KR102599957B1 - Culture solution composition for embryo transfer for improving success rate of assisted reproductive technology and method for manufacturing thereof - Google Patents

Abstract

The present invention relates to a culture composition for embryo transfer and a method for producing the same to increase the success rate of assisted reproduction techniques (test tube fertilization and frozen/thawed embryo transfer) to induce pregnancy in infertile patients. The culture medium composition for embryo transfer is used to treat implantation failure. When applied to assisted reproductive technology in patients with a previous history, the embryo implantation rate and pregnancy rate are significantly improved.

Description

Culture solution composition for embryo transfer for improving success rate of assisted reproductive technology and method for manufacturing thereof}

The present invention relates to a culture medium composition for embryo transfer and a method for producing the same to increase the success rate of assisted reproduction techniques (test tube fertilization and frozen/thawed embryo transfer) to induce pregnancy in infertile patients.

In order for a woman to become pregnant, after fertilization occurs in the fallopian tube, the embryo passes through the fallopian tube and into the endometrium. Implantation of the embryo occurs through the interaction between the endometrium and the embryo that has developed into the blastocyst stage. Afterwards, the embryo develops into a fetus. The endometrium develops into the implantation stage endometrium by the female hormone progesterone for pregnancy. After fertilization, the embryo undergoes a cleavage process and develops into a blastocyst when it is located in the endometrium. Afterwards, the zona pellucida is opened by the embryo's enzymes and the embryo directly contacts the endometrium in the implantation period. In the case of assisted reproduction techniques such as in vitro fertilization and frozen/thawed embryo transfer, embryos are fertilized and cultured outside the body, and 1-2 morphologically good blastocyst stage embryos are transferred on the 5th day after culture. In both cases, the blastocyst stage embryo infiltrates the endometrium and implantation occurs due to the interaction between the embryo and the endometrium. The interaction between the embryo and the endometrium involves various cytokines, integrin receptors, and their substrates. For this function, it is essential to remove the mucus secreted on the endometrium at the contact area for contact between the embryo and the endometrium.

The causes of infertility are diagnosed in various ways. In general, infertility refers to a case where pregnancy is not achieved even after one year of normal marital relations without contraception. It is statistically reported that 40% of cases are due to male factors, 40% are due to female factors, and 20% are due to unidentified causes. Among these, female factors are reported to have various causes, including hormonal abnormalities, fallopian tube malformations, and ovulatory abnormalities, and various papers report infertility due to implantation failure. Among these, implantation failure refers to a case in which pregnancy does not occur even after transplanting a high-quality embryo in assisted reproductive technology, and it is presumed that the cause is a malfunction in the interaction between the embryo and the endometrium.

In the case of a general embryo transfer procedure, the embryo is placed in an embryo transfer catheter along with a culture medium and implanted into the base of the uterus. In this case, the embryo is placed with the culture medium in the endometrial cavity, and implantation is then induced. At this time, the culture medium implanted together is a culture medium that affects the development of the embryo or a general saline solution-based culture medium, and its effect on the interaction between the endometrium and the embryo is not yet known. Therefore, in order to increase the success rate of pregnancy in patients with a history of implantation failure and patients undergoing general in vitro fertilization/embryo transfer, research on culture preparations for transplantation that can mediate the interaction between the embryo and the endometrium during embryo transfer is necessary. there is.

Republic of Korea Open Patent Publication 10-2014-0113917

In order to overcome the limitations of the prior art, the inventor of the present invention has conducted in-depth research on a culture medium preparation for embryo transfer that can increase the success rate of assisted reproductive technology by activating the implantation mechanism by mediating the interaction between the embryo and the endometrium during embryo transfer. The present invention has been completed.

Accordingly, the purpose of the present invention is to provide a culture medium composition for embryo transplantation containing MMP-9 (metalloproteinase-9) and MMP-2 (metalloproteinase-9) as active ingredients.

Another object of the present invention is to provide a method for producing a culture medium composition for embryo transplantation.

In order to achieve the object of the present invention, the present invention provides a culture medium composition for embryo transplantation containing MMP-9 (metalloproteinase-9) and MMP-2 (metalloproteinase-9) as active ingredients.

In one embodiment of the present invention, the weight ratio of metalloproteinase-9 (MMP-9) and metalloproteinase-2 (MMP-2) may be 1:0.5 to 1.5.

In one embodiment of the present invention, the concentration of metalloproteinase-9 (MMP-9) and metalloproteinase-2 (MMP-2) may be 0.05 to 0.15 μg/ml.

