In vitro selection method for mammalian embryos with high implantation rate

Abstract

To provide a relatively simple method for selecting mammalian embryos with a high implantation rate. [Solution] Mammalian embryos with a high probability of implantation are selected in vitro using as an indicator whether the reactive oxygen species (ROS) concentration in the mammalian embryo is a predetermined multiple (specifically, within the range of 1.80 times or more and 3.10 times or less) of the reactive oxygen species concentration in a control embryo cultured in vitro in a culture medium containing neither arginine nor leucine.

Description

[Technical Field]

[0001] The present invention relates to a method for in vitro selection of a mammalian embryo to determine whether the embryo has a high implantation rate, using as an indicator whether the concentration of reactive oxygen species (hereinafter also referred to as "ROS") in the embryo is higher than a specific threshold value (cutoff value). [Background technology]

[0002] Assisted reproductive technologies, such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICS), are essential not only in the medical field for infertility treatment but also in the livestock industry for the production of high-quality livestock and livestock improvement. After mammalian sperm and mammalian eggs are fertilized in vitro, cleavage leads to cell proliferation, resulting in an increase in blastomeres, leading to the 2-cell, 4-cell, and 8-cell stages. The blastomeres then develop into morulae and then into blastocyst-stage mammalian embryos. When mammalian embryos are transplanted, blastocyst-stage embryos (pre-implantation stages) are typically used. However, low conception rates, such as 40-50% in cattle and 25-35% in humans, are problematic. This is thought to be due to the differences between the in vitro culture environment and the in vivo environment, resulting in the presence of embryos with poor developmental ability. Therefore, a method for selecting mammalian embryos with high implantation potential is needed.

[0003] For example, methods for evaluating the growth state of mammalian embryos using the classification methods of Veeck and Gardner are known (Non-Patent Document 1).More recently, a method for selecting mammalian embryos with a high implantation potential using soluble CD146 contained in the culture medium of mammalian embryos as an indicator has been reported (Patent Document 1).

[0004] The present inventors have reported that the implantation rate of mammalian embryos in the arginine and leucine-supplemented group is higher than that of mammalian embryos in the non-supplemented group, and that the implantation rate of mammalian embryos in the arginine-supplemented group is lower than that of mammalian embryos in the non-supplemented group (Non-Patent Document 2). Based on these results, the present inventors considered the possibility that nitric oxide synthesized from arginine by nitric oxide synthase in mammalian embryos may affect the implantation rate of mammalian embryos, and as a result of their investigation, they have reported that mammalian embryos with a high probability of implantation can be selected using the nitric oxide concentration in mammalian embryos as an indicator (Patent Document 2).

[0005] On the other hand, it has generally been thought that accumulation of ROS in mammalian embryos results in oxidative damage, resulting in a decrease in the implantation rate of mammalian embryos (Patent Document 3). However, contrary to expectations, the present inventors have recently reported that mammalian embryos with high implantation potential can be selected using an increase in ROS concentration in mammalian embryos as an indicator (Patent Document 4). However, it was not known whether embryos with high implantation rates could be selected using as an indicator whether the ROS concentration in mammalian embryos is at least a certain multiple (specifically, within a range of 1.80 to 3.10 times) of the ROS concentration in control embryos cultured in vitro in a culture medium containing neither arginine nor leucine. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2018-513979 [Patent Document 2] Patent Publication No. 2020-184994 [Patent Document 3] International Publication No. 2018 / 056461 Brochure [Patent Document 4] Japanese Patent Application Publication No. 2023-177229 [Non-patent literature]

[0007] [Non-Patent Document 1] Baczkowski, T. et al., Reprod, Biol. (2004)4:5-22 [Non-patent document 2] Abstracts of the 109th Annual Meeting of the Japanese Society of Reproductive Biology, P-16 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a relatively simple method for selecting mammalian embryos with a high implantation rate. [Means for solving the problem]

[0009] In order to solve the above problems, the inventors have been diligently investigating various methods and have discovered that embryos with high implantation rates can be selected by using as an indicator whether the ROS concentration in mammalian embryos is higher than a specific cutoff value, thereby completing the present invention.

