Composition for treating immature egg and / or embryo (fertilized egg)

Abstract

The purpose of the present invention is to provide a culture medium for immature eggs that is excellent in quality and safety and that is capable of efficiently maturing immature eggs with respect to immature eggs for vitro maturation-in vitro fertilization-embryo transfer method (IVM-IVF). The present invention is a composition that is for treating immature eggs and / or embryos (fertilized eggs) and that contains a serum-free mesenchymal stem cell culture medium. The treatment composition is suitably used as an immature egg culture agent, an embryo (fertilized egg) culture agent, an implantation adjuvant, an embryo transfer liquid or an implantation rate improver.

Description

Composition for treating immature eggs and / or embryos (fertilized eggs)

[0001] The present invention relates to a composition for treating immature eggs and / or embryos (fertilized eggs).

[0002] "Infertility" refers to a situation in which a healthy man and woman who wish to conceive are unable to conceive for a certain period of time despite having unprotected sexual intercourse, and the Japan Society of Obstetrics and Gynecology defines this "certain period" as "generally one year." It is said that approximately one in ten couples is infertile, but in recent years, the age at which people consider becoming pregnant has been increasing, and it is known that both men and women find it more difficult to conceive with age, so this percentage is said to be even higher.

[0003] Causes of female infertility include ovulatory factors such as ovulation disorders, fallopian tube factors such as blockage of the fallopian tubes, stenosis of the fallopian tubes, and adhesions of the fallopian tubes, uterine factors such as uterine fibroids and endometrial polyps, cervical factors such as cervicitis and abnormal mucus secretion from the cervix, and immune factors such as antisperm antibodies. In general, fallopian tube disorders are considered to be the most common cause of female infertility.

[0004] Infertility treatment is carried out by selecting the most appropriate treatment depending on the cause. The main treatments include timing, ovulation induction, artificial insemination, and advanced reproductive technologies such as in vitro fertilization and microscopic insemination.

[0005] In vitro fertilization and embryo transfer (IVF-ET) can be divided into fresh embryo transfer and frozen-thawed embryo transfer. With fresh embryo transfer, embryo transfer is performed in the cycle in which eggs are retrieved through ovarian stimulation or natural cycle. With frozen-thawed embryo transfer, fertilized eggs or embryos are temporarily frozen after egg retrieval, and then embryo transfer is performed in a subsequent cycle.

[0006] Immature oocyte maturation-in vitro fertilization-embryo transfer (IVM-IVF) involves collecting immature eggs from the ovaries of patients who have not undergone ovarian stimulation or who have only received very low doses of ovulation-inducing drugs, maturing them outside the body (in vitro maturation (IVM)), and then fertilizing the mature eggs via intracytoplasmic sperm injection or insemination. This method is selected when follicles do not mature normally despite ovarian stimulation using ovulation-inducing drugs, or when oocytes must be collected at a premature stage to avoid ovarian hyperstimulation syndrome (OHSS) in patients at high risk for OHSS due to an overreaction of the ovaries to ovulation-inducing drugs. It can also be used when, for some reason, there is a problem with the normal process of egg maturation and the eggs are unable to develop into mature eggs that are sufficiently receptive to sperm.

[0007] Unlike IVF-ET, IVM-IVF requires almost no ovarian stimulation, which means less physical and financial burden from drug administration. It can also be used with frozen cells, which is thought to broaden the range of treatment options for patients. Furthermore, it may be used as an infertility treatment for patients whose ovaries do not produce immature eggs.

[0008] However, existing IVM-IVF methods still pose challenges, such as low immature oocyte maturation efficiency and treatment success rate. Furthermore, many of the media currently used for culturing immature oocytes contain serum (see Non-Patent Document 1). Serum is considered extremely important as a source of growth factors, adhesion factors, hormones, lipids, and minerals required for cell culture in basal medium. However, serum contains many components, including unknown substances. Furthermore, because the components vary depending on the serum lot, performance tends to vary, making it difficult to obtain serum-containing media of consistent quality. Furthermore, because serum is derived from a biological source, there is a risk of infection and allergy, raising safety concerns. Therefore, there is a need for the development of a stable, safe immature oocyte culture medium and a culture method that can improve the maturation efficiency of immature oocytes.

