KR20080072353A - Uses of mmp-9 activities present in blood - Google Patents

Abstract

A method for measuring the activity of MMP(matrix metalloprotease)-9 from blood isolated from superovulated woman is provided to predict implantation rate or pregnancy rate of a fertilized egg conveniently with high patient compliance. A method for predicting implantation rate or pregnancy rate of a fertilized egg comprises a step of measuring the activity of MMP-9 included in blood isolated from superovulated woman, wherein the MMP-9 activity has the positive correlation to the implantation rate or pregnancy rate of the fertilized egg. A kit for predicting the implantation rate or pregnancy rate of the fertilized egg using blood sample of the superovulated woman comprises a substrate of MMP-9, an antibody specifically coupled to the MMP-9 or an antibody specifically coupled to a proteolytic cleavage product of proMMP-9. Further, the substrate of MMP-9 is selected from collagen IV, collagen V, collagen XI, elastin, proteoglycan and gelatin.

Description

Uses of MPM-X Activities Present in Blood

1 is a gel photograph of the activity of MMP-9 and MMP-2 in hyperovulation-induced women's blood measured by a zymography analysis. Lane C is a control group using purified MMP-9, lanes 1-4 are pregnant, and lanes 5-8 are non-pregnant.

The present invention relates to a method for measuring activity of matrix metalloprotease (MMP) -9 using blood, a method for predicting implantation rate or pregnancy rate of fertilized eggs, and a kit used therein.

Since the birth of the first test tube baby in 1979, a variety of assisted reproduction technologies (ART) have been developed and applied to the treatment of infertile patients. In vitro fertilization embryo transfer (IVF-ET), intracytoplasmic sperm injection (ICSI), testicular sperm extraction (TESE), sperm cell injection (round spermatid) injection (ROSI) and embryo freezing methods. At present, reproductive assisted surgery is becoming an important and universal method for treating various causes of infertility, but high cost, complicated process, and low success rate remain problems.

Although reproductive assistive procedures are gradually increasing, pregnancy rates such as in vitro fertilization have generally increased until recently, but there is no increase as expected, and the actual birth home rate is 15-30%. Statistics show that in 1985 the in vitro fertilization rate was 23.8% per cycle, the delivery rate was only 19.3% per cycle, and in 1997 statistics the pregnancy rate increased to 29.3% per cycle and 24.0%. However, there was no difference in the 1996 and 1997 statistics.

Successful human pregnancy in reproductive care depends on follicular development, number of eggs recovered, fertilization, fertilized egg development and implantation (Sakkas et al., 2001, Fertil. Steril. 76 (6): 1150-1156). Implantation of blastocysts in the endometrium is a complex process that depends on structural changes in the development of the endometrium and the fertilized egg. Tissue remodeling requires preparation of the uterus, rupture of the fertilized egg in the endometrium and the appearance of a continuous endometrial matrix. Convincing evidence supports the hypothesis that MMPs (abbreviated as "MMP") are essential for the destruction of extracellular matrix components during this process. In this respect, MMP-9 is thought to be involved in cell meiosis, invasion and tissue remodeling during development. In other words, MMP is known to be involved in the remodeling of the endometrial tissue according to the menstrual cycle by causing changes in the extracellular matrix, and MMP molecules exhibit various expression patterns according to the menstrual cycle and participate in ovulation and implantation process.

Matrix metalloproteinase-9 (MMP-9), also known as 92kDa type IV collagenase / gelatinase B, has many types of collagen (collagen IV, collagen V and collagen XI), elastin, and proteoglas. Lycans and gelatin (Hibbs et al., 1985, J. Biol. Chem. 260: 2493-2500; Murphy et al., 1991, Biochem. J. 277: 277-279). Moreover, previous researchers have reported that human cervical fibroblasts (Sato, H. and Seiki, M., 1993, Oncogene 8: 395-405), trophoblasts (Shimonovitz et al., 1994, Am. J.). Obstet.Gynecol. 171: 832-838) and MMP-9 secreted from endometrial stromal cells (Huang et al., 1998, J. Clin. Endocrincol. Metab. 83: 1721-1729). It has been shown to be stimulated by this hormone and cytokine.

