Composition for treating uterus, agent for improving uterus environment, implantation aid, embryo transplantation solution, sperm transplantation solution, and agent for improving implantation rate

Abstract

The purpose of the present invention is to provide an excellent infertility treatment capable of improving the implantation rate of an embryo (fertilized egg) by improving the uterus environment. The present invention is a composition for treating uterus, which contains a mesenchymal stem cell culture supernatant, and which is used as a uterus environment-improving agent, an implantation aid, an embryo transplantation solution, a sperm transplantation solution or an implantation rate-improving agent. The mesenchymal stem cells are preferably derived from adipose tissue, umbilical cord tissue or bone marrow tissue.

Description

Technical Field

[0001] The present invention relates to a composition for uterine treatment, an agent for improving uterine environment, an implantation assisting agent, an embryo transfer fluid, a sperm transfer fluid, and an agent for improving implantation rate. Background Art

[0002] "Infertility" means that healthy men and women with the intention of pregnancy have sexual intercourse without contraception but do not become pregnant within a certain period of time. The Japan Society of Obstetrics and Gynecology defines this "certain period" as "usually 1 year". It is said that about one in every ten couples is infertile, and this proportion is actually higher because the age at which people consider pregnancy has been increasing in recent years, and it is known that both men and women have difficulty getting pregnant as they age.

[0003] The causes of female infertility include ovulation factors such as ovulation disorders, fallopian tube factors such as blocked fallopian tubes, narrowed fallopian tubes, and adhesions of fallopian tubes, uterine factors such as uterine fibroids and endometrial polyps, endocervical canal factors such as cervicitis and abnormal secretion of cervical mucus, and immune factors such as antisperm antibodies.

[0004] If endometrial blood flow is poor due to uterine fibroids, congenital uterine morphological abnormalities, etc., or adhesions occur in the uterus due to previous surgery or inflammation, etc., it will hinder the attachment and growth of embryos reaching the uterus, making it difficult to become pregnant.

[0005] In the endocervical canal, when ovulation is approaching, the mucus filling the inside of the endocervical canal changes to create an environment in which sperm can enter the uterus. And sperm reach the uterus and acquire the ability to penetrate the zona pellucida on the surface of the egg. However, if the secretion of this mucus is small or this mucus is not suitable for the above-mentioned penetration of sperm, etc., it will cause sperm to be difficult to reach the uterus, difficult to complete fertilization, and difficult to become pregnant.

[0006] In addition, women who produce antisperm antibodies such as sperm immobilizing antibodies due to certain immune abnormalities also secrete the antibodies into the cervical mucus. Even sperm with good motility are difficult to pass through. In addition, sperm immobilizing antibodies are also secreted into the fallopian tubes. Even if sperm are injected deep into the uterine cavity by artificial insemination, it will prevent them from passing through the fallopian tubes. Therefore, at the time of fertilization, sperm immobilizing antibodies will prevent sperm from binding to eggs and sometimes cause infertility.

[0007] In the treatment of infertility, the most suitable treatment method is selected according to the cause for treatment. The main treatment methods include timing selection method, ovulation induction method, artificial insemination, and then assisted reproductive therapies (ART: Assisted Reproductive Technology) such as in vitro fertilization and micromanipulation fertilization. It is said that the probability of having a child through assisted reproductive therapy is on average 11.7% in all treatments, but it varies according to age, being about 20% before the age of 32, decreasing with age, and being 7 - 8% after the age of 40. The current situation is that the pregnancy rate can be said to be not high. In addition, infertility treatment brings a huge physical and mental burden and is also costly. Therefore, the development of new infertility treatment methods is expected.

[0008] Mesenchymal stem cells are progenitor cells with multipotential differentiation ability first isolated from bone marrow by Friedenstein (1982) (see Non-Patent Document 1). It has been clarified that mesenchymal stem cells exist in various tissues such as bone marrow, umbilical cord, and adipose tissue, and mesenchymal stem cell transplantation is expected as a new treatment method for various intractable diseases (see Patent Documents 1 - 2). Recently, it has been learned that cells with the same function exist in mesenchymal stromal cells of adipose tissue, placenta, umbilical cord, egg membrane, etc. Therefore, mesenchymal stem cells are sometimes also referred to as mesenchymal stromal cells.

