a day until birth was confirmed. A photograph of the individuals that gave birth is shown in Figure 10.

[0076] These results suggest that Brilliant Blue FCF (BB), Evans Blue (EB), and Fast Green FCF (FG) have no effect on fertility. [Industrial Applicability]

[0077] The embryo transfer culture medium of the present invention, which has improved visibility at the transfer site, allows not only experienced but also less experienced practitioners to properly perform embryo transfer, which will contribute to the development of industries such as the medical and livestock industries, and to the stimulation of basic research activities.

Claims

1. A pigment containing at least one pigment selected from the group consisting of Fast Green FCF, Evans Blue, Brilliant Blue FCF, Patent Blue, Naphthol Green B, and indigo carmine, and salts, hydrates, or derivatives thereof, and having a color tone (1) In the RGB color model, the R component value is less than 80, the sum of the G and B component values ​​is 100 or more and 360 or less, and the G component value or the B component value is greater than the R component value; or (2) In the RGB color model, the R component value is 80 or more and 150 or less, the sum of the G component value and the B component value is 150 or more and 360 or less, and the G component value or the B component value is greater than the R component value; A culture medium for embryo transfer characterized by:

2. The culture medium for embryo transfer described in claim 1, characterized in that the dye is at least one selected from the group consisting of Fast Green FCF, Brilliant Blue FCF, and their salts, hydrates, or derivatives.

Images