KR20100049153A - Compositions for enhancing embryo implantation or pregnancy - Google Patents

Abstract

PURPOSE: A composition containing MMP-9(metalloproteinase-9) is provided to increase implantation and pregnancy rate. CONSTITUTION: A composition for implantation or pregnancy of animal fertilized egg contains MMP-9 as an active ingredient. The animal is mammal. The mammal includes human, bovine, pig, horse, sheep, rabbit, elephant, monkey, hamster, or rat. The fertilized egg is obtained by in vitro fertilization. The egg is treated with MMP-9 and transplanted before transplantation. The pregnancy is in vitro fertilization-assisted pregnancy or artificial fertilization-assisted pregnancy.

Description

Composition for promoting implantation or fertility of the fertilized egg {Compositions for Enhancing Embryo Implantation or Pregnancy}

The present invention relates to a composition for implantation or pregnancy promotion of an animal fertilized egg.

Like ovulation and implantation, the pregnancy process is characterized by excessive tissue remodeling. Implantation of blastocysts in the human uterus is a complex process that depends on large structural changes in the endometrium and growing embryos (Sakkas et al., 2001). Tissue remodeling is essential for uterine preparation, embryo bridging of the epithelial basement membrane and subsequent penetration of the endometrial matrix. Convincing evidence has been provided during this process to support the hypothesis that matrix metalloproteinases (MMPs) are essential for the degradation of extracellular matrix components (ECMs). From this, it is contemplated that MMP-9 is involved in cell migration, invasion and tissue remodeling in the reproductive process. Matrix metalloproteinase (MMP) -9, also known as 92 kDa type IV collagenase / gelatinase B, is a variety of extracellular substrates including many types of collagen (IV, V and X), elastin, proteoglycans and gelatin Decompose the components (Hibbs et al., 1985; Murphy et al., 1991; Moon et al., 2003; Moon et al., 2004; Chung et al., 2004). Moreover, previous studies have demonstrated that MMP-9 secretion from human cervical fibroblasts, trophoblasts and endometrial cells is stimulated by cytokines and hormones (Shimonovitz et al., 1994; Huang et al., 1998). MMP-9 has been proposed to be involved in other stages of cyclical changes in female reproduction, such as menstrual cycle, ovulation, implantation, female reproduction, mammary gland degeneration after labor and lactation. Ovulation is a process that results from an increase in LH before ovulation from the pituitary gland, which allows mature eggs to be released from the follicles of the ovary before ovulation. Although this process requires tissue remodeling and MMP-9 is important for generating proteolytic activity at the time of ovulation, the relationship between MMP-9 and human follicular fluid or between MMP-9 and medium is unknown. none. Earlier, the inventors have investigated whether expression of MMP-9 from preovulatory follicular fluid and medium is associated with successful pregnancy during in vitro fertilization cycles.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have tried to improve the implantation and pregnancy efficiency of fertilized eggs of animals such as humans, cows, horses and the like. As a result, the present invention was completed by confirming that the treatment of fertilized eggs exogenously MMP-9 significantly increased the efficiency of implantation and pregnancy.

Accordingly, it is an object of the present invention to provide a composition for implantation or fertility promotion of fertilized eggs in animals.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to an aspect of the present invention, the present invention provides a composition for implantation or fertility of the fertilized egg of an animal comprising MMP-9 (metalloproteinase-9) as an active ingredient.

The present inventors have tried to improve the implantation and pregnancy efficiency of fertilized eggs of animals such as humans, cows, horses and the like. As a result, it was confirmed that the treatment of fertilized eggs exogenously treated with MMP-9 significantly increased the efficiency of implantation and pregnancy.

The compositions of the present invention are applied to a variety of animal species to increase implantation and pregnancy efficiency. The animal to which the composition of the present invention can be applied is not particularly limited, and may be applied to, for example, humans, cattle, pigs, horses, sheep, goats, rabbits, elephants, monkeys, mice, hamsters and rats.