[0009] Prior Art Documents

[0010] Patent Documents

[0011] Patent Document 1: Japanese Patent Laid-Open No. 2012 - 157263

[0012] Patent Document 2: Japanese Patent Application Laid-Open for International Publication No. 2012 - 508733

[0013] Non-Patent Documents

[0014] Non-Patent Document 1: Pittenger F.M. et al., Science 284, pp. 143 - 147, 1999 Summary of the Invention

[0015] Problems to be Solved by the Invention

[0016] In order to improve the implantation rate, a method called ERA test for examining the implantation ability of the endometrium is known. The ERA test calculates the timing of easy implantation by observing the gene expression profile of endometrial tissue, rather than improving the implantation rate itself. There are few methods for improving the uterine environment to be easily implantable, and the development of new improvement methods is expected. The object of the present invention is to provide excellent infertility treatment under the above circumstances, which can improve the implantation rate of embryos (fertilized eggs) by improving the uterine environment.

[0017] Solution for solving problems

[0018] In order to solve the above problems, in-depth research has been carried out. As a result, the inventors of the present invention have found that the culture supernatant of mesenchymal stem (stromal) cells (MSC) can improve the implantation rate, thus completing the present invention. According to the present invention, the uterine environment can be improved and the implantation rate of embryos (fertilized eggs) can be increased. That is, the gist of the present invention is as follows.

[0019] [1] A composition for uterine treatment, which comprises the culture supernatant of mesenchymal stem cells.

[0020] [2] The composition for uterine treatment according to [1], which is used as a uterine environment improver, an implantation assisting agent, an embryo transfer fluid, a sperm transfer fluid or an implantation rate improver.

[0021] [3] The composition for uterine treatment according to [1] or [2], wherein the mesenchymal stem cells are derived from adipose tissue, umbilical cord tissue or bone marrow tissue.

[0022] [4] The composition for uterine treatment according to [1], wherein the culture supernatant of mesenchymal stem cells is the culture supernatant of mesenchymal stem cells obtained by culturing mesenchymal stem cells with a serum-free medium.

[0023] Effects of the invention

[0024] According to the present invention, the uterine environment can be improved and the implantation rate can be increased. For the uterus treated with the composition for uterine treatment of the present invention, for example, it is expected to promote decidualization and increase the implantation rate of embryos (fertilized eggs) in artificial insemination and in vitro fertilization, etc. Brief description of the drawings

[0025] Figure 1 A figure showing the blood vessel induction effect in the uterus brought about by the culture supernatant of mesenchymal stem cells.

[0026] Figure 2 A figure showing the endometrial changes (PAS staining (glycogen) and immunostaining of cell proliferation marker Ki67) brought about by the culture supernatant of mesenchymal stem cells.

[0027] Figure 3 A figure showing the endometrial changes (HE staining) brought about by the culture supernatant of mesenchymal stem cells.

[0028] Figure 4 A figure showing the endometrial changes (PAS staining (glycogen)) brought about by the culture supernatant of mesenchymal stem cells.

[0029] Figure 5A figure showing the endometrial morphology after transplantation of a fertilized egg into the uterus treated with the culture supernatant of mesenchymal stem cells.

[0030] Figure 6 A figure showing the effect of the culture supernatant of mesenchymal stem cells in improving the implantation rate (pregnancy rate) during in vitro fertilized egg transplantation.

[0031] Figure 7 A figure showing the effect of the culture supernatant of mesenchymal stem cells in promoting the proliferation of endometrial epithelial cells.

[0032] Figure 8 A figure showing that the culture supernatant of mesenchymal stem cells does not exert a promoting effect on the proliferation of endometrial mesenchymal stromal cells.

