Composition comprising beta core fragment hcg as active ingredient for treating infertility

Abstract

The present invention relates to a composition comprising beta core fragment hCG (βcf hCG) as an active ingredient for treating infertility. It has been confirmed that βcf hCG stimulates endometrial development in the early stage of pregnancy, is highly expressed in placental tissue, and is involved in maintenance of fetal development in the early stage of pregnancy. In addition, βcf hCG increases endometrial thickness, enhances expression of genes required for uterine development, and promotes growth of uterine-derived cell lines, thereby playing an important role in implantation and maintenance in the early stage of pregnancy. Accordingly, the composition can be usefully applied for treating recurrent miscarriage in female infertility and for improving embryo implantation rates during in vitro fertilization procedures, thereby increasing pregnancy success rates.

Description

A composition for treating infertility containing the beta-core fragment HCG as an active ingredient

[0001] The present invention relates to a composition for treating infertility comprising a beta-core fragment hCG (βcf hCG) as an active ingredient.

[0002] Recently, along with the aging society, infertility is on the rise as the age of mothers giving birth increases. Furthermore, pregnancy rates are significantly declining due to increased stress experienced by women resulting from environmental pollution caused by industrialization and their increased participation in the workforce. Commonly known causes of female infertility include ovulation disorders, failure of embryo transfer, and implantation failure. While infertility issues caused by ovulation and embryo transfer are largely resolved through In Vitro Fertilization (IVF), there is currently no clear solution for infertility caused by implantation failure.

[0003] Maintaining a successful pregnancy requires embryonic development, endometrial development, implantation, placental formation, hormone regulation, and the exchange of nutrients and gases between the mother and the fetus. In the early stages of pregnancy, the fetus undergoes morphological changes from a spherical blastocyst to a filamentous shape to implant in the endometrium, forming a successful non-invasive placenta.

[0004] Implantation is an early pregnancy phenomenon in which a fertilized blastocyst, developing into an embryo, attaches to the endometrial layer of the mother's uterus. For a successful pregnancy, the uterus must maintain an endometrial thickness of at least 12 mm during the fertile period, and continuous expansion and normal cell growth must occur throughout the pregnancy. Proper development of the endometrium is essential to prevent miscarriage and successfully maintain the pregnancy. To date, specific regulatory factors in the processes of implantation and endometrial development have not been clearly identified.

[0005] Meanwhile, human chorionic gonadotropin (hCG) is a glycoprotein hormone produced during pregnancy. hCG is produced in the trophoblast of the placental chorionic cells and functions to maintain implantation until 10 weeks of pregnancy, when placental function is fully developed, by causing the continued production of progesterone in early pregnancy.

[0006] The active form in the body, complete hCG (I-hCG), has a molecular weight of approximately 37 kDa and consists of 244 amino acids. I-hCG is largely composed of two subunits, alpha (α) and beta (β); the alpha subunit contains 92 amino acids, and the beta subunit contains 145 amino acids. I-hCG is generally present in the blood during pregnancy and is the primary structure exhibiting biological activity. In addition to the active form mentioned above, various dissociation and degradation products of hCG are found in blood and urine, existing as fragmented hCG (N-hCG), free beta hCG (free β-hCG), free alpha hCG (free α-hCG), and beta-core fragment hCG (βcf hCG).

[0007] βcf hCG is an hCG protein primarily found in the urine of pregnant women, having a molecular weight of approximately 14 kDa. I-hCG is predominantly secreted at 4 weeks of the Last Menstrual Period (LMP), and βcf hCG begins to gradually increase from 5 weeks of the LMP, eventually being secreted at a concentration superior to I-hCG after 5 weeks and 6 days or 6 weeks. In previous research by the inventors, it was revealed that the above-mentioned βcf hCG can be used as a marker for pregnancy diagnosis.

[0008] Against this background, the inventors completed the present invention by investigating specific maintenance factors for the implantation of early fertilized eggs in the uterus and confirming that the beta-core fragment hCG (βcf hCG) is an absolutely necessary and decisive factor for early pregnancy and maintenance.