MMP-9 has been suggested to be involved in complex cyclical changes in women, such as menstrual cycle, ovulation, implantation, delivery and retraction of the mammary gland after lactation (Jeziorska et al., 1996, J. Reprod. Fertil. 107: 43-51; Librach et al., 1991, J. Cell, Biol. 113: 437-449; Vadillo-Ortega et al., 1995, Am. J. Pathol. 146: 148-156). Ovulation is a process caused by the rise of Luteinizing Hormone (LH) from the pituitary gland. As a result, mature eggs are released from the ovarian follicles. In this process, comprehensive tissue formation is required, and MMP-9 is important for causing the proteolytic process necessary for ovulation.

On the other hand, there are currently few methods for accurately predicting or measuring the fertility rate by artificial insemination, and there are few methods for predicting or measuring at the molecular level. The method presented by the present inventors only shows how to accurately predict or measure the fertility rate by artificial insemination. Korean Patent Nos. 520287 and 570501 filed by the present inventors disclose that expression and / or activity of MMP-9 in follicular fluid is closely related to implantation rate and pregnancy rate. The patent document shows that implantation and fertilization of fertilized eggs are achieved only when MMP-9 activity is present in the follicular fluid.

However, since the patent invention uses a follicular fluid of a follicle forcibly collected from a woman undergoing the procedure, there is a problem in that the method is inconvenient to perform the method and the patient compliance is poor.

Throughout this specification many patent documents and articles are referenced and their citations are indicated. The disclosures of cited patent documents and articles are incorporated herein by reference in their entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have tried to develop a method of predicting implantation rate or fertility rate of fertilized eggs, which is simpler and improves patient compliance. As a result, the present invention was completed by finding that the activity of MMP-9 is measured from blood of hyperovulation-induced women and that the activity of MMP-9 is positively correlated with fertilization rate or fertility rate of fertilized eggs.

Accordingly, it is an object of the present invention to provide a method for measuring the activity of matrix metalloprotease (MMP) -9 using blood isolated from hyperovulation induced women.

Another object of the present invention is to provide a method of predicting implantation rate or pregnancy rate of fertilized eggs using blood isolated from hyperovulation-induced women.

Still another object of the present invention is to provide a kit for predicting implantation rate or pregnancy rate of fertilized eggs using blood samples of overovulation-induced women.

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

According to one aspect of the invention, the invention provides a method for measuring the activity of matrix metalloprotease (MMP) -9 contained in blood isolated from hyperovulation induced females.

According to another aspect of the invention, the present invention comprises measuring the activity of matrix metalloprotease (MMP) -9 contained in blood isolated from hyperovulation induced female, the activity of MMP-9 is the implantation rate of fertilized eggs Or a method of predicting implantation rate or pregnancy rate of a fertilized egg, characterized by having a positive correlation with pregnancy rate.

According to another embodiment of the present invention, the present invention specifically binds to a substrate of matrix metalloprotease (MMP) -9, an antibody specifically binding to MMP-9, or a proteolytic cleavage product of proMMP-9. Provided are kits for predicting implantation rate or fertility rate of fertilized eggs, including an antibody, by using a blood sample of an overovulation induced female.

The present inventors have tried to develop a method of predicting implantation rate or fertility rate of fertilized eggs, which is simpler and improves patient compliance. As a result, MMP-9 activity was measured in blood isolated from hyperovulation-induced women, and it was found that the activity of MMP-9 was positively correlated with fertilization rate or pregnancy rate of fertilized eggs.

The methods and kits of the present invention indicate their use in the phrase "prediction of fertilization rate or fertility rate of fertilized eggs", but it has the same meaning as "prediction, measurement or diagnosis of pregnancy potential".

The methods and kits of the invention can be applied to a variety of mammals, for example to humans, dogs, cattle, horses, pigs, sheep and mice, and most preferably to humans.

As used herein, the term “activity” as used in connection with MMP-9 means not only the enzyme activity of MMP-9, but also the expression level of MMP-9 and the amount of MMP-9 in the activated form.

The present invention is applied to the measurement of the activity of various forms of MMP-9 and used to predict the implantation rate or pregnancy rate of fertilized eggs. 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, the MMP-9 measured in the method of the present invention is 97 kDa, 83 kDa or 67 kDa of MMP-9.

In the present invention, the object of measurement is blood of a woman who has been induced to ovulate. As used herein, the term "blood" has the meaning encompassing all whole blood, serum or plasma taken from an animal (most preferably human).