[0033] Figure 9-1 A figure showing the implantation rate in the in vitro fertilization of Example 6.

[0034] Figure 9-2 A figure showing the pregnancy rate in the in vitro fertilization of Example 6. Detailed Description

[0035] The uterine treatment composition of the present invention will be described in detail below. In addition, the uterine environment improver, implantation assistant, embryo transplantation fluid, sperm transplantation fluid, and implantation rate improver of the present invention will also be described.

[0036] [Uterine Treatment Composition]

[0037] The uterine treatment composition of the present invention is characterized by containing the culture supernatant of mesenchymal stem cells. By containing the culture supernatant of mesenchymal stem cells, the uterine treatment composition of the present invention can improve the uterine environment and increase the implantation rate. The uterus treated with the uterine treatment composition of the present invention is expected to promote decidualization and increase the implantation rate of embryos (fertilized eggs) in artificial insemination and in vitro fertilization, etc. The uterine treatment composition of the present invention may contain other components within the range that does not impair the effects of the present invention in addition to the culture supernatant of mesenchymal stem cells as an essential component.

[0038] (Culture Supernatant of Mesenchymal Stem Cells)

[0039] In the present invention, the culture supernatant of mesenchymal stem cells refers to the culture supernatant obtained when culturing mesenchymal stem cells.

[0040] In the present invention, mesenchymal stem cells refer to cells that have the ability to differentiate into one or more cells belonging to mesenchyme (osteocytes, cardiomyocytes, chondrocytes, tendon cells, adipocytes, etc.) and can proliferate while maintaining this ability. The term "mesenchymal stem cells" used in the present invention refers to the same cells as mesenchymal stromal cells, and no special distinction is made between the two. Additionally, it is sometimes abbreviated as "mesenchymal cells". As tissues containing mesenchymal stem cells, for example, adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amnion, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, tooth germ, etc. can be cited. As mesenchymal stem cells in the present invention, mesenchymal stem cells derived from adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amnion, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, tooth germ, etc. can be cited. Among them, mesenchymal stem cells derived from adipose tissue, umbilical cord, and bone marrow are preferred, and mesenchymal stem cells derived from adipose tissue and umbilical cord are more preferred.

[0041] As the species of mesenchymal stem cells in the present invention, human, horse, bovine, sheep, pig, dog, cat, rabbit, mouse, and rat can be cited.

[0042] The mesenchymal stem cells in the present invention and the subject to be treated (test subject) can be of the same origin or of different origins.

[0043] The mesenchymal stem cells can be, for example, cells provided by PromoCell GmbH, Lonza Group Ltd., Biological Industries, Veritas Genetics, R&D Systems, Inc., and Corning Incorporated, etc., or cells prepared by those skilled in the art by known methods. Additionally, the mesenchymal stem cells can be primary cells isolated from donor tissues or cell line-derived cells.

[0044] In the present invention, the culture medium for culturing mesenchymal stem cells is not particularly limited as long as it can culture mesenchymal stem cells while maintaining a good state, for example, a culture medium that can preferably enable human mesenchymal stem cells to proliferate while maintaining the ability to differentiate into osteocytes, chondrocytes, and adipocytes is preferred.

[0045] The culture medium used in the present invention can be prepared by adding one or more serum substitutes such as serum, and / or albumin, transferrin, fatty acids, insulin, sodium selenite, cholesterol, collagen precursors, trace elements, 2-mercaptoethanol, 3'-mercapto glycerol, etc. to a basal medium. Additionally, in these culture media, amino acids such as glutamine, sugars such as glucose, metal salts such as sodium chloride and magnesium sulfate, trace metals such as selenium, lipids such as cholesterol and unsaturated fatty acids, vitamins such as pantothenic acid, proteins such as albumin, insulin, transferrin, growth factors, proliferation factors, cytokines, polysaccharides, low-molecular compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and other substances can be further added as needed.

