JP2021168603A - Blastocyst formation promoting agent - Google Patents

Abstract

To provide a novel blastocyst formation promoting agent.SOLUTION: A blastocyst formation promoting agent comprises yeast fermented garlic or an extract thereof.SELECTED DRAWING: None

Description

The present invention relates to an agent for promoting the formation of a fertilized egg into a blastocyst.

Between the time when the eggs of mammals including humans are fertilized in the oviduct and the time when they are implanted in the uterus, the fertilized eggs divide into cells like 2, 4, and 8 and move in the oviduct while morula. It differentiates into embryos, blastocysts, and blastocysts. In in vitro fertilization, sperm are added to an egg in a medium, and the blastocyst or blastocyst is cultured in vitro and returned to the endometrium.

Here, in in vitro fertilization, it is necessary to donate healthy eggs and sperms in order to improve the pregnancy rate, but the rate of formation into blastocysts in the stage from fertilization to implantation is extremely important. This improvement in the blastocyst formation rate is also important for improving the normal pregnancy rate.
In particular, in in vitro fertilization of mice, a phenomenon in which cell division does not proceed after dividing up to 2 cells, 2 cell block, is known. , The pregnancy rate does not improve (Non-Patent Document 1).

On the other hand, odorless garlic containing no alliin or allicin enhances sperm count and sperm motility (Patent Document 1), and garlic or garlic extract reduces inflammation of the male reproductive tract and increases testosterone concentration in the testis (Patent). It is known that S-allylcysteine suppresses a decrease in sperm count and sperm motility (Patent Document 3).

Japanese Unexamined Patent Publication No. 11-346712 Japanese Patent Publication No. 2008-544994 Japanese Unexamined Patent Publication No. 2012-17295

Int. J. Dev. Biol. 53: 129-134 (2009)

An object of the present invention is to provide an agent for promoting the formation of fertilized eggs into blastocysts and a method for producing in vitro fertilization blastocysts or blastocysts using the same.

Therefore, as a result of various studies to find a component that acts on the formation of the fertilized egg into the blastocyst, the present inventors have an action that the yeast fertilized garlic or its extract promotes the formation from the fertilized egg into the blastocyst. It was found that it has an effect of improving the implantation rate of fertilized eggs and, by extension, improving the pregnancy rate. We also found that yeast-fermented garlic or its extract is also useful as a medium component from fertilized eggs to blastocyst formation after in vitro fertilization.

That is, the present invention provides the following inventions [1] to [4].
[1] A blastocyst formation promoter containing yeast fermented garlic or an extract thereof.
[2] The blastocyst formation-promoting agent according to [1], which is a formation-promoting agent from a fertilized egg to a blastocyst.
[3] In vitro fertilization medium containing yeast fermented garlic or an extract thereof.
[4] A method for producing an in vitro fertilized blastocyst or blastocyst, which comprises fertilizing an egg and a sperm in a medium containing yeast fertilized garlic or an extract thereof, and then culturing the fertilized egg in the medium.

According to the present invention, it is possible to provide a blastocyst formation promoter that promotes the formation of a fertilized egg into a blastocyst. When the blastocyst formation promoter of the present invention is applied at the time of fertilization and / or after fertilization, blastocyst formation after fertilization is promoted, so that the implantation rate and thus the pregnancy rate are improved. Further, the blastocyst forming agent of the present invention is useful as a medium component in in vitro fertilization, and culturing fertilized eggs in a medium containing this medium component promotes the formation of in vitro fertilization blastocysts or blastocysts. And the implantation rate in in vitro fertilization is improved.

It is a figure which shows the influence on the two-cell embryo formation and the influence on the blastocyst formation by the addition of the extract of yeast fermented garlic.

The active ingredient of the blastocyst formation promoter of the present invention is yeast fermented garlic or an extract thereof.

The garlic used as a raw material for yeast-fermented garlic is preferably whole plant / above-ground part or bulb of Allium sativum, and more preferably bulb. In the present invention, it is important to use yeast-fermented garlic or an extract thereof instead of directly using garlic or an extract thereof from the viewpoint of obtaining an excellent blastocyst formation promoting effect.

Saccharomyces cerevisiae, Saccharomyces cerevisiae, Aspergillus oryzae, Aspergillus sojae, Aspergillus awamori, Aspergillus glaucus, Penicillium roqueforti, Aspergillus roqueforti Etc., but Saccharomyces cerevisiae is more preferable.