The sample used in the present invention is the blood of the female, in particular the blood of a controlled ovarian hyperstimulation female. Induction of hyperovulation can be carried out through various methods. For example, clomiphene, hMG (human menopausal gonadotropin, mixed hormone of follicle stimulating hormone (FSH) and luteinizing hormone (LH)], human chorionic gonadotropin (hCG), gonadotropon-releasing hormone (GnRH), and GnRH There is a method of inducing hyperovulation using an agonist or antagonist. In the present invention, after injecting such a superovulation inducer into a woman, an increase in the concentration of MMP-9, an increase in the activity of MMP-9, and / or an active form of MMP-9 in the blood of the woman is detected. According to the method of the present invention, after a certain time after induction of hyperovulation, blood is collected and the activity of MMP-9 in the blood is measured. The time point at which blood is collected is not particularly limited, and blood is collected after a certain time (eg, one day) has elapsed after induction of hyperovulation. Preferably, blood is collected 2-5 days after induction of hyperovulation.

In a woman's blood during hyperovulation induction, an increase in the concentration of MMP-9, an increase in the activity of MMP-9, and / or an increase in the concentration of an active form of MMP-9 occurs, which is positively associated with the implantation rate or pregnancy rate of the fertilized egg. It is related.

According to a preferred embodiment of the present invention, the activity of MMP-9 in the method of the present invention can be measured by a zymography or immunoassay method.

When the method of the present invention is measured by a zymography method, an exemplary embodiment of the present invention is as follows: The zymography method is capable of measuring MMP-9 activity based on the activity of an enzyme that hydrolyzes gelatin. Can be. In this case, gelatin, the substrate of MMP-9, is inserted into a gel such as polyacrylamide. For a detailed description of zymography, see Rawdanowicz et al. J. Clin. Endocrin. Metab. 79: 530-536 (1994), Riley et al., Mol. Hum. Reprod. 5: 376-381 (1999) and Hibbs et al., J. Biol. Chem. 260: 2493-2500 (1985), which is incorporated herein by reference. Zymography analysis can be normalized using purified MMP-9 as a control. The degree of degradation of gelatin is directly related to the activity of MMP-9 in the blood.

In addition, the method of the present invention can be carried out according to various immunoassay methods known in the art.

The immunoassay includes, but is not limited to, Western blotting, radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or hardwood assay, and sandwich assay. Methods of the immunoassay include Enzyme Immunoassay , ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology , Vol. 1, Walker, JM ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

When the method of the present invention is carried out in an ELISA manner, certain embodiments of the present invention comprise the steps of: (i) coating a sample to be analyzed on the surface of a solid substrate; (Ii) reacting said sample with an antibody against MMP-9 as a primary antibody; (Iii) reacting the resultant of step (ii) with the secondary antibody to which the enzyme is bound; And (iii) measuring the activity of the enzyme.

Suitable as the solid substrate are hydrocarbon polymers (eg polystyrene and polypropylene), glass, metal or gel, most preferably microtiter plates.

Enzymes bound to the secondary antibody include, but are not limited to, enzymes catalyzing color reaction, fluorescence, luminescence or infrared reaction, for example, alkaline phosphatase, β-galactosidase, hose Radish peroxidase, luciferase and cytochrome P ₄₅₀ . When alkaline phosphatase is used as the enzyme binding to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS) as a substrate Chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis) if colorimetric substrates such as -B1-phosphate) and enhanced chemifluorescence (ECF) are used, and horse radish peroxidase is used -N-methylacridinium nitrate), resorupin benzyl ether, luminol, Amflex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), TMB (3,3,5,5-tetramethylbenzidine) Substrates such as ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]) and o -phenylenediamine (OPD) can be used.

When the method of the invention is carried out in a capture-ELISA mode, certain embodiments of the invention comprise (i) coating an antibody against MMP-9 as a capturing antibody to the surface of a solid substrate; (Ii) reacting the capture antibody with a sample (egg culture); (Iii) reacting the result of step (ii) with a detecting antibody having a label that generates a signal and which specifically reacts with CD 105 or CD 166; And (iii) measuring the signal resulting from the label.

The detection antibody carries a label which generates a detectable signal. The label may include chemicals (eg biotin), enzymes (alkaline phosphatase, β-galactosidase, horse radish peroxidase and cytochrome P ₄₅₀ ), radioactive substances (eg C ¹⁴ , I ¹²⁵ , P ³² And S ³⁵ ), fluorescent materials (eg, fluorescein), luminescent materials, chemiluminescent and fluorescence resonance energy transfer (FRET). Various labels and labeling methods are described in Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999.

The antibody used in the immunoassay method is a Zymogen form of MMP-9, that is, a proMMP-9 of 97 kDa, an intermediate active form MMP-9 of 83 kDa, an active form MMP-9 of 67 kDa, or proMMP-9. Antibodies that specifically bind to proteolytic products, i.e., products of 9 kDa or 16 kDa, can be used.

