WO2008094016A1

WO2008094016A1 - Uses of mmp-9 activities present in blood

Info

Publication number: WO2008094016A1
Application number: PCT/KR2008/000632
Authority: WO
Inventors: Seung-Woo Kang; Cheorl-Ho Kim
Original assignee: Benebiosis Co., Ltd.
Priority date: 2007-02-02
Filing date: 2008-02-01
Publication date: 2008-08-07
Also published as: KR20080072353A

Abstract

The present invention relates to a method for measuring a MMP (matrix metalloprotease)-9 activity in blood isolated from a female undergoing controlled ovarian hyperstimulation, a method for predicting an implantation rate or a pregnancy rate of a fertilized egg in a female subject, which comprises measuring a MMP-9 activity in blood isolated from a female undergoing controlled ovarian hyperstimulation, wherein a MMP-9 activity has a positive correlation with an implantation rate or a pregnancy rate of a fertilized egg, and a diagnostic kit for predicting an implantation rate or a pregnancy rate of a fertilized egg in a female subject using a blood sample, which comprises a substrate of MMP-9, an antibody binding specifically to the MMP-9 or an antibody binding specifically to a proteolytic cleavage product of proMMP-9. This invention firstly elucidates the fact that the MMP-9 activity in blood is correlated closely to the implantation and pregnancy rates of a fertilized egg with high percentage. According to the present invention, the implantation and pregnancy rates of a fertilized egg are capable of predicting with a relative simplicity and an improvement of patient compliance, due to the utilization of blood isolated from the female.

Description

USES OF MMP-9 ACTIVITIES PRESENT IN BLOOD

FIELD OF THE INVENTION

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

DESCRIPTION OF THE RELATED ART

Since the first test-tube baby was born in 1979, various methods for assisted reproduction technology (ART) have been developed and applied to the treatment of sterile patients. Examples of ART are in vitro fertilization-embryo transfer (IVF-ET), intracytoplasmic sperm injection (ICSI), testicular sperm extraction (TESE), round spermatid injection (ROSI) and embryo freezing. While ART is an important and general method for the treatment of sterile patients, there remain problems to be overcome, such as high costs, complicated procedure and low chance of success.

The use of ART is on the increase. The rate of pregnancy in IVF has increased but is not up to the expectation. The actual take home baby rate is 15-30%. According to the U.S. statistics, the pregnancy rate and delivery rate per cycle by using IVF in 1996 was 23.8% and 19.3% respectively. In 1997 statistics, these rates increased to 29.3% and 24.0% each. However, there was no substantial difference between 1996 statistics and 1997 statistics.

The successful human pregnancy is dependent upon follicular development, number of oocytes retrieved, fertilization, embryo development and implantation, during an assisted reproductive technology (ART) cycle (Sakkas D, et al., 2001, Fertil. Steril. 76(6): 1150-1156). Among them, blastocyst implantation into the human uterus is a complex process dependent on profound structural changes in the endometrium and the developing embryo. Tissue remodeling is requisite to uterine preparation, embryonic breaching of the epithelial basement membrane and subsequent penetration of the endometrial stroma. It has been thought that matrix metalloproteinases (MMPs) are essential for the breakdown of extracellular matrix (ECM) components during this process. In light of these, it would be suggested that MMP-9 is involved in cells meiosis, invasion and tissue remodeling during development. Namely, it was known that MMP contributes to a change of extracellular matrix to induce endometrial tissue remodeling on menstrual cycle. Furthermore, MMP molecules are observed to show diverse expression patterns on menstrual cycle and implicated in ovulation and implantation.

MMP(matrix metalloproteinase)-9, known as 92 kDa type IV collagenase/gelatinase B, degrades many kinds of collagens (IV, V and XI), elastin, proteoglycan and gelatin (Hibbs et al., 1985, J. Biol. Chem. 260:2493-2500; Murphy et al., 1991, Biochem. J. 277:277-279). Moreover, previous studies have demonstrated that the secretion of MMP-9 from 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 endometrial stromal cells (Huang et al., 1998, J. Clin. Endocrincol. Metab. 83:1721-1729) is stimulated by cytokines and hormones.