[0009] J.Mamm.Ova.Res.,1998, vol.15, 109-112

[0010] In order to perform IVM-IVF, it is necessary to mature immature eggs in vitro to the stage where they can be used for in vitro fertilization. In light of the above-mentioned circumstances, the present invention aims to provide an immature egg culture medium with stable quality and high safety. Another aim is to provide an excellent method for maturing immature eggs and infertility treatment that can efficiently mature immature eggs in vitro, thereby providing more treatment opportunities for patients and further improving the maturation of embryos (fertilized eggs) and the implantation efficiency during embryo transfer.

[0011] As a result of intensive research to solve the above-mentioned problems, the present inventors discovered that a serum-free medium for mesenchymal stem (stromal) cells (MSCs) can mature immature eggs, and thus completed the present invention. According to the present invention, it is possible to efficiently mature immature eggs, promote embryo (fertilized egg) development, and increase the implantation rate. That is, the gist of the present invention is as follows: [1] A composition for treating immature eggs and / or embryos (fertilized eggs), comprising a serum-free mesenchymal stem cell medium. [2] The treatment composition according to [1], which is used as an immature egg culture agent, an embryo (fertilized egg) culture agent, an implantation aid, an embryo transfer solution, or an implantation rate improver. [3] The treatment composition according to [1] or [2], wherein the serum-free mesenchymal stem cell medium comprises EGF, bFGF, albumin, transferrin, and insulin.

[0012] The composition for treating immature eggs and / or embryos (fertilized eggs) of the present invention can efficiently mature immature eggs in IVM-IVF, promote embryo maturation, and improve implantation rates.

[0013] FIG. 1 shows a scheme for a test to examine the developmental potential of mature eggs after in vitro maturation and in vitro fertilization.

[0014] The composition for treating immature eggs and / or embryos (fertilized eggs) of the present invention will be described in detail below. Also described will be an immature egg culture agent, an embryo (fertilized egg) culture agent, an immature egg maturation promoter, an embryo (fertilized egg) formation promoter, a blastocyst formation promoter, an implantation aid, an embryo transfer solution, and an implantation rate improver of the present invention.

[0015] [Composition for treating immature eggs and / or embryos (fertilized eggs)] The composition for treating immature eggs and / or embryos (fertilized eggs) of the present invention (hereinafter also simply referred to as "treatment composition") is characterized by containing a mesenchymal stem cell medium. By containing a mesenchymal stem cell medium, the treatment composition of the present invention can efficiently mature immature eggs when cultured in immature egg in vitro maturation-in vitro fertilization-embryo transfer (IVM-IVF), promote embryo (fertilized egg) development, and improve implantation rates. The treatment composition of the present invention is suitably used not only for culturing immature eggs, but also for culturing embryos (fertilized eggs), transplanting embryos (fertilized eggs), assisting in embryo (fertilized egg) implantation, improving the implantation rate of embryos (fertilized eggs), and can be used to improve the success rate and efficiency of artificial insemination and in vitro fertilization in humans, livestock breeding (improving the success rate and efficiency of in vitro fertilized egg production and transplantation), animal breeding and species maintenance (for example, preserving endangered species, maintaining or crossbreeding pet breeds), and the treatment and improvement of various diseases whose primary or secondary cause is impaired egg maturation.

[0016] (Mesenchymal stem cell medium) In the present invention, the mesenchymal stem cell medium is a medium used for culturing mesenchymal stem cells.

[0017] In the present invention, mesenchymal stem cells refer to cells that have the ability to differentiate into one or more types of cells belonging to the mesenchymal system (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 stromal cells, and does not particularly distinguish between the two.

[0018] In the present invention, the medium used for culturing mesenchymal stem cells is not particularly limited as long as it is a medium that can culture mesenchymal stem cells while maintaining their state in good condition. However, it is preferable that the medium be a medium that can proliferate human mesenchymal stem cells while maintaining their ability to differentiate into bone cells, chondrocytes, and adipocytes.

[0019] The mesenchymal stem cell culture medium of the present invention may be prepared by adding to a basal medium one or more serum substitutes, such as albumin, transferrin, fatty acids, insulin, sodium selenite, cholesterol, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc. Furthermore, these media may further contain, as necessary, substances such as 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, growth factors, proteins such as cytokines, polysaccharides, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc.

[0020] Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, MCDB201 medium, and mixed media thereof.

[0021] The mesenchymal stem cell medium contained in the treatment composition of the present invention is preferably a xeno-free medium that does not contain xenogeneic components such as serum, from the viewpoint of using it to treat immature eggs. Examples of such media include 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 Bullet Kit, MSCGM™ Mesenchymal Stem Cell Growth Medium Bullet Kit (manufactured by Lonza), and Xeno-Free Medium for Human Mesenchymal Stem Cells (MSC NutriStem (registered trademark) XF, Biological Examples of such a medium include media provided as pre-prepared media for mesenchymal stem cells (stromal cells), such as MesenCult-ACF Plus (manufactured by Veritas), StemXVivotm 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).