In one embodiment of the present invention, the composition may further include tauroursodeoxycholic acid.

In one embodiment of the present invention, the concentration of tauroursodeoxycholic acid may be 0.3 to 0.7 μg/ml.

In one embodiment of the present invention, the composition may further include an ingredient selected from the group consisting of laminin, hyaluronan, fibronectin, and combinations thereof.

In one embodiment of the present invention, based on the composition, the concentration of laminin is 5 to 10 μg/ml, the concentration of hyaluronan is 15 to 20 μg/ml, and the fibronectin ( The concentration of fibronectin may be 0.5 to 1 μg/ml.

In addition, the present invention provides a buffer solution containing 5 to 10 μg/ml of laminin, 0.5 to 1 μg/ml of fibronectin, 15 to 20 μg/ml of hyaluronan, and 0.05 to 1 μg/ml. A method for producing a culture medium composition for embryo transplantation is provided, comprising mixing 0.15 μg/ml of matrix metalloproteinase (MMP) and 0.3 to 0.7 μg/ml of tauroursodeoxycholic acid.

When the culture medium composition for embryo transfer of the present invention is applied to assisted reproduction in patients with a history of implantation failure, the implantation rate and pregnancy rate of the embryo are significantly improved, so it can be usefully used in related medical industries.

The inventor of the present invention completed the present invention as a result of in-depth research on a culture medium preparation for embryo transfer that can increase the success rate of assisted reproductive technology.

Accordingly, the present invention provides a culture medium composition for embryo transplantation containing MMP-9 (metalloproteinase-9) and MMP-2 (metalloproteinase-2) as active ingredients.

The composition of the present invention can significantly increase the efficiency of implantation and pregnancy in assisted reproductive technology by combined treatment of MMP-9 (metalloproteinase-9) and MMP-2 (metalloproteinase-2).

The composition of the present invention is applied to various animal species to increase implantation and pregnancy efficiency. Animals to which the composition of the present invention can be applied are not particularly limited, and can be applied to, for example, humans, cows, pigs, horses, sheep, goats, rabbits, elephants, monkeys, mice, hamsters and rats. Preferably, the compositions of the present invention are applied to mammals. More preferably, the composition of the present invention is applied to humans, cattle, pigs, horses, sheep, goats, rabbits, elephants and monkeys, and most preferably to humans.

In the present invention, the MMP is a proteolytic enzyme that requires a metal at the catalytic site of the active center, and the metal contains many zinc ions and in rare cases, cobalt ions. It is abundant in the intercellular space and is widely distributed inside cells, cell membranes, body fluids, and secretory fluids.

MMP-9, called 92 kDa type IV collagenase/gelatinase or gelatinase B, is involved in extracellular matrix degradation and pathology in normal physiological processes such as embryonic development, reproduction, angiogenesis, bone development, wound healing, etc. It is known to be involved in this process.

MMP-2, also called 72 kDa type IV collagenase and gelatinase A, is known to be involved in the degradation of extracellular matrix in normal physiological processes such as embryonic development, reproduction, and tissue remodeling, and in disease processes such as arthritis and metastasis. there is.

MMP-2 and MMP-9 used as active ingredients in the present invention include not only those with intrinsic activity but also those with potential activity.

When the composition of the present invention is applied to humans, preferably the fertilized egg is fertilized in vitro. For example, treat and transfer MMP-9 and MMP-2 for an appropriate amount of time before implanting in vitro fertilized eggs.

The composition of the present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is commonly used in preparation and includes, but is limited to, saline solution, sterile water, Ringer's solution, buffered saline solution, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, etc. It doesn't work.

In addition, if necessary, other common additives such as antibiotics, antioxidants, and buffers may be further included, and the content and type are within the range known to those skilled in the art as long as they do not affect the stability or efficacy of the active ingredient. You can select appropriately.

In addition, if necessary, diluents, dispersants, surfactants, binders, lubricants, etc. can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets. Regarding suitable pharmaceutically acceptable carriers and formulations, the formulations can be preferably formulated according to each ingredient using the method disclosed in Remington's literature. Preferably the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium.

The appropriate dosage of the composition of the present invention can be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, body weight, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity.

Specifically, the composition of the present invention is used by 'normal embryo transfer method'. ‘Ordinary embryo transfer method’ refers to a method of placing 1 to 2 embryos in an amount of 10 to 20 μl of the composition in an embryo transfer catheter and transplanting them into the patient’s uterus. The appropriate amount is selected by the number of embryos and the transplanter. It can be. It can also be used for culturing embryos to be transferred before implantation, and the period and amount can be appropriately selected by the user.