[0009] [Prior Art Literature]

[0010] [Non-patent literature]

[0011] (Non-patent literature 0001) Spencer TE et al., Reproduction, 2004, 128(6):657-668.

[0012] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of infertility comprising a beta-core fragment hCG (βcf hCG) as an active ingredient.

[0013] Another object of the present invention is to provide a method for preventing or treating infertility, comprising the step of administering the pharmaceutical composition to a subject.

[0014] Another objective of the present invention is to provide a pharmaceutical composition for promoting pregnancy comprising a beta-core fragment hCG (βcf hCG) as an active ingredient.

[0015] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the present invention, and the present invention is defined only by the scope of the claims.

[0016] The terms used in this specification are for describing embodiments and are not intended to limit the invention. In this specification, the singular form includes the plural form unless specifically stated otherwise in the text. The terms "comprises" and / or "comprising" used in this specification do not exclude the presence or addition of one or more other components in addition to the components mentioned. Throughout the specification, the same reference numerals refer to the same components, and "and / or" includes each of the mentioned components and all combinations of one or more. Although terms such as "first," "second," etc., are used to describe various components, these components are not limited by these terms. These terms are used merely to distinguish one component from another. Therefore, the first component mentioned below may be the second component within the technical scope of the invention.

[0017] Unless otherwise defined, all terms used herein (including technical and scientific terms) may be used in a meaning commonly understood by those skilled in the art to which the present invention pertains. Additionally, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise.

[0018] The present invention provides a pharmaceutical composition for the prevention or treatment of infertility comprising a beta-core fragment hCG (βcf hCG) as an active ingredient.

[0019] The human chorionic gonadotropin (hCG) of the present invention is a glycoprotein hormone produced during pregnancy. hCG is produced in the trophoblast of the placental chorionic villi and functions to maintain implantation until the 10th week of pregnancy, when placental function is completed, by causing the continued production of progesterone in the early stages of pregnancy.

[0020] The active form in the body, complete hCG (I-hCG), has a molecular weight of about 37 kDa and consists of 244 amino acids. I-hCG is composed of two subunits, alpha (α) and beta (β); the alpha-subunit (α-hCG) contains 92 amino acids, and the beta-subunit (β-hCG) contains 145 amino acids.

[0021] I-hCG is generally present in the blood during pregnancy and is the main biologically active structure. In addition to the active form, various dissociation and degradation products of hCG are found in blood and urine, and exist as fragmented hCG (N-hCG), free beta hCG (free β-hCG), free alpha hCG (free α-hCG), beta core fragment hCG (βcf hCG), etc.

[0022] The beta-core fragment hCG (βcf hCG) of the present invention is an hCG protein that is mainly present in the urine of pregnant women and has a molecular weight of about 14 kDa. At 4 weeks of the Last Menstrual Period (LMP), I-hCG is predominantly secreted, and from 5 weeks of the LMP, βcf hCG begins to gradually increase and is secreted at a concentration that is predominantly higher than I-hCG after 5 weeks and 6 days or 6 weeks.

[0023] The beta-core fragment hCG of the present invention comprises amino acids 6-40 and 55-92 of β-hCG. The amino acid sequence of said β-hCG comprises SEQ ID NO. 1:

[0024] SEQ ID NO: 1: Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val Leu Gln Gly Val Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg Asp Val Arg Phe Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asn Pro Val Val Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu Cys Arg Arg Ser Thr Thr Asp Cys Gly Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp Pro Arg Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln

[0025] (SKEPLRPRCRPINATLAVEKEGCPVCITVNTTICAGYCPTMTRVLQGVLPALPQVVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCALCRRSTTDCGGPKDHPLTCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ).