Measurement of the final enzyme activity or signal in the ELISA method and the capture-ELISA method can be carried out according to various methods known in the art. Detection of this signal indicates the presence of MMP-9 activity in the sample, ie blood. If biotin is used as a label, the signal can be easily detected with streptavidin and luciferin if luciferase is used.

When the activity of MMP-9 is measured in the blood according to the method of the present invention, it is predicted that overovulation-induced women are highly likely to be implanted and conceived by future reproductive assistance.

The kit of the present invention for predicting fertilization rate or fertility rate of fertilized eggs specifically binds to a substrate of MMP-9, an antibody specifically binding to MMP-9 and / or a proteolytic cleavage product of proMMP-9. It includes an antibody to make.

According to a preferred embodiment of the invention, the substrate of MMP-9 is collagen IV, collagen V, collagen XI, elastin, proteoglycan or gelatin, most preferably gelatin.

The antibody included in the kit of the present invention is a polyclonal or monoclonal antibody, preferably a monoclonal antibody.

Antibodies to proteolytic products of MMP-9 or proMMP-9 can be prepared by methods commonly practiced in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology , 6: 511-519 (1976)). , Recombinant DNA methods (US Pat. No. 4,816,56) or phage antibody library methods (Clackson et al, Nature , 352: 624-628 (1991) and Marks et al, J. Mol . Biol . , 222: 58, 1 -597 (1991). General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual , Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques , CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY , Wiley / Greene, NY, 1991, which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing immortalized cell lines with antibody-producing lymphocytes, and the techniques required for this process are well known to those skilled in the art and can be readily implemented. Polyclonal antibodies can be obtained by injecting the MMP-9 antigen into a suitable animal, collecting antisera from the animal, and then isolating the antibody from the antisera using known affinity techniques.

According to a preferred embodiment of the invention, the antibody that specifically binds to MMP-9 is an antibody that specifically binds to 97 kDa, 83 kDa or 67 kDa of MMP-9. Antibodies that specifically bind to the proteolytic products of proMMP-9 are those that specifically bind to products of 9 kDa or 16 kDa.

The features and advantages of the present invention are summarized as follows:

(Iii) The present invention first identified the fact that high MMP-9 activity in blood is closely related to the implantation rate and pregnancy rate of fertilized eggs.

(Ii) According to the present invention, since blood (particularly, peripheral blood) is used, implantation rate and pregnancy rate of fertilized eggs can be predicted easily and with high patient compliance.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident.

Example

Example  1: Screening for Research Subjects

In this study, 54 patients with various infertility problems were included in Dongguk University Hospital's IVF program. The causes of infertility were tubal factors (n = 15), endometriosis (n = 11), ovulation (n = 9), and unknown infertility (n = 19). The patients were between 21 and 33 years old (mean 31.3 years). Male infertility factors, ie, atypical and rare spermatozoa, were excluded from this study.

Example  2: preparation of the egg

Hyperovulation was performed with long-term therapy with gonadotropin releasing hormone agonist (GnRH-a). In the mid-luteal phase, 900 buserelin (Hoechst, Germany) was administered nasal spray to inhibit the pituitary gland, and then hMG (human menopausal gonadotropin, Pergonal, Serono or IVF-M, LG, Korea) was administered from 3-5 days after follicular phase. 10,000 mg of human chorionic gonadotropin (hCG, IVF-C, LG, when the follicle diameter is more than 17 mm by adjusting the dose of hMG according to the follicle response using vaginal ultrasound) Korea) was administered to induce ovulation. After 36 hours of hCG injection, follicles were inhaled using choking ultrasound and eggs were collected. The collected oocytes were analyzed using anatomical microscope (SMZ-10, Nicon, Japan) in a manipulator (IVF chamber, USA) controlled at 37 ° C and 5% CO ₂ . After confirming the maturity of the culture, it was transferred to a culture plate (3037, Falcon, USA) containing P1 medium containing 10% SSS (Serum Substitute Supplement) and cultured in a CO ₂ incubator until fertilization. Eggs used for fertilization were adjusted to 5 or less per culture dish.