[0010] That is, the present invention is as follows. [1] A method for in vitro selection of mammalian embryos with high implantation potential, comprising the following steps (a) to (c): (a) contacting a fluorescent indicator for detecting reactive oxygen species with the obtained mammalian embryo to be tested in vitro; (b) measuring the concentration of reactive oxygen species in the test mammalian embryo using the amount of fluorescence from the fluorescent indicator for detecting reactive oxygen species in the test mammalian embryo as an index; (c) selecting the test mammalian embryo as a mammalian embryo with high implantation ability when the reactive oxygen species concentration measured in the step (b) is a predetermined fold or more of the reactive oxygen species concentration in a control embryo cultured in vitro in a culture medium containing neither arginine nor leucine, wherein the predetermined fold is within the range of 1.80-fold or more and 3.10-fold or less; [2] The method according to [1] above, further comprising, before step (a), a step (p) of in vitro culturing the subject mammalian embryo in a culture medium containing arginine and leucine. [3] The method according to [1] or [2] above, wherein the subject mammalian embryo is a mammalian embryo at the blastocyst stage. [4] The method according to any one of [1] to [3] above, wherein in step (b), the concentration of reactive oxygen species in the subject mammalian embryo is measured using an imaging cytometer. [Effects of the Invention]

[0011] According to the present invention, mammalian embryos with a high implantation rate (possibility of implantation) can be selected relatively easily. Therefore, the present invention is useful in fields requiring assisted reproductive technologies (e.g., in vitro fertilization, intracytoplasmic sperm injection), such as the medical field for infertility treatment, the livestock field for producing high-quality livestock (e.g., cattle with marbled characteristics), and the livestock breeding field for livestock improvement. [Brief explanation of the drawings]

[0012] [Figure 1] ROS fluorescence and bright-field images of mammalian embryos. In the bright-field images, "L" indicates a mammalian embryo from group L, which has relatively low ROS fluorescence intensity, and "H" indicates a mammalian embryo from group H, which has relatively high ROS fluorescence intensity. [Figure 2] FIG. 1 shows the results of measuring ROS concentrations in group L embryos (n=6) and group H embryos (n=6) transplanted into the uterus of five recipient mice (R1 to R5). [Figure 3] FIG. 1 shows the results of measuring the implantation rates of embryos from group L (n=6) and embryos from group H (n=6) transplanted into the uterus of five recipient mice (R1 to R5). DETAILED DESCRIPTION OF THE INVENTION

[0013] The method of the present invention includes the steps of: (a) contacting a fluorescent indicator for detecting reactive oxygen species with the obtained mammalian embryo to be tested in vitro; (b) measuring the concentration of reactive oxygen species in the mammalian embryo to be tested using an imaging cytometer, using the amount of fluorescence (in other words, fluorescence brightness) derived from the fluorescent indicator for detecting reactive oxygen species in the mammalian embryo to be tested as an index; and (c) determining that the mammalian embryo to be tested is a mammalian embryo to be a mammalian embryo when the concentration of reactive oxygen species measured in the step (b) is a predetermined fold or more higher than the concentration of reactive oxygen species in a control embryo cultured in vitro in a culture medium containing neither arginine nor leucine. a step (c) of selecting a mammalian embryo with high implantation ability from the embryos selected in step (c) as a mammalian embryo with high implantation ability, wherein the predetermined multiplication factor is within the range of 1.80 to 3.10; and a step (c) of selecting a mammalian embryo with high implantation ability from the embryos selected in step (c) as a mammalian embryo with high implantation ability, wherein the predetermined multiplication factor is within the range of 1.80 to 3.10. When the mammal is a human, the selection method does not include so-called medical procedures performed by a doctor, such as a step of diagnosing the condition of the human embryo in vitro and selecting a human embryo to be implanted in the uterus of a woman, or a step of implanting a human embryo selected in step (c) as an embryo with high implantation ability into the uterus of a woman. On the other hand, when the mammal is a non-human mammal, the selection method may further include a step of implanting a non-human mammal embryo selected in step (c) as an embryo with high implantation ability into the uterus of a non-human mammal embryo of the same species as the non-human mammal.