[0026] The amino acid sequence of the above beta-core fragment hCG includes SEQ ID NOs. 2 and 3:

[0027] SEQ ID NO: 2: Arg Pro Arg Cys Arg Pro Ile Asn Ala Thr Leu Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr Val Asn Thr Thr Ile Cys Ala Gly Tyr Cys Pro Thr

[0028] (RPRCRPINATLAVEKEGCPVCITVNTTICAGYCPT),

[0029] SEQ ID NO: 3: Val Val Cys Asn Tyr Arg Asp Val Arg Phe Glu Ser Ile Arg Leu Pro Gly Cys Pro Arg Gly Val Asn Pro Val Val Ser Tyr Ala Val Ala Leu Ser Cys Gln Cys Ala Leu

[0030] (VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL).

[0031] The beta-core fragment hCG of the present invention can be secreted from the placenta and the corpus luteum of the ovary.

[0032] The placenta of the present invention is an organ that delivers nutrients and oxygen between the mother and the fetus during pregnancy and removes waste products. It is formed from the early stages of pregnancy and is created by the combination of the mother's uterine lining and the fetal chorionic membrane. The placenta is responsible for blood exchange between the fetus and the mother and plays an important role in maintaining the pregnancy by secreting hormones (estrogen, progesterone, hCG).

[0033] The corpus luteum of the present invention is a temporary endocrine organ formed in the ovary after ovulation. As the follicle is ovulated, the remaining part is modified to form the corpus luteum, which secretes mainly progesterone and a small amount of estrogen. These hormones help the fertilized egg implant and play an important role in maintaining the pregnancy.

[0034] In a specific embodiment of the present invention, it was confirmed that the beta-core fragment hCG is expressed very specifically in placental tissue and is associated with changes in the expression of epithelial granular tissue involved in the implantation of a fertilized egg in the endometrial tissue of a non-pregnant uterus. This indicates that it stimulates endometrial development in early pregnancy and is involved in maintaining fetal development.

[0035] The composition of the present invention can be administered to a subject before embryo implantation, specifically within 50 to 90 hours before implantation, and more specifically within 60 to 80 hours.

[0036] Administration of the composition of the present invention may include, but is not limited to, intrauterine administration.

[0037] The intrauterine administration of the present invention involves direct injection into the uterus and is a method of administration used for infertility treatment, assisted reproductive technology (IVF), or the treatment of specific uterine diseases. Since the pharmaceutical composition is delivered directly to the endometrium, this administration method enables more effective local treatment.

[0038] The composition of the present invention can increase the thickness of the uterine endometrium.

[0039] In a specific embodiment of the present invention, as a result of treating with a composition containing the beta-core fragment hCG, it was confirmed that the thickness of the endometrium increased by 19.72% on day 1 after administration and by 23.03% on day 3 after administration compared to the control group (saline administration group). It was also confirmed that it increased compared to the I-hCG administration group. This indicates that the beta-core fragment hCG plays a decisive role in increasing the thickness of the endometrium.

[0040] The composition of the present invention can promote the proliferation of endometrial cells.

[0041] In a specific embodiment of the present invention, it was confirmed that cell growth increased when a composition containing the beta-core fragment hCG was treated for 24 hours to cell lines derived from placenta and uterine tissue.

[0042] The composition of the present invention can increase the expression of genes related to uterine receptivity development and implantation.

[0043] The above genes may include, but are not limited to, HOXA10, HOXA11, DEDD, Cyclin D3, CDK4 / 6, EGR, PIGF, HAND2, or MSX1.

[0044] HOXA10 plays a role in improving endometrial receptivity by regulating cholesterol synthesis in endometrial stromal cells. HOXA11 is involved in endometrial proliferation, differentiation, and embryonic development. DEDD plays an essential role in establishing a uterine environment suitable for early pregnancy. Cyclin D3 and CDK4 / 6 are closely associated with cell cycle regulation for endometrial cell proliferation. HAND2 plays a crucial role in establishing a suitable implantation environment for pregnancy, creating a uterine environment that sensitizes progesterone when implantation begins. PIGF influences the induction of embryonic implantation and development, as well as endometrial development. EGR is an immediate early gene important for cell growth and differentiation, expressed in rapid response to stimuli. MSX1 controls uterine receptivity by regulating uterine epithelial function during embryonic implantation.