[0046] Examples of the above-mentioned basal medium include IMDM medium, Medium 199 medium, Eagle's modified basal medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, MCDB201 medium, and mixed media thereof.

[0047] From the viewpoint of uterine treatment, the medium for culturing mesenchymal stem cells used in the present invention is preferably a medium (free of heterologous substances) that does not contain xenogeneic components such as serum. As such a medium, for example, Mesenchymal Stem Cell Growth Medium 2 (Ready-to-use, manufactured by PromoCell), Mesenchymal Stem Cell Growth Medium XF (Ready-to-use, manufactured by PromoCell), MSCGM BulletKit tm, MSCGM tm Mesenchymal Stem Cell Growth Medium BulletKit tm (manufactured by Lonza), Serum-Free Medium for Human Mesenchymal Stem Cells (MSC NutriStem (registered trademark) XF, manufactured by Biological Industries), MesenCult-ACF Plus (manufactured by Veritas), StemXVivo tm Serum-Free Human MSC Expansion Media (manufactured by R&D Systems, Corning), Serum-Free Medium for Adipose-Derived Stem Cells (KBM ADSC-4, manufactured by Kohjin-bio), and Serum-Free Medium for Mesenchymal Stem Cells (R: STEM Medium for hMSC High Growth, manufactured by Rohto) can be cited as media provided as media prepared in advance for the use of mesenchymal stem cells (mesenchymal stromal cells).

[0048] As the above-mentioned serum, for example, human serum, fetal bovine serum (FBS), bovine serum, calf serum, goat serum, horse serum, pig serum, sheep serum, rabbit serum, rat serum, etc. can be cited, but are not limited to these. When using serum, it can be added at 5 v / v% to 15 v / v%, preferably 10 v / v%, relative to the basal medium.

[0049] Examples of the above fatty acids include linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoleic acid, palmitic acid, stearic acid, etc., but are not limited to these. Examples of lipids include phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, etc., but are not limited to these. Amino acids include, for example, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-glycine, etc., but are not limited to these. Examples of proteins include Ecotin, reduced glutathione, fibronectin, β2-microglobulin, etc., but are not limited to these. Examples of polysaccharides include glycosaminoglycans, and among glycosaminoglycans, hyaluronic acid, heparan sulfate, etc. can be particularly exemplified, but are not limited to these. Examples of growth factors include platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor β (TGF-β), hepatocyte growth factor (HGF), epidermal growth factor (EGF), connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), etc., but are not limited to these.

[0050] (Preparation of Mesenchymal Stem Cell Culture Supernatant)

[0051] The supernatant of mesenchymal stem cells obtained by the following method can be used as the mesenchymal stem cell culture supernatant in the present invention. In addition, substances obtained by removing unnecessary components from the supernatant by means such as dialysis and ultrafiltration, fractions obtained by fractionating the supernatant using a column, etc., fractions selected using antibodies against specific molecules, fractions obtained by centrifugation, etc. can also be used as the mesenchymal stem cell culture supernatant in the present invention.

[0052] As the medium used for obtaining the culture supernatant, the same medium as that used for culturing mesenchymal stem cells can be used. The method for obtaining the culture supernatant is not particularly limited as long as it is suitable for culturing each mesenchymal stem cell. For example, it is a method of culturing mesenchymal stem cells at a temperature of 20°C to 37°C, in an environment of 2% to 7% CO2 and 5% to 21% O2, preferably at room temperature to 37°C and in an environment of 5% CO2, and obtaining the culture supernatant thereof.

[0053] For the mesenchymal stem cell culture supernatant of the present invention, as long as the mesenchymal stem cells are in contact with the medium, the washing solution obtained by washing the mesenchymal stem cells with the medium can also be used as the culture supernatant in the present invention. The contact time between the mesenchymal stem cells and the medium is, for example, within 14 days, preferably within 10 days, more preferably within 7 days, and even more preferably within 5 days. The culture for obtaining the culture supernatant can be monolayer culture attached to a flask or suspension-stirring culture attached to microbeads, etc.