[0014] The fluorescent indicator for detecting reactive oxygen species may contain a fluorescent probe whose luminescence intensity and lifetime change in correlation with the amount of ROS present in a mammalian embryo when contacted with the embryo. Such a fluorescent probe is preferably cell membrane permeable and translocates into the mammalian embryo. Examples of such fluorescent probes include HPF (hydroxyphenyl fluorescein), APF (aminophenyl fluorescein), OxiORANGE®, HySOx, HYDROP®, HYDROP-EX® (all manufactured by Goryo Chemical), CellROX Deep Red, CellROX Green, CellROX Orange (all manufactured by Thermo Fisher Scientific), and DCFH-DA (2',7'-Dichlorodihydrofluorescin diacetate). Since the amount of fluorescence from the fluorescent indicator for detecting reactive oxygen species correlates with the ROS concentration in the mammalian embryo, the ROS concentration in the mammalian embryo can be measured using this amount of fluorescence as an indicator.

[0015] Examples of the reactive oxygen species include radical species (e.g., superoxide, hydroxyl radical, hydroperoxy radical, peroxy radical, alkylperoxyl radical, alkoxy radical, nitrogen dioxide, nitric oxide, thiyl / perthiyl radical, etc.), and non-radical species (e.g., hydrogen peroxide, singlet oxygen, lipid hydroperoxide, hypochlorous acid, ozone, peroxynitrite, etc.).

[0016] As used herein, "in vitro selection of mammalian embryos with high implantation ability" refers to selecting mammalian embryos with high implantation ability from a population of mammalian embryos present outside the body of a mammal. Specifically, it refers to selecting mammalian embryos with high implantation ability from a population of mammalian embryos obtained by in vitro fertilization of mammalian sperm and mammalian eggs by methods such as in vitro fertilization or intracytoplasmic sperm injection, followed by in vitro culture as needed. Therefore, "in vitro selection of mammalian embryos with high implantation ability" does not include in vivo fertilization of mammalian sperm and mammalian eggs by injecting mammalian sperm into the uterus of a mammal at the time of ovulation (artificial insemination [IUI]) and selecting mammalian embryos with high implantation ability from mammalian embryos present in the body of a mammal. Furthermore, as used herein, "mammalian embryos with high implantation ability" specifically refers to mammalian embryos with a higher implantation ability (implantation rate) than mammalian embryos having a reactive oxygen species concentration less than the above-mentioned predetermined value.

[0017] Examples of the mammals include humans, and non-human mammals such as rodents such as mice, rats, hamsters, and guinea pigs, lagomorphs such as rabbits, ungulates such as pigs, cows, goats, horses, and sheep, carnivores such as dogs and cats, and non-human primates such as monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees, among which mice, cows, and humans are particularly preferred.

[0018] The above-mentioned embryo may be any embryo at any developmental stage from an embryo immediately after fertilization (i.e., a fertilized egg) to an embryo in a pre-implantation state (i.e., a blastocyst-stage embryo), and specific examples include a fertilized egg (zygote), a 2-cell stage embryo, a 4-cell stage embryo, an 8-cell stage embryo, a morula, and a blastocyst stage embryo, with a blastocyst stage embryo being a preferred example. Furthermore, the above-mentioned embryos include embryos produced by methods such as in vitro fertilization and intracytoplasmic sperm injection (ICS) between a sperm and an egg, as well as fertilized nuclear transfer embryos produced by injecting (microinjecting) the nucleus of a donor cell into an enucleated egg (recipient egg) or an enucleated fertilized embryo (recipient embryo).

[0019] In the above step (a), the fluorescent indicator for detecting reactive oxygen species is contacted in vitro with the obtained mammalian embryo to be tested. Conditions such as the contact time (incubation time), temperature, carbon dioxide (CO2) concentration, and oxygen (O2) concentration during contact, and solvent used for contact can be appropriately selected in consideration of the properties of the fluorescent indicator for detecting reactive oxygen species, without adversely affecting the survival and development of the mammalian embryo.