MMP-9 has been suggested to intervene at different stages of the cyclical changes in female reproduction such as in the menstrual cycle, ovulation, implantation, parturition and involution of the mammary glands 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 triggered by the preovulatory surge of Luteinizing Hormone (LH) from the pituitary gland, which results in the liberation of the mature ovum from the preovulatory ovarian follicle. This process requires extensive tissue remodeling, and MMP-9 is important for generating the proteolytic activity needed at the time of ovulation.

However, little is known about methods for accurately predicting or measuring pregnancy rates of in vitro fertilization. Furthermore, molecular approaches for predicting or measuring pregnancy rates have not yet reported. The method suggested by the present inventors is considered the only approach for exactly predicting or measuring pregnancy rates of in vitro fertilization. Korean Pat. Nos. 520287 and 570501 filed by the present inventors disclose that the MMP-9 expression and/or activity in follicular fluid were closely related to implantation and pregnancy rates. The patents describe that implantation and pregnancy could be achieved only when the MMP-9 activity in follicular fluid is detected.

However, the methods disclosed in the patents have serious disadvantages such as operation inconvenience and reduced patient compliance due to the utilization of follicular fluid retrieved artificially from a human female undergoing operation.

Throughout this application, several patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications is incorporated into this application in order to more fully describe this invention and the state of the art to which this invention pertains.

DETAILED DESCRIPTION OF THIS INVENTION

The present inventors have made intensive researches to develop a method for predicting an implantation or a pregnancy rate of a fertilized egg with a relative simplicity and an improvement of patient compliance. As a result, the present inventors have found that a MMP-9 activity is measured in blood isolated from a female undergoing controlled ovarian hyperstimulation and has a positive correlation with the implantation or pregnancy rate of the fertilized egg.

Accordingly, it is an object of this invention to provide a method for measuring a MMP (matrix metalloprotease)-9 activity in blood isolated from a female undergoing controlled ovarian hyperstimulation.

It is another object of this invention to provide a method for predicting an implantation rate or a pregnancy rate of a fertilized egg in a female subject using the blood isolated from a female undergoing controlled ovarian hyperstimulation.

It is still another object of this invention to provide a diagnostic kit for predicting an implantation rate or a pregnancy rate of a fertilized egg in a female subject using a blood sample of a female undergoing controlled ovarian hyperstimulation.

Other objects and advantages of the present invention will become apparent from the detailed description to follow taken in conjugation with the appended claims and drawings.

In one aspect of the present invention, there is provided a method for measuring a MMP (matrix metalloprotease)-9 activity in blood isolated from a female undergoing controlled ovarian hyperstimulation.

In another aspect of the present invention, there is provided a method for predicting an implantation rate or a pregnancy rate of a fertilized egg in a female subject, which comprises measuring a MMP (matrix metalloprotease)-9 activity in blood isolated from a female undergoing controlled ovarian hyperstimulation, wherein the MMP-9 activity has a positive correlation with the implantation or pregnancy rate of a fertilized egg.

In still another aspect of the present invention, there is provided a diagnostic kit for predicting an implantation rate or a pregnancy rate of a fertilized egg in a female subject using a blood sample of a female undergoing controlled ovarian hyperstimulation, which comprises a substrate of MMP (matrix metalloprotease)-9, an antibody binding specifically to the MMP-9 or an antibody binding specifically to a proteolytic cleavage product of proMMP-9.

The present inventors have made intensive researches to develop a method for predicting an implantation rate or a pregnancy rate of a fertilized egg with a relative simplicity and an improvement of patient compliance. As a result, the present inventors have found that a MMP-9 activity is measured in blood isolated from a female undergoing controlled ovarian hyperstimulation and has a positive correlation with the implantation rate or pregnancy rate of a fertilized egg.