Yeast-fermented garlic can be obtained, for example, by heat-treating garlic to 80 ° C. or higher and then fermenting it with yeast. First, as the heat treatment, the growth of various germs can be suppressed at 80 ° C. or higher, but 85 ° C. or higher is preferable, and 80 ° C. to 120 ° C. is more preferable. The heat treatment time is preferably 5 minutes to 1 hour, and 5 minutes to 30 minutes is sufficient. Yeast fermentation is preferably carried out at 30-40 ° C. for 2-5 days with the addition of yeast. Fermentation is preferably carried out by adding sugars such as glucose. The progress of fermentation can be confirmed by the change in odor peculiar to garlic and the decrease in pH (decrease to pH 6 or less).

The obtained yeast fermented garlic can be used as it is, but it can also be pulverized and used as a powder, or can be used as an extract. Examples of the yeast-fermented garlic extract include extracts extracted from yeast-fermented garlic with an organic solvent such as water, hot water, and alcohol, but water or hot water extract is more preferable. The extraction means is not particularly limited, and a solvent such as water may be added to the yeast fermented garlic or a pulverized product thereof, and the components dissolved in the solvent may be collected. In solvent extraction, heating or the like may be performed, or Soxhlet extraction or the like may be adopted.

Yeast-fermented garlic or its extract has little effect from fertilization to division into two-cell embryos and significantly promotes the formation of two-cell embryos into blastocysts or blastocysts, as shown in Examples below. Has an action. Such an action was completely unpredictable from the action of garlic or the components contained in garlic.
Therefore, yeast fermented garlic or an extract thereof is useful as a blastocyst formation promoter, particularly as a blastocyst formation promoter from a fertilized egg. In addition, yeast fertilized garlic or its extract is also useful as a component of an in vitro fertilization medium, and if a fertilized egg is cultured in a medium containing yeast fertilized garlic or an extract thereof, an in vitro fertilization blastocyst or blastocyst can be obtained. Blastocyst formation is promoted.

When the blastocyst formation promoter of the present invention is applied at the time of fertilization and / or after fertilization, the implantation rate after fertilization and thus the pregnancy rate are improved. Examples of the means for applying the blastocyst formation promoter include means for ingesting, applying or administering as a drug, food composition, external composition to mammals including humans, and means for applying to fertilized eggs in in vitro fertilization. Therefore, the blastocyst formation promoter of the present invention includes the form of an additive to a fertilized egg culture medium, a pharmaceutical product, a food composition, and an external composition.

The yeast fermented garlic or its extract is preferably contained in these blastocyst formation promoters in an amount of 0.001 to 50% by mass, more preferably 0.001 to 20% by mass as a dry solid content. ..

When the blastocyst formation promoter of the present invention is used as a pharmaceutical product, a food composition, or an external composition, it may contain an arbitrary component used in the formulation of ordinary foods, pharmaceuticals, external preparations, and the like. As such an optional component, if it is an orally administered composition, for example, an excipient such as lactose or sucrose, a binder such as starch, cellulosic, arabic rubber, hydroxypropylcellulosic, or carboxymethyl cellulosic. Disintegrants such as sodium and carboxymethyl cellulose calcium, surfactants such as soy lecithin and sucrose fatty acid esters, sweeteners such as multitor and sorbitol, acidulants such as citric acid, phosphates, etc. Binders, film-forming agents such as shelac and zein, lubricants such as talc and waxes, flow promoters such as light anhydrous citric acid and dry aluminum hydroxide gel, diluents such as physiological saline and glucose aqueous solutions, Preferable examples include a flavoring agent, a coloring agent, a bactericidal agent, a preservative, and a fragrance.

For external compositions, hydrocarbons such as squalane, vaseline and microcrystalin wax, esters such as jojoba oil, carnauba wax and octyldodecyl oleate, triglycerides such as olive oil, beef fat and coconut oil, Fatty acids such as stearic acid, oleic acid and retinoic acid, higher alcohols such as oleyl alcohol, stearyl alcohol and octyldodecanol, anionic surfactants such as sulfosuccinate and sodium polyoxyethylene alkyl sulfate. , Amphoteric surfactants such as alkylbetaine salts, cationic surfactants such as monoalkylammonium salts and dialkylammonium salts, sorbitan fatty acid esters, fatty acid monoglycerides, their polyoxyethylene adducts, polyoxyethylene alkylates. Nonionic surfactants such as tell, polyoxyethylene fatty acid ester, polyvalent alcohols such as polyethylene glycol, glycerin, 1,3-butanjiol, thickening / gelling agent, antioxidant, It can contain an ultraviolet absorber, a coloring agent, an antiseptic, and the like.
The production can be carried out by means suitable for various formulations according to a conventional method. Further, as the form of the blastocyst formation promoting agent of the present invention, tablets, soft capsules, hard capsules, granules, drinks, vaginal external agents, jelly-like external agents and the like are preferable.