[0020] The contact time in step (a) is not particularly limited and is, for example, within the range of 1 minute to 24 hours, preferably 5 minutes to 12 hours, more preferably 10 minutes to 6 hours, even more preferably 10 minutes to 3 hours, and even more preferably 10 minutes to 1 hour.

[0021] The temperature during contact in step (a) is, for example, within the range of 0 to 40°C, preferably 4 to 40°C, more preferably 14 to 39°C, even more preferably 20 to 39°C, and even more preferably 30 to 38°C. The CO2 concentration during contact in step (a) is, for example, within the range of 1 to 10%, preferably 2 to 8%, and more preferably approximately 5% (4 to 6%). The O2 concentration during contact in step (a) may be either normoxic (18 to 22% O2) or hypoxic (0 to 10% O2). When mammalian embryos are cultured in vitro before contact with the fluorescent indicator for reactive oxygen species detection, the contact conditions in step (a) can be the same as those for in vitro culture.

[0022] Examples of solvents used for the contact include saline, buffered saline (e.g., PBS, Tris Buffered Saline (TBS), HEPES-buffered saline), Ringer's solution, lactate Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, 5% aqueous glucose solution, and culture medium.

[0023] In the above step (a), conditions such as culture medium, culture time, temperature, carbon dioxide (CO2) concentration, and oxygen (O2) concentration can be appropriately selected so as to achieve culture conditions suitable for culturing mammalian embryos outside the mammalian body.

[0024] In the present invention, mammalian embryos can be obtained by known methods for producing fertilized eggs in vitro from mammals, such as injecting mammalian sperm into at least one collected mammalian egg using a glass pipette (injection pipette for microinjection) under a microscope (i.e., intracytoplasmic sperm injection); or by allowing mammalian sperm and mammalian eggs to fertilize naturally in a culture medium (i.e., in vitro fertilization), to obtain embryos immediately after fertilization, and then culturing them in vitro to various developmental stages as necessary. Furthermore, in the present invention, the number of mammalian embryos may be one or more (e.g., 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, 60 or more, 100 or more).

[0025] In the above step (b), the method for measuring the reactive oxygen species concentration in the test mammalian embryos may be any method capable of measuring the amount of fluorescence derived from a fluorescent indicator for detecting reactive oxygen species in the test mammalian embryos while the test mammalian embryos are still alive (live imaging method), and a suitable example of such a method is a method using an imaging cytometer. Here, "imaging cytometer" refers to an image analysis system that can automatically acquire fluorescence images using a microscope and analyze the acquired fluorescence images. Specific examples of the measurement method in the above step (b) include automatically detecting and quantifying the amount of fluorescence derived from a fluorescent indicator for detecting reactive oxygen species emitted in each of one or more mammalian embryos and acquiring the fluorescence image; acquiring analytical data that displays values ​​related to the fluorescence intensity, such as the total fluorescence intensity or the maximum fluorescence intensity, for each embryo based on the fluorescent image using a histogram, dot plot, or the like; and acquiring quantitative results related to the fluorescence intensity based on the analytical data.

[0026] Examples of the imaging cytometer include a confocal laser microscope system (A1R HD25, manufactured by Nikon Corporation), a confocal laser microscope system (TCS SP8, manufactured by Leica Microsystems), a spinning disk confocal system (SpinSR10, manufactured by Olympus Corporation), and a multi-angle live imaging system (LightsheetZ.1, manufactured by ZEISS).

[0027] The control embryo may be a mammalian embryo cultured in vitro in a culture medium containing neither arginine nor leucine, and the conditions for ex vivo culture are preferably the same as those for the ex vivo culture of the test mammalian embryo, except that the culture medium contains arginine and leucine. The reactive oxygen species concentration value in the control embryo may be the reactive oxygen species concentration value in one control embryo, but is preferably the average or median of the reactive oxygen species concentrations in two or more control embryos. The reactive oxygen species concentration in the control embryo is preferably calculated by treating a comparative mammalian embryo in the same manner as the test mammalian embryo.