[0054] The amount of the mesenchymal stem cell culture supernatant contained in the uterine treatment composition of the present invention is 0.1% to 100% by weight of the whole uterine treatment composition, preferably 0.5% to 95% by weight, more preferably 1% to 90% by weight, still more preferably 3% to 80% by weight, and particularly preferably 5% to 50% by weight. By making the amount of the mesenchymal stem cell culture supernatant contained in the uterine treatment composition of the present invention within the above numerical range, the uterine treatment composition of the present invention has excellent effects of improving the uterine environment and increasing the implantation rate.

[0055] In addition to the mesenchymal stem cell culture supernatant, the uterine treatment composition of the present invention may contain other components within the range that does not hinder the effects of the present invention. Examples of other components include protective agents such as dimethyl sulfoxide (DMSO) and serum albumin, antibiotics, vitamins, carriers, excipients, disintegrants, buffers, emulsifiers, stabilizers, preservatives, antiseptics, and physiological saline.

[0056] The species that is the subject of the uterine treatment composition of the present invention may be any mammal, and examples include humans, horses, cows, sheep, pigs, dogs, cats, rabbits, mice, rats, and rare animals.

[0057] The uterine treatment composition of the present invention is used as a liquid agent that directly contacts the uterus, such as an implantation adjuvant, an embryo transfer fluid, a uterine environment improver, a sperm transfer fluid, and an implantation rate improver.

[0058] An implantation adjuvant refers to a preparation used to help a fertilized egg better contact and implant in the endometrium, that is, "implantation". An embryo transfer fluid refers to a solution used to place an embryo (fertilized egg) when the embryo (fertilized egg) returns to the uterus after developing to a certain extent, that is, "embryo transfer". A uterine environment improver refers to a preparation used to improve the uterine environment to a state more suitable for pregnancy. The uterine environment improver of the present invention can be used for artificial insemination / in vitro fertilization (to improve success rate and efficiency), livestock breeding (to improve the success rate and efficiency of artificial insemination, breeding / species maintenance (such as the maintenance of endangered species, the maintenance of pet strains or hybridization), and the treatment / improvement of various diseases with uterine disorders as the main or secondary cause (such as uterine disorders after malignant tumor surgery or chemotherapy). A sperm transfer fluid refers to a solution used to suspend sperm when injecting sperm into the uterus during artificial insemination. An implantation rate improver is a preparation used to increase the implantation rate of an embryo (fertilized egg).

[0059] The composition for uterine treatment of the present invention acts on the uterus and can increase the expression of implantation-related genes in endometrial epithelial cells and induce decidualization. Examples of the above-mentioned implantation-related genes include Lif (Leukemia Inhibitory Factor), FOXO1 (Forkhead box protein O1), Hoxa-10 (Homeobox A10), Integrin β-3 (Integrinβ3, Int3b), etc.

[0060] The composition for uterine treatment of the present invention can be prepared by mixing other necessary components into the above-mentioned mesenchymal stem cell culture supernatant by a conventional method.

[0061] Examples

[0062] The following examples and test examples are listed to illustrate the present invention in detail, but the present invention is not limited by these examples, etc.

[0063] [Example 1: Preparation of culture supernatant and detection of components contained in the culture supernatant]

[0064] Umbilical cord mesenchymal stem cells (C-12971 Human Mesenchymal Stem Cells from Umbilical Cord Matrix (hMSC-UC), manufactured by PromoCell) were cultured in RIM medium (serum-free medium for mesenchymal stem cells, containing EGF, bFGF, albumin, transferrin, and insulin, manufactured by Rohto) or RS medium (serum-free medium for mesenchymal stem cells, containing EGF, bFGF, albumin, transferrin, and insulin, manufactured by Rohto) for 3 days to obtain culture supernatants (hereinafter referred to as "3D RIM culture supernatant" and "3D RS culture supernatant" respectively). In addition, after 3 days of culture, the medium was replaced and cultured for another 1 day to obtain culture supernatants (hereinafter referred to as "1D RIM culture supernatant" and "1D RS culture supernatant" respectively). Various factors contained in each culture supernatant were measured by ELISA, and as a result, it was confirmed that all contained PGE2, IL-6, HGF, MCP1, MMP2, BDNF, and CD63+ exosomes.