[0045] The infertility of the present invention may be one or more selected from the group consisting of nonimplantation of ovum, female infertility associated with anovulation, female infertility of tubal origin, female infertility of uterine origin, female infertility of cervical origin, and female infertility associated with male factors, but is not limited thereto.

[0046] The pharmaceutical composition of the present invention may include a beta-core fragment hCG as an active ingredient, and in addition to the active ingredient, may additionally include one or more active ingredients that have the same or similar function, that is, are effective in treating infertility.

[0047] The term "pharmaceutical composition" of the present invention may be described interchangeably with "pharmaceutical composition."

[0048] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives.

[0049] The above additive may further include suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions.

[0050] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is one that is commonly used in formulations and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0051] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, or sterile injectable solutions, each according to conventional methods. Specifically, when formulating, it may be prepared using diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants that are commonly used.

[0052] The pharmaceutical composition of the present invention may additionally include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc., in addition to the above components.

[0053] The pharmaceutical composition of the present invention may be administered parenterally, specifically by intrauterine injection, but is not limited thereto.

[0054] The appropriate dosage of the pharmaceutical composition of the present invention depends on factors such as the formulation method, mode of administration, age, body weight, sex, pathological condition, food, time of administration, route of administration, rate of excretion and response responsiveness, and the degree of cancer in the patient, and can be appropriately selected by a person skilled in the art.

[0055] The pharmaceutical composition of the present invention may be prepared in a unit volume form or contained in a multi-volume container by formulation using pharmaceutically acceptable carriers and / or excipients according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0056] The subjects of administration of the present invention include, but are not limited to, humans, monkeys, cattle, horses, pigs, sheep, chickens, cats, dogs, mice, rabbits, etc.

[0057] In addition, the present invention provides a method for preventing or treating infertility, comprising the step of administering the above pharmaceutical composition to a subject suspected of or having developed infertility.

[0058] The terms, pharmaceutical composition, and infertility of the present invention are as described above.

[0059] The treatment method of the present invention comprises administering a pharmaceutical composition of the present invention in a pharmaceutically effective amount into a subject suspected of or having developed infertility. The subject refers to all mammals including dogs, cattle, horses, rabbits, mice, rats, chickens, or humans, but the subject of the present invention is not limited by the above examples.

[0060] The above pharmaceutical composition may be administered parenterally, and for local treatment, may be administered by a suitable method including intrauterine administration if necessary. The preferred dosage of the above pharmaceutical composition of the present invention may vary depending on the subject's condition and body weight, the severity of the disease, the form of the drug, the route of administration, and the duration, but can be appropriately selected by those skilled in the art.

[0061] In addition, the present invention provides a pharmaceutical composition for promoting pregnancy comprising a beta-core fragment hCG (βcf hCG) as an active ingredient.

[0062] The term of the present invention, beta core fragment hCG, is as described above.

[0063] The pregnancy of the present invention is a process in which a fertilized egg implants in the inner wall of the uterus, receives nutrition from the mother, and develops into a fetus, and the normal pregnancy process proceeds in the order of fertilization, implantation, and fetal development.

[0064] The fertilization of the present invention refers to the formation of a fertilized egg when an egg ovulated from a woman's ovary meets a sperm in the upper part of the fallopian tube. Eggs have the ability to survive and fertilize for 1 to 2 days after ovulation, and sperm survive for 2 to 3 days in the uterus; sperm can also survive for up to one week after ejaculation. Since sperm reach the vicinity of the ovary along the fallopian tube within 2 to 3 hours after ejaculation, sperm generally reach the upper part of the fallopian tube first, and when an egg ovulated from the ovary reaches this location, only one of the sperm combines with the egg to form a fertilized egg.

[0065] The implantation of the present invention refers to the phenomenon in which a fertilized egg, formed by the meeting of a sperm and an egg in the upper part of the fallopian tube, moves to the uterus while undergoing repeated cleavage and becomes embedded in the inner wall of the uterus in the state of a blastocyst. The period after the implantation of the fertilized egg is called pregnancy. The fertilized egg implanted in the uterus grows for about 9 months while receiving nutrients from the inner wall of the uterus and is born.