[0028] In the present selection method, when two or more mammalian embryos are tested, the reactive oxygen species concentration value in the test mammalian embryos may be the average or median. Examples of the lower limit of the above-mentioned "range of 1.80 times or more and 3.10 times or less" include 1.80 times, 1.85 times, 1.90 times, 1.95 times, 2.00 times, 2.05 times, 2.10 times, 2.15 times, 2.20 times, 2.25 times, 2.30 times, 2.35 times, 2.40 times, 2.45 times, 2.50 times, 2.55 times, 2.60 times, 2.65 times, 2.70 times, 2.75 times, 2.80 times, 2.85 times, 2.90 times, 2.95 times, 3.00 times, 3.10 times, 3.20 times, 3.30 times, 3.40 times, 3.50 times, 3.60 times, 3.70 times, 3.80 times, 3.90 times, 3.95 times, 3.00 times, 3.10 times, 3.20 times, 3.30 times, 3.40 times, 3.50 times, 3.60 times, 3.70 times, 3.85 ...45 times, Examples of the upper limit include 3.10 times, 3.05 times, 3.00 times, 2.95 times, 2.90 times, 2.85 times, 2.80 times, 2.75 times, 2.70 times, 2.65 times, 2.60 times, 2.55 times, 2.50 times, 2.45 times, 2.40 times, 2.35 times, 2.30 times, 2.25 times, 2.20 times, 2.15 times, 2.10 times, 2.05 times, 2.00 times, 1.95 times, 1.90 times, 1.85 times, etc. These lower limit and upper limit values ​​can be combined in any desired manner.

[0029] Preferably, the present selection method further comprises, prior to the above step (a), a step (p) of in vitro culturing the subject mammalian embryo in a culture medium containing arginine and leucine. Herein, when in vitro culturing a mammalian embryo in a culture medium, the lower limit of the culture time is, for example, 12 hours, 16 hours, 20 hours, 24 hours, 30 hours, 36 hours, or 48 hours, and the upper limit of the culture time is, for example, 60 hours, 48 ​​hours, 36 hours, or 30 hours. Therefore, when in vitro culturing a mammalian embryo in a culture medium, the culture time can be, for example, 12 to 60 hours, 16 to 48 hours, 20 to 36 hours, or 20 to 30 hours.

[0030] In this specification, the concentration of arginine in the culture medium is, for example, within the range of 0.001 to 10 mM, and examples include 0.003 to 6 mM, 0.006 to 3 mM, 0.01 to 3 mM, 0.03 to 3 mM, 0.06 to 3 mM, 0.006 to 1 mM, 0.01 to 1 mM, 0.03 to 1 mM, and 0.06 to 1 mM.

[0031] In this specification, the concentration of leucine in the culture medium is, for example, within the range of 0.001 to 10 mM, and examples include 0.003 to 6 mM, 0.006 to 3 mM, 0.01 to 3 mM, 0.03 to 3 mM, 0.06 to 3 mM, 0.006 to 1 mM, 0.01 to 1 mM, 0.03 to 1 mM, and 0.06 to 1 mM.