Example  3: in vitro fertilization

Sperm were collected from the eggs and collected semen in a sample container (Baxter, USA) by masturbation. Sperm concentration and motility were measured according to WHO criteria. The semen was left at room temperature for 10-20 minutes to induce liquefaction. The liquefied semen was transferred to a 15 ml conical tube (2099, Falcon, USA) and centrifuged twice (5 minutes, 3 minutes) at 1,500 rpm with Ham's F-10 with 10% SSS. . After centrifugation, the supernatant except the pellets was removed and sperm was suspended in the incubator for 30 minutes by carefully adding Ham's F-10 with 1 ml of 10% SSS added to prevent the pellets from scattering. Suspended sperm were used for fertilization while stored in 5 ml tubes (2003, Falcon, USA). The sperm used for fertilization were injected into the culture solution containing fertilized eggs at 1 × 10 5 / ml. The next morning, under the dissecting microscope in a manipulator controlled at 37 ° C. and 5% CO ₂ , the oocytes were removed and examined for fertilization using a syringe needle (320310, BD, USA). The magnetic nucleus and the male pronucleus were formed and the two polar bodies were confirmed to have been modified.

Example  4: Pear

Normally, only fertilized eggs were selected and cultured for 48 hours in P1 culture, followed by transplantation with 2-5 eggs over 8-cell stage. In most cases, Tomcat catheter (8890-793021, Sherwoo, USA) was used for transplantation.

Example  5: Administration of Progesterone and Confirmation of Pregnancy

After embryo implantation, 100 mg of progesterone in oil (Progest, Samil, Korea) was injected daily and defined as chemical pregnancy when the concentration of blood β-hCG was 10 mIU / ml or more at about 10 days after transplantation. Clinical gestation was defined as the presence of a sac on the vaginal ultrasonography three weeks after embryo transplantation.

Example  6: Preparation of Blood Sample

1, 2, 3, 4, and 5 days after the hMG administration, and the day immediately before the hCG administration (when two or more are observed that the diameter of the follicles is 17 mm or more) and hCG administration 36 hours Peripheral blood was then collected from hyperovulation-induced women. The collected blood was coagulated by standing at room temperature for 3 hours without treatment with anticoagulant, and then left to stand at 4 ° C. for 16 hours to induce retraction of coagulated blood. Serum was separated by centrifugation at 1,500 rpm for 5 minutes at 4 ° C. and then stored at −20 ° C., which was used for the experiment.

Example  7: Zymography  In the used blood MMP -9 Determination of enzyme activity

Expression of MMP-9 (ie, enzymatic activity) in the blood is described in the design of Rawdanowicz et al. (1994, J. Clin. Endocrin. Metab. 79: 530-536) Riley et al. (1999, Mol. Hum. Reprod. 5: 376-381) was confirmed using a modified zymography. Blood (blood isolated from women 5 days after hMG administration) was determined by SDS-PAGE (7.5% (w / v) gels containing 1 mg / ml gelatin (0.1%); Minigel apparatus; Bio-Rad, Hemel Hempsead). The gel was washed twice in 2.5% (v / v) Triton X-100, and digested with digestion buffer; 200 mM NaCl, 50 mM Tris-HCl, 2.5 mM CaCl ₂ , 1 mM ZnCl ₂ , pH 7.6; Sigma Chemical Co, St Louis, MO) was stored for 18 hours at 37 ℃. Staining of the gel was performed at room temperature with 0.5% (w / v) Coomassie blue R250 (Bio Rad, Richmond, CA) in 30% (v / v) methanol 10% (v / v) glacial acetic acid in H ₂ O). Stained.

Example  8: Zymography  after MMP -9 Quantification of Enzyme Activity

Gelatinase activity was measured using a Gel Documentation semiautomated image analyzer (Core-Bio System, Seoul, Korea), which measures the intensity and surface of the band layer after scanning the gel.

Example  9: Statistics

The results were averaged by at least 3 repetitions and the significance of the mean was performed by Student's t-test. P <0.05 was determined to have statistical significance.

Experiment result

MMP-9 enzymatic activity in serum obtained from hyperovulation induced women was shown in SDS-PAGE zymography and was consistent with MMP-9 activity as demonstrated by zymography (FIG. 1). MMP-9 enzyme activity in pregnant group was significantly higher than non-pregnant group (P <0.01). However, MMP-2 enzyme activity present in 92 kDa size was detected in a constant high enzyme activity in the blood of all test subjects, there was no difference between pregnant and non-pregnant group (Fig. 1).