Preferably, the composition of the present invention is applied to a mammal. More preferably, the compositions of the present invention apply to humans, cows, pigs, horses, sheep, goats, rabbits, elephants and monkeys, and most preferably to humans, cows, pigs and horses.

MMP-9 used as an active ingredient in the present invention includes not only the inherent activity of MMP-9, but also potentially having MMP-9 activity.

MMP-9 is known as 92 kDa type IV collagenase / gelatinase or gelatinase B. Purification of MMP-9 and cDNA sequences encoding the same are disclosed in US Pat. No. 4,992,537. The zymogen of MMP-9, i.e. proMMP-9, when activated has an intermediate active form with a molecular weight of 83 kDa, in which case an inactive proteolytic cleavage product of 9 kDa is produced, which further degrades further. To form an active form of molecular weight 67 kDa and an inactive proteolytic product of 16 kDa.

According to a preferred embodiment of the present invention, MMP-9 used as an active ingredient in the present invention is 92 kDa, 83 kDa or 67 kDa MMP-9, more preferably 83 kDa or 67 kDa MMP-9.

When the composition of the present invention is applied to a human, preferably the fertilized egg is modified in vitro . For example, MMP-9 is treated and implanted for a suitable time prior to implanting fertilized fertilized eggs.

When the composition of the present invention is applied to cattle, it is preferable to treat MMP-9 on the uterine wall rather than directly on fertilized eggs or eggs.

According to a preferred embodiment of the present invention, the pregnancy is an IVF-assisted conception or artificial insemination-pregnancy.

Modified in vitro-engaged pregnancy (IVF-assisted conception) means to the implanted embryos have been modified in vitro to the parent of the implantation and pregnancy. Insemination-involved pregnancy is the treatment of sperm into an egg in the mother to fertilize and conceive.

The composition of the present invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are conventionally used in the formulation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, poly Vinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. The composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The composition of the present invention is preferably administered directly to the fertilized egg or directly to the wall of the uterus.

Suitable dosages of the compositions of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of the patient, food, time of administration, route of administration, rate of excretion and response to reaction. have. Suitable dosages of the compositions of the invention are 1-200 units / fertilized eggs. If the composition of the present invention is administered directly to the uterine wall, a suitable dosage is 10-1000 units / kg.

The compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient, according to methods which can be easily carried out by those skilled in the art. It can be prepared by incorporation into a multi-dose container. The formulation may also be in the form of a solution, suspension or emulsion in oil or aqueous medium.

The composition of the present invention greatly increases the implantation of the fertilized egg or significantly increases the pregnancy rate. As demonstrated in the examples below, the compositions of the present invention can induce an increase in pregnancy rate and implantation rate of about 25%.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental material

MMP-9 (95 kDa gelatinase; EC 3.4.24.35) obtained from human fibroblasts was obtained from Sigma Co. (Catalog number: M4809; St. Louis, MO, USA). The purchased enzyme showed> 95% (SDS-PAGE) purity and was stored in buffered glycerol aqueous solution (phosphate buffer solution, pH 7.4, 0.5 M sodium chloride, 4% DMSO and 10% glycerol).

Ethical Review

The review board of the Korean National Institute of Glue Biology (NRL) approved all experimental procedures in this study. On the other hand, because the review board is postmortem and only requires chart review, approval of the review board is not necessary in this study.

Animal characteristics

For in vitro fertilization embryo transfer (IVF-ET) for reproductive processes, from February 2004 to June 2006, 42 Holstein heifers, 5 Holstein cows (Holstein cows) and 17 Korean native cows were used in this study.

Follicle Preparation

Follicle fluid was obtained from cattle that were stimulated and participated in the IVF program. Oocyte recovery and follicular fluid collection were performed by the ultrasound-guided evaporation method. The total follicular fluid of each follicle was evaporated and sent to a laboratory to collect oocytes. The follicular fluid was labeled and centrifuged at 300 g for 10 minutes to remove blood and granulosa cells.