[0032] As used herein, the culture medium may be any suitable medium for culturing mammalian embryos, including a medium containing serum (e.g., 0.1 to 30% [v / v] FBS, CS, etc.) or a serum-free medium. However, serum-free culture medium is preferred from the viewpoint of minimizing the effects of ionized serum albumin contained in serum. The term "serum-free culture medium" refers to a culture medium that does not contain unadjusted or unpurified serum, and includes a culture medium containing a serum substitute. Specific examples of such serum substitutes include commercially available B27 Supplement (Insulin) (Life Technologies), N2 Supplement (Life Technologies), B27 Supplement (Life Technologies), and Knockout Serum Replacement (Invitrogen). Specific examples of the culture medium include, for example, TCM-199 culture medium (see Proc. Soc. Exp. Biol. Med. 1950; 73: 1-8); mSOF culture medium (see JP 2012-210199 A) for culturing bovine embryos; NCSU-23 culture medium (see Theriogenology 1992; 37: 95-109); PZM culture medium (see Biol. Reprod. 2002; 66: 112-119) for culturing porcine embryos; Eagle's MEM culture medium (see J. Anim. Sci. 1976; 42: 912-917) for culturing ovine embryos; and Ham's F10 culture medium (see Exp. Cell Res. 1963; 29: 515-526); Eagle's MEM culture medium (see J. Anim. Sci. 1976; 42: 912-917); RPMI1640 culture medium (see J. Am. Med. Assoc. 1967; 199: 519-524); etc., when culturing rat embryos, m-KRB culture medium; HECM-1 culture medium (see Hum. Reprod. 1991; 6: 1445-1448); etc., when culturing hamster embryos, Eagle's MEM culture medium (see J.Examples of suitable media for culturing mouse embryos include TYH medium (see Jpn. J. Anim. Reprod. 1971; 16: 147-157); CZB medium (see J. Reprod. Fertil. 1989; 86: 679-688); and KSOM medium (see Isaji et al., J. Reprod. Dev. 2015, 61: 503-510). Examples of suitable media for culturing human embryos include modified HTF medium (see J. Assist. Reprod. Genet. 1995; 12: 97-105); 10% plasma protein fraction (PPF; plasma protein fraction), human albumin (HAS; human albumin), and the like. Examples of suitable fertilizers include cleavage medium (SAGE [registered trademark] Cleavage Medium, Cooper Surgical, Inc., Connecticut, USA) containing sucralose, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate, hydroxybenzoate; albumin; SSS; serum substitute supplement (manufactured by Irvine Scientific), etc.); sperm washing medium (Irvine Scientific, California, USA); fertilization (HTF) medium (SAGE In-Vitro Fertilization, Connecticut, USA); Ferticult (registered trademark) Sperm Washing Flushing Medium (FertiPro NV, Beernem, Belgium); Insemination Medium; NI fertilization medium (NAKA ivf medium, Naka Medical, Japan); and the like.

[0033] The culture medium may contain optional components such as reducing agents (e.g., 2-mercaptoethanol, dithiothreitol (DTT) and the like), iron sources (e.g., transferrin and the like), minerals (e.g., sodium selenite), amino acids (e.g., glutamine, alanine, asparagine, serine, aspartic acid, cysteine, glutamic acid, glycine, proline, tyrosine), vitamins (e.g., choline chloride, pantothenic acid, folic acid, nicotinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, ascorbic acid, niacin ... Examples of optional ingredients include: saccharides (e.g., glucose, etc.), organic amines (e.g., putrescine, etc.), steroids (e.g., progesterone, β-estradiol, etc.), antibiotics (e.g., penicillin, streptomycin, etc.), interleukins (e.g., IL-1, IL-2, IL-3, IL-6, etc.), adhesion factors (e.g., heparin, heparan sulfate, collagen, fibronectin, etc.), organic acids (e.g., pyruvic acid, succinic acid, lactic acid, etc.) or salts thereof, buffers (e.g., HEPES, etc.), etc. As used herein, the term "optional ingredient" refers to an ingredient that may or may not be included.

[0034] Herein, the temperature during in vitro culture of mammalian embryos is usually within the range of about 30 to 40°C, preferably about 37°C. The CO2 concentration during in vitro culture of mammalian embryos is usually within the range of about 1 to 10%, preferably about 5%. The O2 concentration during in vitro culture of mammalian embryos may be either normoxic (18 to 22% O2) or hypoxic (0 to 10% O2).

[0035] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to these examples. In the following examples, sperm pre-culture, insemination, and embryo culture were carried out in an incubator (under conditions of 5% CO2 / 20% O2 and 37°C, the same applies hereinafter). HTF culture medium and KSOM culture medium were prepared according to standard methods. [Example]

[0036] Selection of mammalian embryos with high and low ROS concentrations To investigate the relationship between ROS concentrations in mammalian embryos and their implantation rate, we measured ROS concentrations in mammalian embryos using live imaging according to the following method, selected mammalian embryos with high ROS concentrations and those with low ROS concentrations, and then transplanted each embryo into a mouse uterus to analyze the embryo's implantation ability.