The method and the diagnostic kit of the present invention represent the use as the term "prediction of an implantation or a pregnancy rate of a fertilized egg", however the term has the same meaning as "prediction, measurement or diagnose of the pregnancy chance".

The method and the diagnostic kit of the present invention may be applied to various mammals such as human, dog, cattle, horse, pig, sheep and mouse, most preferably human.

The term "activity" as used herein being associated with MMP-9, refers to MMP- 9 activity as well as MMP-9 expression level and amount of activated form MMP-9.

The present invention has the utilization of the prediction of the implantation or pregnancy rate of a fertilized egg being applied to the measurement of various forms

MMP-9 activity. MMP-9 has been known as 92 kDa type IV collagenase/gelatinase or gelatinase B. A purification of MMP-9 and cDNA sequence encoding the same are disclosed in U.S. Pat. No. 4,992,537. Where proMMP-9 (zymogen of MMP-9) is activated, it is cleaved to generate an intermediate activated form of 83 kDa and an inactivated proteolytic cleavage product of 9 kDa and then further cleaved to produce an activated form of 67 kDa and an inactivated proteolytic cleavage product of 16 kDa.

According to a preferred embodiment, the MMP-9 measured in the present invention is 97 kDa, 83 kDa or 67 kDa of MMP-9.

A measuring object in this invention is blood isolated from a female undergoing controlled ovarian hyperstimulation. The term "blood" as used herein refers to whole blood, serum or plasma obtained from animals (most preferably, human).

A sample used in this invention is among blood of the female, particularly blood of the female undergoing controlled ovarian hyperstimulation.

The controlled ovarian hyperstimulation may be performed by various methods. Examples include methods using clomiphene, hMG [human menopausal gonadotropin, mixture hormone of FSH (follicle stimulating hormone) and LH (luteinizing hormone)], hCG (human chorionic gonadotropin), GnRH (gonadotropon-releasing hormone) or an agonist or antagonist of GnRH. After injecting agents for inducing ovarian hyperstimulation, the increases in MMP-9 concentration, MMP-9 activity, and/or MMP-9 activity of activated form are analyzed according to the present invention. According to the present method, a blood sample is collected after operation of controlled ovarian hyperstimulation and MMP-9 activity in blood is then measured. The period of time for retrieving the blood sample is not limited to. The blood sample may be collected at any time {e.g., 1 day) after controlled ovarian hyperstimulation, preferably 2-5 days after controlled ovarian hyperstimulation. In blood of the female during the controlled ovarian hyperstimulation procedure, the increases in MMP-9 concentration, MMP-9 activity, and/or MMP-9 concentration of activated form are analyzed, which has a positive correlation with the implantation or pregnancy rate of a fertilized egg of this female.

According to a preferred embodiment, the MMP-9 activity in the present method is measured by using zymography or immunoassay method.

Where the present method is measured by zymography, the exemplary embodiment of this invention is described as follows: Zymography method enables to measure MMP-9 activity based on enzyme activity to hydrolyze gelatin. At this time, the substrate of MMP-9, gelatin is present in gels such as polyacrylamide. Details about zymography are disclosed in Rawdanowicz et al. J. Clin. Endcrin. Metab. 79:530- 536(1994), Riley et al., MoI. Hum. Reprod. 5: 376-381(1999) and Hibbs et al., J. Biol. Chem. 260:2493-2500(1985), which are incorporated herein by references. Gelatin degradation level shows a direct correlation with MMP-9 activity in blood.

Furthermore, the present method may be carried out according to various immunoassay methods well-known in one skilled in the art.

Such immunoassay includes western blotting, radioimmunoassay, radioimmuno- precipitation, immuno-precipitation, ELISA (enzyme-linked immunosorbent assay), capture-ELISA, inhibition or competition assay, sandwich assay, but not limited to. The immunoassay can be found in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J. M. ed., Humana Press, NJ, 1984; and Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1999, which are incorporated herein by references.