[0022] Examples of the fatty acids include, but are not limited to, linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, and stearic acid. Examples of lipids include, but are not limited to, phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine. Examples of amino acids include, but are not limited to, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, and L-glycine. Examples of proteins include, but are not limited to, ecotin, reduced glutathione, fibronectin, and β2-microglobulin. Examples of polysaccharides include glycosaminoglycans, and among glycosaminoglycans, particular examples include, but are not limited to, hyaluronic acid and heparan sulfate. Examples of growth factors include, but are not limited to, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), connective tissue growth factor (CTGF), and vascular endothelial growth factor (VEGF).

[0023] The treatment composition of the present invention may contain other components in addition to the mesenchymal stem cell medium, provided that the effects of the present invention are not impaired. Examples of other components include protective agents such as dimethyl sulfoxide (DMSO) and serum albumin, antibiotics, carriers, excipients, disintegrants, buffers, emulsifiers, stabilizers, preservatives, antiseptics, and physiological saline.

[0024] The target species of the treatment composition of the present invention may be any mammal, such as humans, horses, cattle, sheep, pigs, dogs, cats, rabbits, mice, rats, and rare animals, and preferably humans, cattle, dogs, and cats.

[0025] The treatment composition of the present invention is used as a liquid preparation that comes into direct contact with immature eggs or embryos (fertilized eggs), such as an immature egg culture agent, an embryo (fertilized egg) culture agent, an immature egg maturation promoter, an embryo (fertilized egg) formation promoter, a blastocyst formation promoter, an implantation aid, an embryo transfer liquid, or an implantation rate improver.

[0026] An immature egg culture agent is a composition for maturing eggs collected in an immature state in vitro until they can be fertilized with sperm. It is also called an immature egg maturation promoter. It may be in the form of a culture medium (culture medium) used to culture immature eggs, or it may be added to the culture medium (culture medium) and used. An embryo (fertilized egg) culture agent is a liquid for culturing fertilized eggs in vitro until they reach the blastocyst stage, which can be transplanted into the uterus. It is also called an embryo (fertilized egg) formation promoter or blastocyst formation promoter. An implantation aid is a preparation that assists in the successful implantation of a fertilized egg, which is when the fertilized egg contacts the uterine lining and settles there. An embryo transfer solution is a solution in which an embryo (fertilized egg) is placed during embryo transfer, in which the embryo (fertilized egg) is grown to a certain extent and then returned to the uterus. An implantation rate improver is a preparation for improving the implantation rate of an embryo (fertilized egg).

[0027] The treatment composition of the present invention can be prepared by a conventional method by mixing the above-mentioned mesenchymal stem cell culture medium with other necessary ingredients.

[0028] The present invention also includes a method for promoting the maturation of immature eggs, a method for promoting embryo (fertilized egg) formation, a method for promoting blastocyst formation, and a method for improving the implantation rate of fertilized eggs in vitro, using the above-mentioned treatment composition of the present invention.

[0029] The method for promoting maturation of immature eggs of the present invention comprises the step of culturing immature eggs collected from ovaries in the treatment composition (medium) of the present invention for 2 to 90 hours, preferably 5 to 60 hours, and more preferably 10 to 48 hours. Compared to conventional methods, this method can improve the maturation rate of oocytes and promote embryo (fertilized egg) formation and blastocyst formation.

[0030] The method for promoting embryo (fertilized egg) formation and the method for promoting blastocyst formation of the present invention comprise the steps of in vitro fertilizing oocytes matured using the treatment composition of the present invention with sperm, and then culturing the oocytes using the treatment composition of the present invention for 1 to 21 days, preferably 2 to 14 days, and more preferably 4 to 8 days. This method using the treatment composition of the present invention can promote embryo (fertilized egg) formation and blastocyst formation.

[0031] The method for improving the fertilized egg implantation rate of the present invention is a method that, in addition to the above-mentioned methods for promoting maturation of immature eggs, methods for promoting embryo (fertilized egg) formation, and methods for promoting blastocyst formation, includes a step of treating an embryo (fertilized egg) with the treatment composition of the present invention during embryo transfer, in which the embryo (fertilized egg) is transferred into the uterus of a surrogate mother. This method using the treatment composition of the present invention can improve the fertilized egg implantation rate.

