METHODS OR KITS FOR DIAGNOSING RECURRENT SPONTANEOUS ABORTION

Technical Field

The present invention relates to a method and a kit for diagnosing recurrent spontaneous abortion.

Background Art

Recurrent spontaneous abortion is defined as occurrence of three or more consecutive pregnancy losses before the 20th week of gestation. The incidence of recurrent spontaneous abortion in the whole women is about 1%, and 10-15% of clinically recognized pregnancies end in recurrent spontaneous abortion. In addition, it is reported that recurrent spontaneous abortion affects about 5% of couples who want to bear a child.

Much research has been made to find the etiologies of recurrent spontaneous abortion during pregnancy. According to the research results, it has been reported that recurrent spontaneous abortion is associated with autoimmune, genetic or chromosomal abnormalities, anatomical abnormalities, endocrinologic or hormonal abnormalities, blood coagulation-associated protein defects, etc. According to some research about molecular genetic mechanisms associated with recurrent spontaneous abortion, it has been reported that immune-, vascularization-, or apoptosis-associated genes are associated with recurrent spontaneous abortion. In most research, however, the etiologies of recurrent spontaneous abortion and molecular mechanisms related thereto have not yet been identified.

Meanwhile, the present inventors have identified some genes which show differential transcription in the chorions of recurrent spontaneous abortion patients and normal persons, and classified and reported them as immunosuppression, vascularization, embryo implantation, and apoptosis associated genes (Choi, H. K., Choi, B. C ₁ Lee, S. H., Kim, J. W. et al., MoI. Reprod. Dev. 2003, 66, 24-31.; Baek, K. H., Choi, B. C, Lee, J. H., Choi, H. K. et al., Reprod. Fertil. Dev. 2002, 14, 235-240).

However, the identification of proteins specifically expressed in patients with recurrent spontaneous abortion, in addition to the previously identified genes or proteins expressed therefrom, may be useful for developing a diagnostic kit for early diagnosis of patients suffering from recurrent spontaneous abortion or capable of diagnosing recurrent spontaneous abortion.

Detailed Description of the Invention

Technical Problem

The present inventors performed two-dimensional electrophoresis, MALDLI TOF-MS, protein sequencing, etc. using the follicular fluids derived from recurrent spontaneous abortion patients and normal persons, and found proteins whose expression was specifically reduced in the recurrent spontaneous abortion patients. Therefore, the present invention provides a method for diagnosing recurrent spontaneous abortion using a protein whose expression is specifically reduced in a recurrent spontaneous abortion patient.

The present invention also provides a kit for diagnosing recurrent spontaneous abortion, including a molecule capable of measuring the expression level of a gene encoding the protein.

Technical Solution

According to an aspect of the present invention, there is provided a method for diagnosing recurrent spontaneous abortion, the method including measuring the expression level of a gene encoding at least one protein selected from the group consisting of proteins as set forth in SEQ ID NOS: 1-5 in a sample derived from a pregnant woman.

According to another aspect of the present invention, there is provided a kit for diagnosing recurrent spontaneous abortion, including a molecule capable of measuring the expression level of a gene encoding at least one protein selected from the group

consisting of proteins as set forth in SEQ ID NOS: 1-5, wherein the molecule is an antibody, substrate, ligand, or cofactor specifically binding to the at least one protein; or a primer having a complementary sequence specific for the gene encoding the at least one protein.

Brief Description of the Drawings

FIG. 1 shows two-dimensional electrophoretic results of proteins extracted from follicular fluids derived from a recurrent spontaneous abortion (RSA) group and a control group;

FIGS. 2 and 3 show Western blot results for the expression of fibrinogen α, β, and Y in a RSA group and a control group;

FIGS. 4 and 5 show Western blot results for the expression of antithrombin in a RSA group and a control group; FIGS. 6 and 7 show the gene expression levels of complement component C3c chain E, fibrinogen y, antithrombin, and angiotensinogen in a RSA group and a control group; and

FIG. 8 is the results of real-time PCR showing the expression levels of fibrinogen Y and antithrombin in a RSA group and a control group.