Activity of MMP-9 Protease

After 16-20 hours (day 1), 72 hours (day 3) or 120 hours (day 5) of incubation from the fertilized egg-stage, embryos are cultured (day 1, day 3 of culture: P1 medium + 10%). FBS, 5 days of culture: blastocyst medium + 10% SSS). Conditioned medium (CM) was collected as described above and then analyzed for the activity of MMP-2 and -9 by gelatin zymography. Total protein in the sample was measured by spectrophotometer. Samples were adjusted to have the same total protein and volume.

Zymography

MMP-9 in follicular fluid and medium was detected by zymography according to a known method (Lee et al., 2005). Follicle fluid samples were separated under non-reducing conditions by SDS-PAGE (7.5% (w / v gel; Minigel tool; Bio-Rad, Hemel Hempsead)) using a gel containing gelatin (1 mg / ml). After washing with 2.5% (v / v) Triton X-100, digestion buffer (200 mM NaCl, 50 mM Tris-HCl, 2.5 mM CaCl ₂ , 1 μM ZnCl ₂ , pH 7.6; Sigma Chemical Co, St Louis, MO ) For 18 hours at 37 ° C. The gel was stained with a solution of 0.5% (w / v) Coomassie Blue R250 (Bio Rad, Richmond, CA) in 30% (v / v) methanol 10% (v / v). The reaction was performed for 3 hours at room temperature in glacial acetic acid in H ₂ O. Subsequently, decolorization (staining solution without Coomassie blush dye) was performed to visualize specific sites in which gelatin was cleaved by gelatinase.

Densitometry

A semi-automated image analysis tool (National Institutes of Health Image 1.52) was used to quantify MMP-9 activity, which quantifies the surface and intensity of the degradation band after scanning of the gel.

Statistical analysis

Experimental results are presented as mean ± SE values for at least three independent experiments. Statistical differences were assessed by analysis by Student's t-test. P <0.05 values were evaluated as statistically significant.

conclusion

In follicular fluid from all animals, zymography showed expression of gelatinase consistent with MMP-9 at 92 kDa molecular weight, respectively (FIG. 1). High concentrations of MMP-9 were observed in the pregnant group compared to the non-pregnant group. The mean MMP-9 concentration in pregnant cows was 61,759 units.

Relationship between Protease Activity and Successful Pregnancy in Follicle Fluids

From February 2002 to June 2004, 42 embryos were surgically implanted into 42 Holstein young cows, 5 Holstein cows, and 17 Korean native cows. When MMP-9 (10 units / embryo) was administered to cloned embryos, the pregnancy rates of the control and MMP-9 groups were 51.6% and 76.7%, respectively, with significant differences between treatments (p <0.01). The implantation rate was significantly (p <0.01) difference between the control group (53.6%) and the MMP-9 administration group (78.6%) (Table 1). These results suggest that MMP-9 administration may improve fertility rates in in vitro fertilized embryos in Korean native cows.

Addition of MMP-9 Receptor number Number of Oocytes Irradiated Average number of embryos transplanted Pregnancy number (%) Implantation / transplantation embryos (implantation rate,%) - 31 1541 1.3 16/31 (51.6) 22/41 (53.6) + 30 1550 1.4 23/30 (76.7) ^** 33/42 (78.6) ^**