[0032] The present invention will be described in detail below with reference to examples and test examples, but the present invention is not limited to these examples.

[0033] Example: Examination of in vitro maturation of immature oocytes and developmental potential after in vitro fertilization. A test was performed according to the scheme shown in Figure 1. Specifically, immature oocytes were collected from the ovaries of cattle (Japanese Black, Holstein, and crossbreeds thereof) and cultured for 21 hours in R:STEM medium (R:STEM Medium for hMSC High Growth, Regenerative Medicine Product Material Eligibility Confirmation Certificate, PMDA Director-General's Notification No. 492, Drug Substance Registration Master File Application Planned, Serum-free medium for mesenchymal stem cells, containing EGF, bFGF, albumin, transferrin, and insulin, Rohto Co.) supplemented with 1 μg / ml β-estradiol (E-8875; Merck Millipore Co.) and 0.02 mg / ml FSH (Antrin; Kyoritsu Tokyo, Japan), and the maturation rate of the oocytes was examined. As a control, oocytes were cultured for 21 hours in TCM199 medium (Gibco, Grand Island, NY) supplemented with 1 μg / ml β-estradiol (E-8875; Merck Millipore Co.) and 0.02 mg / ml FSH (Antrin; Kyoritsu Tokyo, Japan). The oocytes were fixed overnight in Carnoy's fixative (3 parts ethanol:1 part acetic acid), and the chromosomes were stained with aceto-orcein. Mature oocytes were identified by the appearance of metaphase II meiosis and the release of the first polar body under a phase-contrast microscope (x400). The maturation rates of oocytes matured in R:STEM medium and TCM-199 medium were 86.3% and 77.9%, respectively.

[0034] Next, in vitro fertilization was performed using the eggs matured as described above and ejaculated sperm from Holstein cows. The resulting fertilized eggs were then cultured in SOFaa medium. The normal fertilization and cleavage rates of oocytes matured in R:STEM medium and TCM-199 medium were examined. Normal fertilization was determined by the release of one male and one female pronucleus and two polar bodies under a phase-contrast microscope after Carnoy fixation, as in the case of measuring maturation rates. Cleavage rates were determined by the release of oocytes undergoing cell division under a stereomicroscope (x40) on day 7 after fertilization. The normal fertilization rates of oocytes matured in R:STEM medium and TCM-199 medium were 59.0% and 47.6%, respectively, and the cleavage rates were 97.5% and 91.7%, respectively.

[0035] Blastocysts produced 7 days later from oocytes matured in R:STEM medium and TCM-199 medium were fixed and stained in 100% ethanol containing 25 μg / ml Hoechst 33342 (Dojindo) for 120 minutes at 4°C. The cell numbers were then counted using a confocal microscope (CV1000, Yokogawa Electric Corporation), yielding 188.9 and 75.6 cells, respectively. The number of blastocyst cells produced 8 days later was 143.3 and 128.9 cells, respectively.

[0036] These results demonstrate that culturing immature oocytes in R:STEM medium efficiently matures the oocytes and also promotes the maturation of fertilized oocytes. Culturing immature oocytes in R:STEM medium also promotes the maturation of fertilized oocytes, which is thought to contribute to improving the implantation rate of embryos (fertilized oocytes).

[0037] The composition for treating immature eggs and / or embryos (fertilized eggs) of the present invention can efficiently mature immature eggs, promote embryo maturation, and improve the implantation rate in IVM-IVF. The treatment composition of the present invention is suitable for purposes such as culturing immature eggs, culturing embryos (fertilized eggs), embryo (fertilized egg) transfer, assisting embryo (fertilized egg) implantation, and improving the implantation rate of embryos (fertilized eggs), and can be used for improving the success rate and efficiency of artificial insemination and in vitro fertilization in humans, livestock breeding (improving the success rate and efficiency of in vitro fertilized egg production and transplantation), animal breeding and species maintenance (e.g., preserving endangered species, maintaining or crossbreeding pet breeds), and treating or ameliorating various diseases caused primarily or secondary by disorders of egg maturation.

Claims

1. A composition for treating immature eggs and / or embryos (fertilized eggs), comprising a serum-free mesenchymal stem cell medium.

2. The treatment composition according to claim 1, which is used as an immature egg culture agent, an embryo (fertilized egg) culture agent, an implantation aid, an embryo transfer liquid, or an implantation rate improving agent.

3. The treatment composition according to claim 1 or 2, wherein the serum-free mesenchymal stem cell medium contains EGF, bFGF, albumin, transferrin and insulin.

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