[0065] [Example 2: Vascular induction]

[0066] HTF medium (manufactured by Fujifilm Wako Pure Chemical Corporation) supplemented with 3D RIM culture supernatant or 1D RIM culture supernatant at a final concentration of 10%, and HTF medium without added supernatant (control) as a negative control were injected into the uteri of 8-week-old female mice. As a positive control, 8-week-old female mice were mated with 12-week-old male mice. Two days after injecting these, the uteri were excised, and their images are shown in Figure 1 . In addition, the figure shows the proportion of uteri with vascular induction under each condition.

[0067] As Figure 1 shown, it was confirmed that the addition of cell culture supernatant induced blood vessels and induced uterine changes similar to those caused by coital stimulation.

[0068] [Example 3: Endometrial changes (improvement of uterine environment)]

[0069] HTF medium (manufactured by Fujifilm Wako Pure Chemical Corporation) supplemented with 1D RIM culture supernatant at a final concentration of 10%, and HTF medium without added supernatant (control) as a negative control were injected into the uteri of 8-week-old female mice. As a positive control, 8-week-old female mice were mated with 12-week-old male mice. Two days after injecting these, the uteri were excised, fixed with 4% paraformaldehyde for 12 hours, and then embedded in paraffin. The embedded uteri were sectioned and subjected to PAS staining for glycogen staining and immunostaining using an antibody recognizing the cell proliferation marker Ki67. The stained images are shown in Figure 2 .

[0070] In addition, HTF medium (manufactured by Fujifilm Wako Pure Chemical Corporation) supplemented with 1D RIM culture supernatant at a final concentration of 10%, and HTF medium without added supernatant (control) as a negative control were injected into the uteri of 8-week-old female mice. Four days later, corn oil was injected surgically to induce decidualization of the endometrium. Three days after injecting the corn oil, the uteri were excised, fixed with 4% paraformaldehyde for 12 hours, and then embedded in paraffin. The embedded uteri were sectioned, stained with HE for morphological observation, and stained with PAS to verify glycogen accumulation. The HE-stained images are shown in Figure 3 , and the PAS-stained images are shown in Figure 4 .

[0071] In vitro fertilization was performed using eggs recovered from 3-week-old female mice and sperm recovered from 12-week-old male mice to produce in vitro fertilized eggs. HTF medium (manufactured by Fujifilm Wako Pure Chemical Corporation) supplemented with 1D RIM culture supernatant at a final concentration of 10% and HTF medium without supernatant as a negative control (control) were injected into the uterus of 8-week-old female mice. The in vitro fertilized eggs produced above were transplanted into the fallopian tubes the next day. After 7 days, the uterus was removed, fixed with 4% paraformaldehyde for 12 hours, and then embedded in paraffin. Sections of the embedded uterus were made and the morphology was observed by HE staining. The stained images are shown in Figure 5 .

[0072] As Figures 2 to 5 shown, it was confirmed that by injecting the cell culture supernatant into the uterus, glycogen is decomposed and metabolized after glycogen accumulation, and cell proliferation is promoted, that is, decidualization is promoted, and the uterine environment is improved to make the endometrium more receptive to implantation.

[0073] [Example 4: In vitro fertilization - 1]

[0074] In vitro fertilization was performed using eggs recovered from 3-week-old female mice and sperm recovered from 12-week-old male mice to produce in vitro fertilized eggs. HTF medium (manufactured by Fujifilm Wako Pure Chemical Corporation) supplemented with 1D RIM culture supernatant at a final concentration of 10% and HTF medium without supernatant as a negative control (control) were injected into the uterus of 8-week-old female mice. Twenty in vitro fertilized eggs produced were transplanted into the fallopian tubes the next day. After 7 days, Evans blue solution was injected into the tail vein of the mice, and then the uterus was removed and the number of implantations was counted. In addition, the implantation rate (pregnancy rate) was measured and is shown in Figure 6 .