Significant difference from each group, ** P <0.01

Discussion

Changes in women's endocrine during the reproductive cycle and pregnancy lead to extensive tissue remodeling in the womb (Wewer et al., 1986; Aplin et al., 1988). For example, various basal membrane components such as type IV collagen, laminin, fibronectin and proteoglycans in human uterus undergo changes in the menstrual cycle and throughout pregnancy (Aplin et al., 1988). Similarly, mouse endometrial cells undergoing decidulization also remodel extracellular matrix components (Wewer et al., 1986) and MMP-9 and TIMPs are involved in the degradation of the matrix during ovulation, implantation and decidualization. It is thought to be a key mediator (Leco et al., 1996; Alexander et al., 1996). Many studies suggest that MMPs play an important role in tissue remodeling and tissue repair in a variety of physiological and pathological conditions such as morphogenesis, ovulation, angiogenesis, arthritis, wound healing and tumor infiltration. However, nothing is known about the physiological regulation of these protease activation mechanisms in MMPs and in vivo. The presence of MMP-9 in follicular fluid with substrate specificity, including the basement membrane component Collagen IV, can be inferred that this enzyme would be required for tissue remodeling during follicle growth and development. Ovulation may be the result of regulatory enzymatic degradation and loss of collagen in the follicular wall (Luck and Zhao, 1995). Collagen degrading enzymes that break down the formation of fibrillar collagen, which impart structural integrity to the ovary substrate, are thought to play a key role in ovulation in other species, including fibrillar collagenase, such as MMP-1. Also in mouse and human preimplantation embryos, EC cells, and blastocyst growth, expression of MMP-9 was detected by gyography, immunocytochemistry and PCR. The blastocyst culture in the presence of fibroblast growth factor-4 increased the secretion of MMP-9. MMP-9 was developmentally regulated in mouse blastocysts and trophoblast cells during embryo implantation. Reponen et al., Although reported significantly higher expression of MMP-9, made similar observations. These events require degradation of the extracellular matrix and cell migration. MMPs are an important family of zinc-dependent enzymes with a wide range of substrate specificities that can degrade extracellular matrix (Hulboy et al., 1997). In order to determine the protein activity of MMP-9, CM from embryo medium was analyzed by gelatin zymography method. MMP-9 activity was present in the CM of the sample analyzed (FIG. 1). As we have observed previously (Lee et al., 2005), MMP-9 is essential before and after implantation, and successful pregnancy is due to infiltration of the trophoblast into the decidual membrane after implantation and subsequent excretion of the extraravillous trophoblast. It relies on additional penetration into the walls of the female spiral arterioles.

In the above experimental results, when MMP-9 was administered for cloning embryo transplantation, the pregnancy rate of the control group and the MMP-9 group was significantly increased from 51.6% to 76.7% and there was a significant difference between treatments (p <0.01). Moreover, the implantation rate was significantly (P <0.01) difference between the control group (53.6%) and the MMP-9 administration group. These results indicate that MMP-9 administration can improve pregnancy rate in vitro fertilized embryo transplantation in Korean native cows.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

References

1.Sakkas D, Percival G, D'Arcy Y, Sharif K, Afnan M. (2001) Assessment of early cleaving in vitro fertilized human embryos at the 2-cell stage before transfer improves embryo selection. Fertil Steril 76 (6): 1150-1156.

2.Hibbs MS, Hasty KA, Seyer JM, Kang AH, Mainardi CL (1985) Biochemical and immunological characterization of the secreted forms of human neutrophil gelatinase. J Biol Chem 260: 2493-2500.

3.Murphy G, Cockett MI, Ward RV and Docherty AJ. (1991) Matrix metalloproteinase degradation of elastin, type IV collagen and proteoglycan. A quantitative comparison of the activities of 95 kDa and 72 kDa gelatinases, stromelysins-1 and -2 and punctuated metalloproteinase (PUMP). Biochem J. 277: 277-279.

4. Moon SK, Cha BY, Lee YC and CH Kim (2003) In vitro cellular aging is associated with enhanced proliferative capacity, G1 cell cycle modulation, and matrix metalloproteinase-9 regulation in mouse aortic smooth muscle cells. Arch Bichem. Biophys. 418 (1), 39-48.

5. Moon SK, Cha BY, Lee YC, Ko JH, Runge MS, Patterson C, and Kim CH. 2003. Age-related changes in matrix metalloproteinase-9 regulation in cultured mouse aortic smooth muscle cells. Experimental Gerontol. In Press.