[0066] The composition of the present invention may include the beta-core fragment hCG alone or may further include a substance effective in promoting pregnancy as an active ingredient, and in addition to the active ingredient, may further include additional ingredients, namely pharmaceutically acceptable or nutritionally acceptable carriers, excipients, diluents, or auxiliary ingredients depending on the formulation, method of use, and purpose of use.

[0067] The above composition can be used to promote pregnancy and can be used in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

[0068] The present invention relates to a composition for treating infertility comprising a beta-core fragment hCG (βcf hCG) as an active ingredient. The βcf hCG is confirmed to stimulate endometrial development in early pregnancy and to be highly expressed in placental tissue, thereby participating in the maintenance of early fetal development. It also plays an important role in early implantation and maintenance of pregnancy by increasing the thickness of the endometrium, increasing the expression of genes necessary for uterine development, and promoting the growth of uterine-derived cell lines. It can be usefully employed to treat recurrent miscarriage in female infertility and to increase the pregnancy success rate by improving the implantation rate of fertilized eggs during the in vitro fertilization process.

[0069] Figure 1 shows the major expressing tissues of βcf hCG, indicating that it is specifically expressed in placental tissue.

[0070] Figure 2 shows the change in endometrial thickness when βcf hCG is administered to the uterus of experimental animals.

[0071] Figure 3 shows that the endometrial thickness of βcf hCG increased on days 1 and 3 of administration compared to the control group (saline administration group) and the comparison group (Highly purified intact hCG).

[0072] Figure 4 shows the results of observing changes in the expression of genes related to uterine receptivity development and implantation in a uterus treated with βcf hCG.

[0073] Figure 5 shows the growth of cell lines compared to Highly purified intact hCG after treating placenta-derived cell lines with βcf hCG at a concentration of 0.1 to 1 pmol / mL for 24 hours and 48 hours.

[0074] Figure 6 shows the results on days 1 to 3 after administering βcf hCG into the uterine cavity of mice.

[0075] Figure 7 shows the results of measuring the cell viability of uterine-derived cell lines after treatment with βcf hCG.

[0076] The contents of the present invention will be explained in more detail below through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, but includes variations of equivalent technical concepts.

[0077] Example 1. Cell culture and βCF hCG treatment

[0078] Mouse primary endometrial cells were obtained from uterine tissue of B6D2F1 mice. The excised uterus was finely chopped and enzymatically dissociated in a mixture of Collagenase V (Sigma; C9263-100MG; 0.4 mg / ml) and dispase IID4693 (1.25 IU / ml) at 37°C for 1 hour. The cells were serially separated through a strainer (40 μm; SPL, 93040).

[0079] Filtrate containing endometrial cells was collected by centrifugation and cultured with DMEM / F12 supplemented with 20% heat-inactivated FBS, 1% L-glutamine (Gibco, Grand Island, NY, USA), and 1% penicillin-streptomycin. Up to 10 passages were used in the experiment. For the analysis of the effects of βcf hCG, cells were treated with βcf hCG at a concentration of 50 μg / 100 μl for 2 to 72 hours.

[0080] Example 2. Intrauterine administration of βcf hCG in animals

[0081] The therapeutic effects of βcf hCG were analyzed and evaluated using 10-week-old female and male B6D2F1 mice (Orientbio, Gapyeong, Gyeonggi-do). All animal husbandry and experimental procedures were carried out in accordance with the policies and regulations of the Institutional Animal Care and Use Committee. Mice were housed in a standard environment with 12 hours of light:12 hours of darkness conditions at controlled indoor temperatures (20-22 ℃ and 40-60% humidity).

[0082] To evaluate the effect of βcf hCG on endometrial receptivity, female mice were randomly selected, and βcf hCG (40 pmol / ml, 20 μl) was administered into one side of the mouse uterine cavity (Fig. 8). For the control group, saline injection was performed into the opposite side of the uterine horn. For further analysis, samples were taken from both sides of the uterine horn at designated time points. The βcf hCG dose applied in the experiment was selected based on clinical studies involving infertile women. Many studies have administered 500 IU of hCG intrauterinely during IVF-ET. This dose was converted according to animal-to-human dose conversion guidelines and applied in the experiment.