[0075] As Figure 6 shown, the implantation rate (pregnancy rate) of in vitro fertilized eggs was increased by treating the uterus with the cell culture supernatant.

[0076] [Example 5: In vitro culture of endometrial mesenchymal stromal cells]

[0077] The uterus was removed from 12-week-old C57BL6 female mice given estradiol to induce estrus. After the excised uterus was cut open, it was treated with an HBSS solution (CaCl2·2H2O 0.185 g / L, MgSO4 0.098 g / L, KCl 0.400 g / L, KH2PO4 0.060 g / L, NaHCO3 0.350 g / L, NaCl 8.000 g / L, Na2HPO4 0.048 g / L, D-glucose 1.0 g / L) containing 25 mg / mL trypsin preparation (P1750; Sigma-Aldrich) and 0.25% (v / v) trypsin EDTA (nacalai tesque) to separate epithelial cells. In addition, after inoculating the cell suspension into a collagen-coated culture dish (IWAKI), it was treated with HBSS containing 0.1 mg / mL collagenase type I (Worthington) and 0.05% (v / v) trypsin EDTA to separate mesenchymal stromal cells. These cells were inoculated into a collagen-coated culture dish and cultured in a modified DMEM and Ham’s F-12 medium (10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin) containing 10% of the 1D RIM culture supernatant or a modified DMEM and Ham’s F-12 medium without the culture supernatant as a control at 37 °C, 5% CO2, 95% atmosphere, and a humid environment for 48 hours.

[0078] After washing with HBSS, the cells were detached using 0.25% (v / v) trypsin, and the cell count was measured using an automatic cell counter TC20TM.

[0079] The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of the cultured cells were measured using an extracellular flux analyzer (XF HS Mini; Agilent Technologies). After detaching the cultured cells, they were suspended in RPMI medium (manufactured by Agilent Technologies, supplemented with 10 mM D-glucose, 1 mM pyruvate, 2 mM L-glutamine, 1% FBS) and inoculated into the wells for analysis.

[0080] The extracellular flux analysis was based on 9 minutes per cycle (mixing for 3 min / waiting for 3 min / measuring for 3 min). In the ECAR measurement, after 5 cycles of measurement, D-glucose (final concentration 10 mM; Agilent Technologies) was injected and 3 cycles of measurement were performed. The basal ECAR was the value measured in the 5th cycle. Glycolysis was calculated by subtracting the value of the basal ECAR from the value measured in the 8th cycle.

[0081] In the measurement of OCR, the following steps were performed:

[0082] (1) 5 cycles with only RPMI medium [initial OCR]

[0083] (2) 3 cycles of ATP synthesis inhibition caused by oligomycin (Sigma - Aldrich) at a final concentration of 1 μM [post - oligomycin OCR]

[0084] (3) 3 cycles of mitochondrial uncoupling caused by carbonyl cyanide 4 - (trifluoromethoxy)phenylhydrazone (FCCP; Sigma - Aldrich) at a final concentration of 5 μM [post - FCCP OCR]

[0085] (4) 3 cycles of inhibition of the electron transport chain caused by rotenone (Sigma - Aldrich) / antimycin A (Sigma - Aldrich) at final concentrations of 1 μM each [post - rotenone / antimycin - A OCR].

[0086] The measured values of OCR were calculated using the final cycle values of each condition (the values of the 5th, 8th, 11th, and 14th cycles).

[0087] Each index was calculated through the following steps:

[0088] Basal OCR = initial OCR - post - rotenone / antimycin - A OCR

[0089] ATP Production = Basal OCR - post - oligomycin OCR

[0090] As Figure 7 shown, the addition of the supernatant significantly promoted the proliferation of endometrial epithelial cells. On the other hand, endometrial mesenchymal stromal cells did not show a proliferation - promoting effect due to the addition of the supernatant ( Figure 8 ), and its effect is selective for endometrial epithelial cells.