The MMP-9 enzyme activity in the blood of hyperovulation-induced women was 6,248 densitometric units. Table 1 below, according to the degree of enzymatic activity of MMP-9 was classified into four groups arbitrarily divided the pregnancy rate and implantation rate. The MMP-9 enzyme activity of the first group was divided into 3,000-4,000 units, the second group was 4,000-5,000 units, the third group was 5,000-6,000 units, and the fourth group was divided into 6,000-70.000 units.

variable MMP-9 Densitometer Unit 3,000-4,000 4,000-5,000 5,000-6,000 6,000-7,000 Implantation rate (%) 0 0 38.9 39.1 Pregnancy Rate (%) 0 0 48.7 49.5

Interestingly, both implantation and pregnancy rates were 0% when the MMP-9 enzyme activity level was below 5,000 units. In contrast, implantation rates and pregnancy rates were 38.9% and 48.7%, respectively, when the MMP-9 enzyme activity levels were 5,000-6,000 units. In addition, when MMP-9 enzyme activity was 6,000-7,000 units, implantation rate and pregnancy rate were 39.1% and 49.5%, respectively. Under 5,000 units of MMP-9 enzyme activity, neither implantation nor conception occurred.

Based on the above results, it can be seen that MMP-9 activity in blood is directly related to implantation rate and pregnancy rate.

Example  10: in blood by capture-ELISA MMP -9 Analysis

MMP-9 in serum was measured using a Biotrak Assay kit (Amersham Pharmacia Biotech, USA). MMP-9 was measured according to the product instructions using an ELISA plate to which the reagent provided in the kit and the MMP-9 specific antibody were attached. 100 μl of serum diluted in 1/10 was added to the plate and reacted at room temperature for 2 hours. The wells of the plates were then washed with 300 μl of phosphate buffer. Then, 100 μl of peroxidase-conjugated antibody was dispensed into each well and allowed to react for 2 hours at room temperature. After the reaction, the mixture was washed with a phosphate buffer solution, and 100 μl of a TMB (3,3,5,5-tetramethylbenzidine) / hydrogen peroxide (20% in dimethylformamide) substrate solution was dispensed and reacted at room temperature for 30 minutes. When the color reaction occurred, the absorbance of each well was measured with an ELISA reader (Molecular Devices, USA) at a wavelength of 630 nm.

Experimental results showed that the absorbance at 630 nm of ovulation-induced peripheral blood of hyperovulation-induced women (OD ₆₃₀ ) 1, 2, 3, 4 and 5 days after hMG administration and immediately before hCG administration and 36 hours after hCG administration. In comparison, OD ₆₃₀ was 2-6 times higher in the pregnant group than in the non-pregnant group.

Therefore, it can be seen that the amount of MMP-9 in blood is directly related to implantation rate and pregnancy rate.

As described in detail above, the present invention provides a method for measuring the activity of matrix metalloprotease (MMP) -9 contained in blood, a method of predicting fertilization rate or pregnancy rate of fertilized egg using blood, and implantation rate or fertility rate of fertilized egg It provides a prediction kit of. The present invention is the first invention to find out that MMP-9 activity confirmed in blood is closely related to the implantation rate and pregnancy rate of a high rate of fertilized eggs. According to the present invention, since blood (particularly, peripheral blood) is used, the implantation rate and pregnancy rate of fertilized eggs can be predicted easily and with high patient compliance.

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.

Claims (8)

A method for measuring the activity of matrix metalloprotease (MMP) -9 in blood isolated from hyperovulation induced women. Measuring the activity of matrix metalloprotease (MMP) -9 contained in blood isolated from hyperovulation-induced females, wherein the activity of MMP-9 has a positive correlation with fertilization rate or pregnancy rate of fertilized eggs. Method of prediction of fertilization rate or fertility rate of fertilized eggs. The method of claim 1 or 2, wherein the activity of the MMP-9 is measured by a zymography or immunoassay method. The method of claim 1 or 2, wherein the MMP-9 is 97 kDa, 83 kDa or 67 kDa MMP-9. Implantation rate or fertility rate of fertilized eggs, including substrates of matrix metalloprotease (MMP-9), antibodies that specifically bind to MMP-9 or antibodies that specifically bind to proteolytic cleavage products of proMMP-9 A kit for predicting using hyperovulation-induced blood samples from women. 6. The kit of claim 5, wherein said MMP-9 is 97 kDa, 83 kDa or 67 kDa MMP-9. 6. The kit of claim 5, wherein the substrate of MMP-9 is selected from the group consisting of collagen IV, collagen V, collagen XI, elastin, proteoglycans and gelatin. 6. The kit of claim 5, wherein the proteolytic product of proMMP-9 is a product of 9 kDa or 16 kDa.

Images