[0083] Example 3. Histological analysis

[0084] Hematoxylin and Eosin (H&E) staining was performed to confirm morphological changes upon hCG administration. The obtained tissues were fixed with 4% paraformaldehyde and dehydrated using 80–100% ethanol and Histoclear (National Diagnostics; HS-202). Then, the tissues were embedded vertically in paraffin.

[0085] Paraffin blocks were sectioned into 5 μm sections, stained with hematoxylin (BioGNOST; HEMH-49 / 20) for 30 seconds, and stained with eosin (BioGNOST; EOYA-10-17 / 20) for 20 seconds. After mounting in Permount medium (Fisher chemical; 196934), images were captured using an Olympus CKX53 microscope (Olympus Life Science), and the total number of glands, endometrial area, and endometrial thickness were evaluated using Image J.

[0086] Example 4. qRT-PCR Analysis

[0087] Tissues were homogenized in 500 μl of labozol reagent (Cosmo; CMRZ001), and 1 μg of total RNA was converted to complementary DNA using SuperScript™ IV (invitrogen; 18091050). Amplification was performed using SYBR Green (Roche, Basel, Switzerland) on a CFX Connect Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA).

[0088] Standard curves for each primer were generated by plotting the log value of the template start amount against the cycle threshold obtained during amplification for each dilution, which exhibited an R2 value greater than 0.98 and an amplification efficiency of approximately 100%. All reactions were performed in triplicate.

[0089] Example 5. Statistical Analysis

[0090] The comparison groups were analyzed using unpaired Student's t-tests for the parameter distributions. For multiple comparisons, standard one-way ANOVA using Dunnett's multiple comparison test was used. For all cases, a p-value < 0.05 was considered statistically significant.

[0091] Experimental Example 1. Identification of major βcf hCG-expressing tissues

[0092] It was confirmed in mouse (E16 mouse) fetuses on day 16 of pregnancy that βcf hCG is expressed very specifically in placental tissue compared to intact hCG (Fig. 1).

[0093] βcf hCG was shown to be associated with changes in the expression of epithelial granular tissue involved in the implantation of fertilized eggs in the endometrial tissue of non-pregnant uterus.

[0094] This suggests that, histologically, βcf hCG stimulates the development of the endometrium in early pregnancy and is highly expressed in placental tissue during pregnancy, thus being involved in maintaining fetal development in early pregnancy.

[0095] Experimental Example 2. Endometrial development induced by βcf hCG

[0096] When 200 μL of βcf hCG at 40 pmol / mL was artificially administered vaginally to the uterus of experimental animals, the thickness of the uterine endometrium increased significantly compared to the control group (Fig. 2).

[0097] In addition, it was confirmed that granulosa cell activity significantly increased in the endometrium treated with βcf hCG.

[0098] In granulosa cells, aromatase is activated by FSH (follicle-stimulating hormone), promoting the biosynthesis of estrogen from androstenedione produced by LH (luteinizing hormone).

[0099] As shown in Table 1, Table 2, Figures 2 and 3 below, it was confirmed that the endometrial thickness of the group treated with βcf hCG increased by approximately 19.72 to 23% or more compared to the control group. On the other hand, the group treated with intact hCG increased by 15% compared to the control group.

[0100] [Table 1]

[0101]

[0102] [Table 2]

[0103]

[0104] Experimental Example 3. Analysis of gene expression of factors necessary for uterine development using βcf hCG

[0105] It was confirmed that genes promoting the growth of endometrial cells were expressed in uteruses treated with βcf hCG. Increased expression of genes related to uterine receptivity development and implantation was confirmed in uteruses treated with βcf hCG (Fig. 4).