[0037] 1. Method [Insemination] In vitro insemination of mammalian sperm and eggs was carried out according to the following steps [1] to [7]. [1] Five-month-old male ICR mice were euthanized, and the cauda epididymis was removed and collected. [2] 400 μL of HTF culture medium was added dropwise to a sperm pre-culture dish to prepare a drop of HTF culture medium, which was then covered with liquid paraffin (manufactured by Nacalai Tesque). [3] While grasping the tail of the collected epididymis, the epididymal duct was incised with scissors, and the resulting sperm mass was transferred to a drop of HTF culture medium using a 1 mL tuberculin syringe with a 26G needle (Terumo Corporation), and the sperm were cultured (pre-cultured) for 2 hours before insemination. [4] Pregnant mare serum gonadotropin (PMSG) was administered intraperitoneally to 5-month-old female ICR mice. 48 hours after PMSG administration, human chorionic gonadotropin (hCG) was administered to induce superovulation. 12 to 16 hours after hCG administration, the mice were euthanized and the oviducts were collected. [5] 200 μL of HTF culture medium was added dropwise to an inoculation dish to prepare a drop of HTF culture medium, which was then covered with liquid paraffin. [6] The recovered oviduct was transferred to liquid paraffin in an insemination dish, and while holding it with tweezers, the oviduct wall at the ampulla was torn using a 1 mL tuberculin syringe with a 26G needle, and the resulting egg mass was transferred to a drop of HTF culture medium. [7] The pre-cultured sperm was adjusted to 700 sperm / μL using a micropipette (manufactured by Nichiryo Co., Ltd.) and transferred into a drop of HTF culture medium containing egg masses in an insemination dish for insemination.

[0038] [In vitro embryo culture] In vitro culture of mammalian embryos was carried out according to the following procedures [1] and [2]. [1] Four hours after insemination, the fertilized eggs were collected, transferred to HTF medium, and cultured for development. [2] After approximately 90 hours of developmental culture, embryos that had progressed to the blastocyst stage were cultured for 24 hours in KSOM medium containing 0.2 mM arginine and 0.2 mM leucine. Control embryos were also cultured for 24 hours in KSOM medium containing neither arginine nor leucine (hereafter referred to as "control embryos").

[0039] [Measurement of ROS concentration in embryos] The ROS concentration in mammalian embryos was measured according to the following procedures [1] to [3]. In the examples of Patent Document 4, the ROS concentration in multiple embryos (embryo population) was measured on a glass-bottom dish. However, during the procedure of recovering embryos for transfer, the embryos moved, making it difficult to recover embryos with the desired ROS concentration. Therefore, to recover embryos with the desired ROS concentration, embryos were transferred one by one to each array of a live imaging microarray, as shown in the following procedure [2], and the ROS concentration in the embryos was measured. [1] An ROS detection reagent (CellROX Orange, Thermo Fisher Scientific) was added to the culture medium containing the embryos to a final concentration of 5 μM, and the mixture was incubated in an incubator for 20 minutes. [2] After washing the embryos with fresh KSOM culture medium, each embryo was transferred to each array of a live imaging microarray (#MGA-125-02, 125 μm × 125 μm; Microsurfaces). [3] Using a confocal laser microscope system (TCS SP8, Leica Microsystems), excitation was performed at 545 nm, and an orange fluorescence image (ROS fluorescence image) at a wavelength of 565 nm (left panel of Figure 1) and a bright-field image of the embryo (right panel of Figure 1) were obtained. The fluorescence intensity in the obtained ROS fluorescence image was quantified, and the relative value, with the average fluorescence intensity in the ROS fluorescence image of control embryos (n = 30) set to 1, was calculated as the ROS concentration in the embryo.