Where the present method is performed by the ELISA method, the specific example of the present method comprises the steps of: (i) coating a surface of solid substrates with a sample to be analyzed; (ii) incubating the sample with a primary antibody to MMP-9 protein; (iii) incubating the resultant of step (ii) with a secondary antibody conjugated with an enzyme; and (iv) measuring the activity of the enzyme.

The solid substrate useful in this invention includes carbohydrate polymer {e.g., polystyrene and polypropylene), glass, metal and gel, most preferably microtiter plates. The enzyme conjugated with the secondary antibody includes the enzyme catalyzing colorimetric, fluorometric, luminescence or infra-red reactions, but not limited to, e.g., including alkaline phosphatase, β-galactosidase, horseradish peroxidase, luciferase and Cytochrome P450. Where using alkaline phosphatase, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-Bl- phosphate and ECF (enhanced chemifluorescence) may be used as a substrate for color-developing reactions, in the case of using horseradish peroxidase, chloronaphtol, aminoethylcarbazol, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), resorufin benzyl ether, luminol, Amplex Red reagent (10-acetyl-3,7- dihydroxyphenoxazine), TMB (3,3,5,5-tetramethylbenzidine), ABTS (2,2'-Azine-di[3- ethylbenzthiazoline sulfonate]) and o-phenylenediamine (OPD) may be used as a substrate. Where the present method is performed in accordance with the capture-ELISA method, the specific example of the present method comprises the steps of: (i) coating a surface of a solid substrate with a capturing antibody capable of binding specifically to the MMP-9 protein (ii) incubating the capturing antibody with a sample o

{e.g., the cultured embryo) to be analyzed; (iii) incubating the resultant of step (ii) with a detecting antibody which is capable of binding specifically to MMP-9 and conjugated with a label generating a detectable signal; and (iv) detecting the signal generated from the label conjugated with the detecting antibody. The detecting antibody has a label generating a detectable signal. The label includes, but not limited to, a chemical {e.g., biotin), an enzymatic {e.g., alkaline phosphatase, β-galactosidase, horseradish peroxidase and Cytochrome P450), a radioactive {e.g., C14, 1125, P32 and S35), a fluorescent {e.g., fluorescein), a luminescent, a chemiluminescent and a FREET (fluorescence resonance energy transfer) label. Various labels and methods for labeling antibodies are well known in the art (Ed Harlow and David Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999).

The antibody used in the immunoassay method may be capable of using an antibody binding specifically to zymogen form of MMP-9 {i.e. 97 kDa of proMMP-9), 83 kDa of intermediate activated form MMP-9, 67 kDa of activated form MMP-9, or proteolytic cleavage product of proMMP-9 {i.e. 9 kDa or 16 kDa of product).

The measurement of enzyme activity and signal intensity in such ELISA and capture-ELISA method can be carried out by various processes well known in the art.

The detection of this signal is indicative of the presence of MMP-9 activity in sample, i.e. blood. Where biotin and luciferase are used as labels, the signal detection may be achieved by use of streptavidin and luciferin, respectively.

When the MMP-9 activity in blood is measured according to the present method, it is predicted that the female undergoing controlled ovarian hyperstimulation have the much higher chances of implantation and pregnancy by assisted reproduction technology (ART) hereafter.

The diagnostic kit for predicting the implantation or pregnancy rate of a fertilized egg in the female subject comprises the substrate of MMP (matrix metalloprotease)-9, the antibody binding specifically to the MMP-9 and/or the antibody binding specifically to the proteolytic cleavage product of proMMP-9.

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

The antibody comprised in the diagnostic kit of this invention is a polyclonal or a monoclonal antibody, preferably the monoclonal antibody.

In this invention, the antibody capable of binding to MMP-9 or the proteolytic cleavage product of proMMP-9 could be prepared by conventional techniques such as a fusion method (Kohler and Milstein, European Journal of Immunology, 6:511- 519(1976)), a recombinant DNA method (USP 4,816,56) or a phage antibody library (Clackson et al, Nature, 352:624-628(1991) and Marks et al, J. MoI. Biol., 222:58, 1- 597(1991)). The general procedures for antibody production 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 references. For example, the preparation of hybridoma cell lines for monoclonal antibody production is done by fusion of an immortal cell line and the antibody producing lymphocytes. This can be done by techniques well known in the art. Polyclonal antibodies may be prepared by injection of the MMP-9 antigen to suitable animal, collecting antiserum containing antibodies from the animal, and isolating specific antibodies by any of the known affinity techniques.