When the blastocyst formation promoter of the present invention is used as an additive for an in vitro fertilization medium, an in vitro fertilization medium having an improved formation rate from a fertilized egg to a blastocyst can be obtained. The amount of yeast fermented garlic or its extract added to the in vitro fertilization medium is preferably 0.001 to 50% by mass, more preferably 0.001 to 20% by mass as the dry solid content.

Cultivate sperm in a sperm preculture medium containing yeast fertilized garlic or an extract thereof, then cultivate in a medium containing no extract, fertilize the eggs and sperm, and then cultivate the fertilized eggs in the medium. For example, in vitro fertilization vesicles or scutellum vesicles can be efficiently obtained.

Here, examples of the medium for in vitro fertilization include a medium used for normal in vitro fertilization. For example, sperm pre-culture includes CARD FERTIUP ^TM sperm pre-culture medium (Kyudo, Saga, Japan). Preculture of egg mass includes CARD MEDIUM ^TM high-performance in vitro fertilization medium (Kyudo, Saga, Japan). CARD mHTF in vitro fertilization medium (Kyudo, Saga, Japan) is used for culturing fertilized eggs. Examples of 2-cell embryo cultures include KSOM-AA (MERCK, NJ, USA).

The fertilization means is carried out, for example, by pre-culture of sperm and subsequent in vitro fertilization. Examples of the sperm preculture medium include FERTIUP ^TM sperm preculture medium (Kyudo, Saga, Japan). Sperm were pre-cultured in a medium containing yeast fermented garlic or an extract thereof under 37 ° C., 5% CO ₂ , 95% air conditions for 30 minutes. Then, the cells are ^{cultured in FERTIUP TM} sperm preculture medium under 37 ° C., 5% CO ₂ , 95% air conditions for 60 minutes. Examples of the egg preculture medium include CARD MEDIUM ^TM (Kyudo, Saga, Japan) and the like. The pre-cultured sperm and egg mass are in vitro fertilized in an in vitro fertilization medium such as CARD mHTF in vitro fertilization medium under 37 ° C., 5% CO ₂ , and 95% air conditions.

As a culturing means, for example, pre-cultured sperm and egg mass are in vitro fertilized, and _{after 3 hours under 5% CO 2} and 95% air conditions at 37 ° C., the fertilized egg is subjected to CARD mHTF in vitro fertilization medium (Kyudo, Saga). , Japan), and incubate overnight at 37 ° C, 5% CO ₂ , 95% air conditions. The next day, 2-cell stage embryos are transferred to KSOM-AA (MERCK, NJ, USA) medium and cultured at 37 ° C. under 5% CO ₂ and 95% air conditions for 3 days.

The fertilized egg (4-8 cell stage, blastocyst or blastocyst) thus obtained is returned to the uterus. Normal embryo transfer is performed in the uterus at the 4-8 cell stage. In addition, the implantation rate is higher in blastocyst transplantation, in which blastocysts are developed and then transplanted. However, there is a problem that not all cultured embryos become blastocysts. According to in vitro fertilization using the blastocyst formation promoter of the present invention, the formation of 4 to 8 cells or blastocysts from the fertilized egg is promoted, so that a fertilized egg that is good for transplantation is formed and implantation. The rate, and thus the pregnancy rate, improves.

Next, the present invention will be described in more detail with reference to examples.

Production Example 1 (Yeast fermented garlic powder)
100 g of stripped raw garlic was made into a paste using a mixer and then heated to 75-85 ° C. for 30 minutes. Then, yeast (saccharomyces cerevisiae) was inoculated with a platinum loop, glucose was added, and fermentation was carried out at 35 ° C. for about 3 days. After heating to 85 ° C. for 30 minutes to stop fermentation, freeze-drying was performed in a freeze-dryer for 48 hours to obtain yeast-fermented garlic powder (yield 30 g).

Test example (test method)
Test animals In this experiment, semen was collected from two male C57BL / 6J and BALB / c mice (both 12 weeks old). In addition, eggs were collected from 6 female C57BL / 6J mice (4 weeks old).