In the present invention, the weight ratio of MMP-9 (metalloproteinase-9) and MMP-2 (metalloproteinase-2) may be 1:0.5 to 1.5, and more preferably 1:1. The present invention confirmed that when MMP-9 and MMP-2 were treated at the above weight ratio, the implantation rate and pregnancy rate were significantly improved (see Experimental Example 2).

In the present invention, the concentration of metalloproteinase-9 (MMP-9) and metalloproteinase-2 (MMP-2) may be 0.05 to 0.15 μg/ml. It was confirmed that MMP-9 and MMP-2 showed significantly improved implantation and pregnancy rates in the above concentration range (see Experimental Example 2).

In the present invention, the composition may further include tauroursodeoxycholic acid. The taurourusodeoxycholic acid has the compound name 2-[[(4R)-4-[(3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10 ,13-dimethyl2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl] pentanoyl]amino]ethanesulfonic acid or 3α,7β-dihydroxy-5β-cholanoyltaurine. It is a type of bile acid produced in the liver using cholesterol as a precursor, and can exist in various salt forms and pharmaceutically acceptable salt forms, for example, in the form of alkali metal salts such as sodium and potassium.

It was confirmed that when the taurourusodeoxycholic acid was additionally added to the composition of the present invention, the implantation rate and pregnancy rate were significantly improved (see Experimental Example 2).

In the present invention, the concentration of tauroursodeoxycholic acid may be 0.3 to 0.7 μg/ml, and more preferably 0.5 μg/ml. The present invention confirmed that the implantation rate and pregnancy rate were significantly improved when treated with taurourusodeoxycholic acid in the above concentration range (see Experimental Example 2).

In the present invention, the composition may further include ingredients selected from the group consisting of laminin, hyaluronan, fibronectin, and combinations thereof.

The laminin is the main protein of basal lamina, a part of the basement membrane, and is the basis of the protein network of most cells and organs. Laminin is a biologically active part of the basal lamina and plays important functions in cell differentiation, migration, and adhesion, as well as phenotype and survival.

The hyaluronan, also called hyaluronic acid, is a type of glycosaminoglycan commonly found in connective tissues such as cartilage. Glycosaminoglycans are polymers of repeating disaccharides and are sugars without side chains. Hyaluronan is secreted by chondrocytes and mainly exists in a form bound to proteoglycan via a linker protein.

The fibronectin is a high molecular weight glycoprotein and has three domains that bind to proteoglycan, collagen, and integrin, which are components of the extracellular matrix, respectively. A dimer formed through covalent bonding of two fipronectin monomers is involved in forming the extracellular matrix.

Based on the composition, the concentration of laminin is 5 to 10 μg/ml, the concentration of hyaluronan is 15 to 20 μg/ml, and the concentration of fibronectin is 0.5 to 1. It may be μg/ml, and at this concentration, it can effectively induce implantation of embryos and pregnancy.

In another aspect, the present invention includes 5 to 10 μg/ml of laminin, 0.5 to 1 μg/ml of fibronectin, 15 to 20 μg/ml of hyaluronan, A method for producing a culture medium composition for embryo transplantation is provided, comprising mixing 0.05 to 0.15 μg/ml of matrix metalloproteinase (MMP) and 0.3 to 0.7 μg/ml of tauroursodeoxycholic acid.

This will be explained in detail below. All combinations of the various elements disclosed herein fall within the scope of the present invention. Additionally, the scope of the present invention cannot be considered limited by the specific description below.

<Example>

<Materials and preparation>

The culture medium composition for embryo transfer of the present invention is intended to increase the success rate of assisted reproductive techniques to induce pregnancy in a patient, and must mediate the interaction between the embryo and the endometrium. In addition to the ingredients in Table 1 below, a buffered glycerol aqueous solution is added as a buffer solution. (Phosphate buffer solution, pH 7.4, 0.5 M sodium chloride, 4% DMSO, and 10% glycerol) was added to prepare a sample of the culture medium composition for embryo transfer so that the total amount was 1 ml. Reagents were used that were commercially available.