[0091] [Example 6: In vitro fertilization - 2]

[0092] Cord mesenchymal stem cells (C-12971 Human Mesenchymal Stem Cells from Umbilical Cord Matrix (hMSC-UC), manufactured by Promocell) were cultured in RIM medium (a serum-free medium for mesenchymal stem cells suitable for regenerative medicine and other product materials, undergoing consultation and confirmation for suitability and registration application for active pharmaceutical ingredients, etc., containing EGF, bFGF, albumin, transferrin, and insulin, manufactured by Rohto) for 3 days. Then, the medium was changed and further cultured for 1 day to obtain the culture supernatant (hereinafter referred to as "1D RIM culture supernatant" respectively). By using ELISA to measure various factors contained in each culture supernatant, it was confirmed that all contained PGE2, IL-6, HGF, MCP1, MMP2, BDNF, and CD63+ exosomes.

[0093] In vitro fertilization was performed using eggs recovered from 3-week-old female mice and sperm recovered from 12-week-old male mice to produce in vitro fertilized eggs. After transplanting the produced in vitro fertilized eggs into the fallopian tubes of 8-week-old female mice, the day of in vitro fertilized egg transplantation was set as day 1, and on days 0, 3, and 4, HTF medium (manufactured by Fujifilm Wako Pure Chemical Corporation) supplemented with 1D RIM culture supernatant at a final concentration of 10% was injected into the uterus of 8-week-old female mice. After 7 days, Evans blue solution was injected into the tail vein of the mice, and then the uterus was excised to count the number of implantations. In addition, the implantation rate and pregnancy rate were measured and shown in Figure 9-1 (implantation rate) and Figure 9-2 (pregnancy rate).

[0094] As Figure 9-1 and 9-2 shown, by treating the uterus with the cell culture supernatant, the implantation rate (pregnancy rate) of in vitro fertilized eggs was increased.

[0095] In addition, it was confirmed that in the HTF medium group supplemented with 1D RIM culture supernatant, the expression of implantation-related genes (Lif, FOXO1, Hoxa-10, Int3b) in endometrial epithelial cells increased, suggesting the possibility that the implantation preparation of endometrial epithelial cells was promoted. Furthermore, nuclear translocation of FOXO1 was confirmed, so it was considered that decidualization was induced. It can be seen that 1D RIM culture supernatant has the effects of improving the uterine environment, assisting in the implantation of fertilized eggs, and improving the implantation rate. 1D RIM culture supernatant can be suitably used as a uterine environment improver, implantation assistant, embryo transfer fluid, sperm transfer fluid, or implantation rate improver.

[0096] Industrial Applicability

[0097] The composition for uterine treatment of the present invention can be used for artificial insemination and in vitro fertilization (to improve success rate and efficiency), livestock breeding (to improve the success rate and efficiency of artificial insemination), breeding and species maintenance (such as the maintenance of endangered species, the maintenance of pet strains or hybridization), the treatment and improvement of various diseases with uterine disorders as the main or secondary cause (such as uterine disorders after malignant tumor surgery or chemotherapy), etc.

Claims

1. A composition for uterine treatment, which comprises a mesenchymal stem cell culture supernatant.

2. The composition for uterine treatment according to claim 1, which is used as a uterine environment improver, an implantation assisting agent, an embryo transfer fluid, a sperm transfer fluid or an implantation rate improver.

3. The composition for uterine treatment according to claim 1 or 2, wherein The mesenchymal stem cells are derived from adipose tissue, umbilical cord tissue or bone marrow tissue.

4. The composition for uterine treatment according to claim 1, wherein, The mesenchymal stem cell culture supernatant is a mesenchymal stem cell culture supernatant obtained by culturing mesenchymal stem cells with a serum-free medium.

Images