According to a preferred embodiment, the antibody binding specifically to MMP- 9 is the antibody binding specifically to 97 kDa, 83 kDa or 67 kDa of MMP-9. The antibody binding specifically to the proteolytic cleavage product of proMMP-9 is the antibody binding specifically to 9 kDa or 16 kDa of product.

The summary of features and advantages of this invention is as follows:

(i) This invention firstly elucidates the fact that the MMP-9 activity in blood is correlated closely to the implantation and pregnancy rate of a fertilized egg with high percentage.

(ii) According to the present invention, the implantation and pregnancy rates of a fertilized egg are capable of predicting with a relative simplicity and an improvement of patient compliance, due to the utilization of blood (particularly, peripheral blood).

The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a gel photograph measured by zymography analysis of the MMP-9 and MMP-2 activity in blood of the female undergoing controlled ovarian hyperstimulation. Lane C, lane 1-4 and lane 5-8 represent the control group by use of the purified MMP-9, the pregnant group and the non-pregnant group, respectively.

EXAMPLES Example 1: Selection of Research Objects 54 patients who underwent IVF-ET program of the women's Human Infertility of Dongguk University for a variety of infertility problem were included in this study. Causes of infertility were included tubal factors (n=15), endometriosis (n=ll), anovulation (n=9) and unexplained infertility (n=19). The female partners were between 21 and 33 years old (average, 31.3 years old). Couples with male factor infertility such as sperm deficiency were excluded from this study.

Example 2: Preparation of Oocyte Ovarian induction was performed by gonadotropin-releasing hormone agonist (GnRH-a). 900 buserelin (Hoechst, Germany) was sprayed to the nasal cavity for pituitary desensitization during luteal phase and human menopausal gonadotropin (hMG, Pergonal, Serono or IVF-M, LG, Korea) was injected 3-5 days after the start of follicular phase. The amount of hMG is adjusted according to the response of follicle using negative vaginal ultrasound scan. Ovulation was induced with 10,000 IU of human chorionic gonadortopin (hCG, IVF-C, LG, Korea) when the patients had more or two follicles greater than 17 mm in diameter. Oocyte retrieval was performed 36 hr after the injection of hCG by aspiring follicle using vaginal ultrasound. The oocyte retrieved was placed in the manipulator (IVF chamber, USA) with 37⁰C temperature and 5% CO₂ and the maturity of the oocyte was measured by observing the properties of ovarian cell and the existence of GV in the cytoplasm using dissecting microscope (SMZ-10, Nicon, Japan). Then the oocyte was transferred to culture plate (3037, Falcon, USA) which contained Pl media added with 10% SSS (serum substitute supplement; Irvine Science, Santa Ana, Calif., USA) and cultured in the CO₂ cultivator. The number of oocytes used was not more than 5 per culture plate.

Example 3: External Fertilization

After retrieving oocytes, sperm was collected to the specimen container (Baxter, USA) through masturbation. The concentration and mobility of sperms were measured according to the standard of WHO. The sperm was maintained in the room temperature environment for 10 to 20 min to obtain liquid phase sperm. The liquid phase sperm was transferred to 15 ml conical tube (2099, Falcon, USA) and centrifuged using Ham's F-10 containing 10% SSS at 1500 rpm twice for 5 and 3 min each. Supernatant liquid of centrifuged sperm was removed and Ham's F-IO containing 10% SSS was cautiously added to the remaining pellet so that the sperm can float. Floating sperm was stored in the 5 ml tube (2003, Falcon, USA) and used for fertilization. lx lO⁵/ml of sperms were injected to the cultured oocyte. In the following morning, the oocyte retrieved was transferred to the manipulator with 37⁰C temperature and 5% CO₂ and ovarian cell was removed by using syringe needle (320310, BD, USA) while observing with dissection microscope to determine the success of fertilization. Female pronucleus and male pronucleus were observed to be formed and two polycytes existed and from this observation the success of fertilization could be confirmed.