Reagents and Medium CARD FERTIUP ^TM sperm preculture medium (Kyudo, Saga, Japan) was used for sperm preculture. ^{CARD MEDIUM TM} high-performance in vitro fertilization medium (Kyudo, Saga, Japan) was used for pre-culture of the egg mass. CARD mHTF in vitro fertilization medium (Kyudo, Saga, Japan) was used for culturing fertilized eggs. KSOM-AA (MERCK, NJ, USA) was used for culturing 2-cell embryos.

Sperm collection After the test animals were euthanized by cervical dislocation, the epididymal tails on both sides were removed to remove attached blood and fat. The epididymis tail was fixed with tweezers, and the epididymis in the center of the tail was incised with Noes scissors. After scooping the sperm mass from the epididymal tail incision with an anatomical needle and _{pre-culturing (37 ° C, 5% CO 2} , 95% air) in ^{FERTIUP TM} pre-culture medium (Kyudo, Saga, Japan) for 30 minutes. ^{, FERTIUP TM} sperm preculture medium (Kyudo, Saga, Japan) containing yeast fermented garlic extract (1%) was precultured for 60 minutes (37 ° C, 5% CO ₂ , 95% air). Controls were similarly pre-cultured for 30 minutes (37 ° C, 5% CO ₂ , 95% air) and then ^{60 minutes pre-cultured in FERTIUP TM} sperm preculture medium (Kyudo, Saga, Japan) without garlic extract. (37 ° C, 5% CO ₂ , 95% air).

Egg mass collection Female C57BL / 6J mice (4 weeks old ^{) were intraperitoneally injected with 0.1 ml of CARD HyperOva TM} excess ovulation inducer (Kyudo, Saga, Japan), and 48 hours later, 37.5 IU / ml of human chorionic villi. Gonadotropin hCG (Gonadotropin, Asuka Pharmaceutical Co., Ltd., Tokyo, Japan) was intraperitoneally injected to induce excessive ovulation. 16 hours after administration of hCG, over-ovulated female mice were euthanized by cervical spinal cord dislocation to remove the fallopian tubes, and the ampulla of uterine tubes was cleaved in liquid paraffin for cell culture (Nakaraitex, Kyoto, Japan) to ova. Eggs were collected and ^{precultured in CARD MEDIUM TM} (Kyudo, Saga, Japan).

In vitro fertilization and early embryo culture Pre-cultured sperm and egg mass were in vitro fertilized (37 ° C, 5% CO ₂ , 95% air). After 3 hours, the fertilized eggs were washed 3 times with CARD mHTF in vitro fertilization medium (Kyudo, Saga, Japan) and cultured overnight (37 ° C, 5% CO ₂ , 95% air). The next day, 2-cell stage embryos were transferred to KSOM-AA (MERCK, NJ, USA) and cultured for 3 days (37 ° C, 5% CO ₂ , 95% air).

Fertilization rate and blastocyst rate On the day after in vitro fertilization, the fertilization rate (%, number of 2-cell stage embryos / number of all cells) was calculated by counting 2-cell stage embryos, unfertilized eggs, and dead eggs, respectively. .. In addition, 2-cell stage embryos are transferred to KSOM-AA (MERCK, NJ, USA) and cultured for 3 days, and blastocysts, 2-cell stage embryos, and dead embryos are counted, respectively, and the blastocyst rate (number of blastocysts). / Total number of embryos), 2-cell stage embryo rate (2-cell stage number of embryos / all number of embryos), and dead embryo rate (number of dead embryos / total number of embryos) were calculated.

Statistical analysis All data are expressed as mean ± standard error (SEM). Statistical analysis was performed by Student's t-test, and p <0.05 was judged to be statistically significant.

(result)
The yield up to 2-cell embryo and the yield up to blastocyst formation are shown in FIG.
From FIG. 1, the fertilized eggs cultured with the addition of the yeast fermented garlic extract had the same proportion up to the 2-cell embryo as compared with the control. On the other hand, in the fertilized eggs cultured with the addition of the yeast fermented garlic extract, the formation rate to the blastocyst was significantly increased as compared with the control.
Therefore, it was found that yeast-fermented garlic or an extract thereof has an action of promoting the formation of fertilized eggs into blastocysts.

Claims (4)

A blastocyst formation promoter containing yeast fermented garlic or an extract thereof. The blastocyst formation promoter according to claim 1, which is a formation promoter from a fertilized egg to a blastocyst. In vitro fertilization medium containing yeast fermented garlic or an extract thereof. A method for producing an in vitro fertilization blastocyst or blastocyst, which comprises fertilizing an egg and a sperm in a medium containing yeast fertilized garlic or an extract thereof, and then culturing the fertilized egg in the medium.

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