Unit: μg buffer solution laminin fibronectin Hyaluronan MMP-9 MMP-2 TA sample 1 Appropriate amount 7 0.8 17.5 0.1 - - sample 2 Appropriate amount 7 0.8 17.5 0.1 0.05 - sample 3 Appropriate amount 7 0.8 17.5 0.1 0.1 - sample 4 Appropriate amount 7 0.8 17.5 0.1 0.15 - sample 5 Appropriate amount 7 0.8 17.5 0.1 0.1 0.3 sample 6 Appropriate amount 7 0.8 17.5 0.1 0.1 0.5 sample 7 Appropriate amount 7 0.8 17.5 0.1 0.1 0.7

※TA: Tauroursodeoxycholic acid test was conducted on 70 cases of patients who did not become pregnant despite transplanting high-quality embryos (implantation failure) among patients who had previously undergone IVF-embryo transfer among assisted reproductive techniques. The characteristics of patients (10 patients) in each assigned sample are shown in Table 2.

item sample 1 sample 2 sample 3 sample 4 sample 5 sample 6 sample 7 age 35.15(1.12) 34.95
(1.82) 35.01
(1.88) 34.89
(1.31) 34.91
(1.11) 35.11
(1.73) 34.90
(1.55) Number of eggs collected 12.85(3.31) 11.80
(3.72) 12.21
(3.88) 11.95
(2.99) 11.89
(3.08) 11.93
(3.45) 12.51
(3.54) fertility(%) 79.37
(3.79) 74.15
(2.68) 78.51
(3.88) 80.31
(2.91) 77.75
(3.51) 74.98
(3.52) 79.01
(2.90)

※Numbers are average (SD).

<Experimental Example 1: Evaluation of cytotoxicity of the composition of the present invention>

The toxicity of the composition of the present invention to cells was confirmed by MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbromide) analysis.

Specifically, Ishikawa cells, an endometrial cell line, were treated with samples 1 to 7 according to the preparation example for 24 hours. The MTT solution (2 mg/ml) was reacted at 37 degrees in a CO ₂ incubator for about 4 hours, and the supernatant was removed. Living cells were reduced by intracellular oxidoreductase and a purple dye (Formazan) was dissolved in DMSO and measured at a wavelength of 540 nm using a microplate reader. Viable cells were expressed as percentage compared to control. As a result, it was confirmed that the composition of the present invention was not cytotoxic, as shown in Table 3 below.

Cell viability (% of control) control group 100 sample 1 98 sample 2 101 sample 3 103 sample 4 99 sample 5 104 sample 6 101 sample 7 101

<Experimental Example 2: Confirmation of the effect of increasing the implantation rate and pregnancy rate of the transplanted embryo>

In order to confirm the effect of increasing the implantation rate and pregnancy rate of the transferred embryos, the target group was randomly assigned, 10 patients were assigned to each sample, and the sample 1 treatment group was used as the control group to confirm the effect of increasing the implantation rate and pregnancy rate of the transferred embryos in the remaining samples. did.

Transplantation was carried out using the usual embryo transfer method. The collected eggs were cultured for about 4 days after in vitro fertilization, and then 20 μg of the composition of the present invention was placed in an embryo transfer catheter along with the two embryos and implanted into the patient's uterus. The pregnancy rate was calculated by counting positive β-hCG, and the implantation rate was calculated based on the number of embryos transferred to the pregnant patient. The results of the transferred embryos are shown in Table 4.

item sample 1 sample 2 sample 3 sample 4 sample 5 sample 6 sample 7 Pregnancy rate (%) 20 30 50 40 60 70 60 Implantation rate (%) 47.36 53.41 65.38 59.91 72.81 79.18 75.75

As shown in Table 4, the implantation rate and pregnancy rate were significantly increased when MMP-9 and MMP-2 were combined compared to MMP-9 alone, especially when the weight ratio of MMP-9 and MMP-2 was 1:1. It was confirmed that the implantation rate and pregnancy rate significantly increased. Furthermore, when taurourusodeoxycholic acid was additionally treated with MMP-9 and MMP-2, the implantation rate and pregnancy rate were further improved, and taurourusodeoxycholic acid was further improved. It was confirmed that the implantation rate and pregnancy rate increased most significantly when the acid concentration was 0.5 μg/ml.

The above description of the present invention is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. For example, each component described as unitary may be implemented in a distributed manner, and similarly, components described as distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims described below, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.

Claims (5)

Laminin 5 to 10 μg/ml, fibronectin 0.5 to 1 μg/ml, hyaluronan 15 to 20 μg/ml, MMP-9 (metalloproteinase-9) 0.1 μg/ml, A culture medium composition for embryo transplantation, comprising 0.1 μg/ml of MMP-2 (metalloproteinase-2) and 0.5 μg/ml of tauroursodeoxycholic acid as active ingredients.
According to paragraph 1,
The composition is,
A culture medium composition for embryo transplantation, which has the effect of increasing the implantation rate and pregnancy rate of the transplanted embryo. delete delete delete