Example 4: Transfer of Embryo

The embryos which had been successfully fertilized were collected and cultured in the Pl media for 48 hr, and 2-5 embryos with 8th cell period were selected and transferred into the uterine cavity. In most cases, a Tomcat catheter (8890-793021, Sherwoo, USA) was used for transfer.

Example 5: Injection of Luteal Hormone and Confirmation of Fertilization Everyday 100 mg of luteal hormone (progesterone in oil, Progest, Samil, Korea) were injected to the patients by intramuscular injection. 10 days after the transfer, biochemical pregnancies were defined as those pregnancies with a transient elevation of serum β-hCG level (>10 mIU/ml). 3 weeks after the transfer, a clinical pregnancy was defined as the presence of gestational sac detectable by vaginal ultrasound.

Example 6: Preparation of Blood Sample

Peripheral blood samples of women undergoing controlled ovarian hyperstimulation were retrieved on 1, 2, 3, 4 and 5 days after injecting hMG in Example 2, 1 day before injecting hMG (the period of time that not less than two follicles with diameter of not less than 17 mm were observed) and 36 hr after injecting hMG, respectively. The blood samples not treated with anticoagulant were kept to stand for 3 hr at room temperature for coagulation and then kept to stand for 16 hr at 4⁰C, inducing retraction of coagulated blood. Serum was prepared by centrifugation with 1,500 rpm at 4⁰C, and stored in a refrigerator at -2O⁰C. The serum samples were used for experiments.

Example 7: Measuring MMP-9 Activity in Blood by use of Zymography Expression of MMP-9 {i.e. enzyme activity) in blood was detected using zymography as described by Rawdanowicz et al. (1994, J. Clin. Endocrin. Metab. 79:530-536) with minor modifications (Riley et al., 1999, MoI. Hum. Reprod. 5:376- 381). Blood samples (blood isolated from the women 5 days after injecting hMG) were separated by SDS-PAGE (7.5% (w/v) gels; Minigel apparatus; Bio-Rad, Hemel Hempsead) using gels containing gelatin (1 mg/ml) in non-reducing conditions. Gels were washed twice in 2.5%(v/v) Triton X-IOO and incubated in digestion buffer (200 mM NaCI, 50 mM Tris-HCI, 2.5 mM CaCI₂, 1 mM ZnCI₂, pH 7.6; all chemicals from Sigma Chemical Co, St Louis, Mo.) for 18 hr at 37⁰C. The gels were stained in staining solution (0.5% (w/v) Coomassie blue R250 (Bio Rad, Richmond, CA) in 30% (v/v) methanol 10% (v/v) glacial acetic acid in H₂O) at room temperature.

Example 8: Quantification of MMP-9 Activity after Zymography

Gelatinase activities were quantified using Gel Documentation semi-automated image analysis (Core-Bio System, Seoul, Korea) which quantified both the surface and the intensity of the lysis bands after scanning of the gels.

Example 9: Statistical Analysis

Results are presented as the mean±SE of at least three separate experiments. Statistical differences were evaluated by analysis with Student's t-test. Values of P<0.05 were accepted as significant.

Experiment Results The MMP-9 activity was detected in serum obtaining from women undergoing controlled ovarian hyperstimulation by SDS-PAGE zymography (FIG. 1). The activities of MMP-9 increased significantly (P<0.01) in pregnant group compared with the nonpregnant group. The MMP-2 activity was detected with high level at 92 kDa in blood samples of all women. However, no significant differences in MMP-2 expressions were found between pregnant and non-pregnant group (FIG. 1).