[0106] Specifically, HOXA10 plays a role in improving endometrial receptivity by regulating cholesterol synthesis in endometrial stromal cells. HOXA11 is involved in endometrial proliferation, differentiation, and embryonic development. DEDD plays an essential role in establishing a uterine environment suitable for early pregnancy. Cyclin D3 and CDK4 / 6 are closely associated with cell cycle regulation for endometrial cell proliferation. HAND2 plays a crucial role in establishing a suitable implantation environment for pregnancy, creating a uterine environment that sensitizes progesterone when implantation begins. PIGF influences the induction of embryonic implantation and development, as well as endometrial development. EGR is an immediate early gene important for cell growth and differentiation, expressed in rapid response to stimuli. MSX1 controls uterine receptivity by regulating uterine epithelial function during embryonic implantation.

[0107] Consequently, increased expression of the aforementioned gene was confirmed in uteruses treated with βcf hCG. This indicates that it will have a significant impact on the receptive development of the uterus and implantation. Furthermore, it suggests that it can contribute to successful embryo implantation by improving the adaptability of the intrauterine environment during the early stages of pregnancy.

[0108] Experimental Example 4. Growth of placental and endometrial-derived cell lines treated with βCF₆hCG

[0109] When placenta-derived cell lines were treated with βcf hCG at concentrations of 0.1 to 1 pmol / mL for 24 and 48 hours, it was confirmed that the growth of the cell lines increased compared to the control group (Fig. 5). As a result of treatment with βcf hCG, it was also confirmed that the cell viability of endometrial-derived cell lines significantly increased (Fig. 7).

[0110] These results indicate that βcf hCG plays an important role in early pregnancy implantation and maintenance through the development of the uterine lining. This can be developed not only as a treatment to improve recurrent miscarriage in female infertility, but also as a useful application to increase pregnancy success rates by improving the implantation rate of fertilized eggs during in vitro fertilization procedures.

[0111] Although embodiments of the present invention have been described above, the present invention is not limited to the above embodiments and can be manufactured in various different forms, and those skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical concept or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A pharmaceutical composition for the prevention or treatment of infertility comprising a beta-core fragment hCG (βcf hCG) as an active ingredient.

2. In Paragraph 1, A pharmaceutical composition characterized in that the beta-core fragment hCG comprises amino acids 6-40 and 55-92 of β-hCG, and the amino acid sequence of the beta-core fragment hCG comprises SEQ ID NOs. 2 and 3.

3. In Paragraph 1, A pharmaceutical composition characterized in that the above-mentioned beta-core fragment hCG is secreted in the urine of pregnant women or secreted from the placenta and the corpus luteum of the ovary.

4. In Paragraph 1, A pharmaceutical composition characterized by administering the above composition within 50 to 90 hours prior to embryo implantation.

5. In Paragraph 4, A pharmaceutical composition characterized by the above administration including intrauterine administration.

6. In Paragraph 1, The above composition is a pharmaceutical composition characterized by increasing the thickness of the uterine endometrium.

7. In Paragraph 1, The above composition is a pharmaceutical composition characterized by promoting the proliferation of endometrial cells.

8. In Paragraph 1, The above composition is a pharmaceutical composition characterized by increasing the expression of genes related to uterine receptivity development and implantation.

9. In Paragraph 8, A pharmaceutical composition characterized in that the above gene comprises HOXA10, HOXA11, DEDD, Cyclin D3, CDK4 / 6, EGR, PIGF, HAND2, or MSX1.

10. In Paragraph 1, A pharmaceutical composition characterized in that the above-mentioned infertility is one or more selected from the group consisting of nonimplantation of ovum, female infertility associated with anovulation, female infertility of tubal origin, female infertility of uterine origin, female infertility of cervical origin, and female infertility associated with male factors.

11. A method for preventing or treating infertility, comprising the step of administering a pharmaceutical composition of any one of claims 1 to 10 into the uterus.

12. In Paragraph 11, The above method is a method for preventing or treating infertility, characterized by administering it to animals other than humans.

13. A pharmaceutical composition for promoting pregnancy comprising a beta-core fragment hCG (βcf hCG) as an active ingredient.

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