[0040] [Selection of embryos with high and low ROS concentrations] Five 5-month-old female ICR mice (R1–R5) were prepared as recipient mice for embryo transfer. From the embryos whose ROS concentrations were measured as described above, six embryos with relatively low ROS concentrations (group L) and six embryos with relatively high ROS concentrations (group H) were selected for transplantation into the uterus of each recipient mouse (see Figure 2). Table 1 shows the mean and median ROS concentrations in the embryos in groups L and H.

[0041] [Table 1]

[0042] [Embryo transfer] Mice are multiparous animals, and multiple embryos can implant in the uterus. The mouse uterus also has two separate lumens (uterine horns). L group embryos (n=6) and H group embryos (n=6) were transplanted into the left and right uterine horns of the same female mouse, respectively, and the presence or absence of implantation was analyzed. The percentage of embryos that implanted in each group was calculated as the implantation rate.

[0043] As a result, in all recipient mice, the implantation rate of embryos was higher when embryos from group H were transplanted than when embryos from group L were transplanted (see Figure 3 and Table 2). This result indicates that when the ROS concentration in the test embryos is higher than that in the control embryos, the test embryos can be evaluated as having a higher implantation ability than the control embryos.

[0044] For example, if we focus on the average ROS concentration in embryos (see Table 1) and set the cutoff value to 1.90 (i.e., 1.90 times the ROS concentration in control embryos), the probability (sensitivity) of correctly identifying embryos with a high implantation rate (H group) as positive is 100%, and the probability (specificity) of correctly identifying embryos with a low implantation rate (L group) as negative is 60%. If we set the cutoff value to 2.30, the sensitivity is 100% and the specificity is 80%. If we set the cutoff value to 3.00, the sensitivity is 60% and the specificity is 100%.

[0045] Furthermore, for example, focusing on the median ROS concentration in embryos (see Table 1), if the cutoff value is set to 1.80 (i.e., 1.80 times the ROS concentration in control embryos), the sensitivity is 100% and the specificity is 60%. If the cutoff value is set to 2.20, the sensitivity is 100% and the specificity is 80%. If the cutoff value is set to 3.10, the sensitivity is 60% and the specificity is 100%.

[0046] Therefore, if the ROS concentration in the test embryo is a certain multiple (specifically, within the range of 1.80 times or more and 3.10 times or less) of the ROS concentration in the control embryo, it indicates that the test embryo is likely to have a high implantation rate.

[0047] [Table 2] [Industrial Applicability]

[0048] The present invention will be useful in fields requiring assisted reproductive technologies (e.g., in vitro fertilization, intracytoplasmic sperm injection), such as the medical field aimed at infertility treatment, the livestock field aimed at producing high-quality livestock (e.g., cattle with marbled characteristics), and livestock improvement.

Claims

1. A method for in vitro selection of mammalian embryos with high implantation potential, comprising the steps of: (a) selecting a mammalian embryo from the cultured embryo; (a) contacting a fluorescent indicator for detecting reactive oxygen species with the obtained mammalian embryo to be tested in vitro; (b) measuring the concentration of reactive oxygen species in the test mammalian embryo using the amount of fluorescence derived from the fluorescent indicator for detecting reactive oxygen species in the test mammalian embryo as an index; (c) selecting the test mammalian embryo as a mammalian embryo with high implantation ability when the reactive oxygen species concentration measured in the step (b) is a predetermined fold or more of the reactive oxygen species concentration in a control embryo cultured in vitro in a culture medium containing neither arginine nor leucine, wherein the predetermined fold is within the range of 1.80-fold or more and 3.10-fold or less;

2. The method according to claim 1, further comprising, prior to step (a), a step (p) of in vitro culturing the subject mammalian embryo in a culture medium containing arginine and leucine.

3. The method according to claim 1 or 2, wherein the subject mammalian embryo is a mammalian embryo at the blastocyst stage.

4. The method according to claim 1 or 2, wherein in step (b), the reactive oxygen species concentration in the subject mammalian embryo is measured using an imaging cytometer.

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