The MMP-9 activities in blood of women undergoing controlled ovarian hyperstimulation were analyzed. The mean MMP-9 densitometry for pregnant group was 6,248 units. Table 1 shows the pregnancy rate (PR) and implantation rate (IR) according to four arbitrary groups of MMP-9 expression values as follows: group 1, MMP-9 densitometric 3,000-4,000 units; group 2, 4,000-5,000 units; group 3, 5,000- 6,000 units and group 4, 6,000-7,000 units.

Table 1

Interestingly, when the MMP-9 expression levels were less than 5,000 units using the densitometry, the implantation and pregnancy rates were all 0%. In contrast, when the levels of MMP-9 expression were 5,000-6,000 units, the implantation and pregnancy rates were 38.9% and 48.7% respectively. The implantation and pregnancy were found successful only when the MMP-9 activities were higher in blood of women undergoing controlled ovarian hyperstimulation. Moreover, when the MMP-9 expression levels were 6,000-7,000 units using the densitometry, the implantation and pregnancy rates were 39.1% and 49.5% respectively. When the MMP-9 activities were less than 5,000 units, the implantation and pregnancy were all failed.

The results demonstrate that the MMP-9 activities in blood are correlated directly with the implantation and pregnancy rates. Example 10: Analysis of MMP-9 in Blood by Capture-ELISA Method

The MMP-9 level in serum was measured using Biotrak Assay kit (Amersham Pharmacia Biotech, USA). The MMP-9 level was measured using reagents contained in the kit and ELISA plate with bound antibodies to MMP-9 in accordance with manufacturer's protocol. 100 μl of serum diluted by 1/10 was added to the plate and incubated for 2 hr at room temperature. The wells of the plate were washed with 300 μl of phosphate buffer. Afterwards, 100 μl of peroxidase-conjugated antibody was aliquoted to each well and incubated for 2 hr at room temperature. The plate was washed using phosphate buffer and 100 μl of substrate solution of TMB (3,3,5,5- tetramethyl benzidine)/ hydrogen peroxide (20% in dimethylformamide) was aliquoted and incubated for 30 min at room temperature. Where the colorimetric reaction was developed, the absorbance of each well was measured at 630 nm using a ELISA reader (Molecular Devices, USA). The absorbance values at 630 nm (OD₅₃₀) of peripheral blood samples from women on 1, 2, 3, 4 and 5 days after hMG injection, 1 day before injecting hMG or 36 hr after hMG injection were measured and compared. It was revealed that OD₆₃₀ of pregnant group is higher (2-6 fold) than that of non-pregnant group.

These results urge us to reason that the levels of MMP-9 in blood are correlated directly with the implantation and pregnancy rates.

Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

Claims

What is claimed is:

1. A method for predicting an implantation rate or a pregnancy rate of a fertilized egg of a female subject, which comprises measuring the activity of MMP (matrix metalloprotease)-9 in blood isolated from the female subject undergoing controlled ovarian hyperstimulation, wherein the activity of MMP-9 in blood has a positive correlation with the implantation or pregnancy rate of a fertilized egg.

2. The method according to claim 1, wherein the activity of MMP-9 is measured by zymography or immunoassay method.

3. The method according to claim 1, wherein the molecular weight of MMP-9 is 97 kDa, 83 kDa or 67 kDa.

4. A diagnostic kit for predicting an implantation rate or a pregnancy rate of a fertilized egg of a female subject by use of a blood sample of the female undergoing controlled ovarian hyperstimulation, which comprises a substrate of MMP (matrix metalloprotease)-9, an antibody binding specifically to the MMP-9 or an antibody binding specifically to a proteolytic cleavage product of proMMP-9.

5. The diagnostic kit according to claim 4, wherein the molecular weight of MMP-9 is 97 kDa, 83 kDa or 67 kDa.

6. The diagnostic kit according to claim 4, wherein the substrate of MMP-9 is selected from a group consisting of collagen IV, collagen V, collagen XI, elastin, proteoglycan and gelatin.

7. The diagnostic kit according to claim 4, wherein the molecular weight of the proMMP-9 proteolytic cleavage product is 9 kDa or 16 kDa.