6. Chung TW, Lee YC, CH Kim. 2004. Hepatitis B viral HBx stimulates the ERKs and PI-3K / AKT / PKB signal pathways, and activates AP-1 and NF-kB binding sites for enhanced matrix metalloproteinase-9 gene expression: invasive and metastatic potential of liver cancer cell. FASEB J. 18, 1123-1125.

Lee DM, Lee TK, Song HB, Kim CH. 2005. The expression of matrix metalloproteinase-9 (MMP-9) in human follicular fluid is associated with in vitro fertilization pregnancy. British Journal of Obstetrics and Gynecology, In Press.

Shimonovitz S, Hurwitz A, Dushnik M, Anteby E, Geva-Eidar T, Yagel S. (1994) Developmental regulation of the expres-sion of 72 and 92 kd type IV collagenases in human trophoblasts: A possible mechanism for control of tropho-blast invasion. Am J Obstet Gynecol 171: 832-838.

9.Huang HY, Wen Y, Irwin JC, Kruessel JS, Soong YK, Polan ML. (1998) Cytokine-mediated regulation of 92-kilodalton type IV collagenase, tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-3 messenger ribonucleic acid expression in human endometrial stromal cells. J Clin Endocrinol Metab 83: 1721-9.

10. Jeziorska, M. Nagase H. Salamonsen, L. A and Wolley. D.E (1996) Immunolocalization of the matrix metalloproteinases gelatiase B and stromelysin 1 in human endometrium throughout the menstrual cycle. J. Repro. Fertil. 107: 43-51

11.Librach CL, Werb Z, Fitzgerld ML, Chiu K, Corwin NM, Esteves RA, Grobelny D, Galardy R, Damsky CH and Fisher SJ (1991) 92kDa type IV collagenase mediates invasion of human cytotrophoblasts Jounal of Cell Biology 113: 437 -449

12. Vadillo-Ortega, F. Gonzalez-Azila, G, Furth, E.E. et al. (1995) 92-kd type IV collagenase (matrix metalloproteinase-9) activity in human amniochorin increases with labor. Am.J.Pathol. 146: 148-156

15. Wewer UM, Damjanov A, Weiss J, Liotta LA, Damjanov I (1986) Mouse endometrial stromal cells produce basement-membrane components. Differentiation 32: 49-58.

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21. Leco KJ, Edwards DR, Schultz GA (1996) Tissue inhibitor of metalloproteinases-3 is the major metalloproteinases inhibitor in the decidualizing murine uterus. Mol Reprod Dev 45: 458-465.

22. Alexander CM, Hansell EJ, Behrendtsen O, Flannery ML, Kishnani NS, Hawkes SP, Werb Z (1996) Expression and function of matrix metalloproteinases and their inhibitrors at the maternal-embryonic boundary during mouse embryo implantation. Development 122: 1723-1736.

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25.Reponen P, Leivo I, Sahberg C, Apte SS, Olsen BR, Thesleff I, Tryggvason K. (1995) 92-kDa type IV collagenase and TIMP-3 but not 72kDa type IV collagenase or TIMP-1 or TIMP-2 , are highly expressed during mouse embryo implantation. Develop Dynam 202: 388-396.

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Figure 1 shows the results of a zymography analysis of proMMP-9 activity in follicular fluid. Follicle fluid was centrifuged at 300 g for 10 minutes to remove blood and granulosa cells. The follicles were electrophoresed and washed on 0.1% gelatin / 7.5% SDS-PAGE gels and then reacted overnight in zymography reaction buffer to degrade the gelatin. Lane 1, pregnant cow; Lanes 2-4, non-pregnant cattle.

Claims (4)

Implantation or fertility promoting composition of an animal comprising MMP-9 (metalloproteinase-9) as an active ingredient. The composition of claim 1, wherein said animal is a mammal. The composition of claim 1, wherein the fertilized egg is fertilized in vitro . The composition of claim 1, wherein the pregnancy is an IVF-assisted pregnancy or an artificial insemination-pregnant pregnancy.

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