Best Mode for Carrying Out the Invention

The present inventors performed cDNA subtractive hybridization for chorionic tissues derived from recurrent spontaneous abortion patients and normal persons in order to identify genes whose expression is specifically reduced in the recurrent spontaneous abortion patients.

Follicular fluid provides the in vivo environment suitable for oocytes during maturation of the oocytes. According to various research reports, follicular fluid includes important substances associated with follicle growth and oocyte fertilization during fertilization in mammals. Proteins produced by granulosa cells and theca cells are included in the ovary. It has been demonstrated that macrophage inflammatory

protein-3α, follistatin, activin, TGFβ superfamily, etc. are associated with the growth and differentiation of follicular cells. A chemical composition of a solution produced from the mature follicle is a marker for the secretion and metabolism of the follicular cells, which is important since it is associated with the quality of the follicle. This suggests that the composition of proteins present in follicular fluid is important in the growth and maturation of follicular cells and oocytes.

The present inventors isolated recurrent spontaneous abortion-associated proteins from the follicular fluids derived from recurrent spontaneous abortion patients using two-dimensional electrophoresis, i.e., 2D-PAGE, MALDLI TOF-MS, and protein sequencing, and found that the proteins were abnormally expressed in only recurrent spontaneous abortion patients using a biochemical analysis, such as Western blotting, reverse-transcription PCR, or real-time PCR.

The proteins thus obtained are proteins having sequences as set forth in SEQ ID NOS: 1-5, and are summarized in Table 1 below.

Table 1

Therefore, the present invention provides a method for diagnosing recurrent spontaneous abortion, the method including measuring the expression level of a gene encoding at least one protein selected from the group consisting of proteins as set forth in SEQ ID NOS: 1-5 in a sample derived from a pregnant woman.

In the diagnostic method of the present invention, the sample derived from a pregnant woman may be blood, urine, follicular fluid, or the like, preferably blood. Generally, pregnant women are medically examined periodically or as needed in the obstetrics and gynecology department from when the fact of pregnancy is confirmed,

and a clinician extracts a sample of blood, urine, follicular fluid, or the like to perform various examinations. For example, examinations such as genetic disorders are performed using an extracted blood in the obstetrics and gynecology department. The diagnostic method of the present invention can be employed in determining recurrent spontaneous abortion using an extracted/isolated blood.

The diagnostic method of the present invention includes measuring the expression level of a gene encoding at least one protein selected from the group consisting of proteins as set forth in SEQ ID NOS: 1-5. The proteins as set forth in SEQ ID NOS: 1-5 and their gene sequences are known in GenBank, etc. The measurement of the expression level of the gene may be achieved by measuring the expression level of the mRNA or protein (i.e., at least one of the proteins as set forth in SEQ ID NOS: 1-5) of the gene using a method commonly known in the biotechnology field. The expression levels of the proteins as set forth in SEQ ID NOS: 1-5 may be measured by a method such as Western blotting. When the expression level of a target protein is measured by Western blotting, if the expression level of the protein in a pregnant woman is about 50% or less, preferably about 40% or less, more preferably about 30-35% of the expression level of the protein in a normal person, it can be determined that the pregnant woman is at the risk of recurrent spontaneous abortion.

The mRNA expression level of a gene encoding at least one of the proteins as set forth in SEQ ID NOS: 1-5 may be measured by a method such as reverse-transcription PCR or real-time PCR. When the expression level of mRNA encoded by a target gene is measured by reverse-transcription PCR or real-time PCR, if the mRNA expression level in a pregnant woman is 30% or less of the mRNA expression level in a normal person, it can be determined that the pregnant woman is at the risk of recurrent spontaneous abortion. For example, if the mRNA level of a gene encoding compliment component C3c chain E as set forth in SEQ ID NO: 1 in a pregnant woman is 30% or less, preferably 10% or less, more preferably 5% or less of that in a normal person, it can be determined that the pregnant woman is at the risk of recurrent spontaneous abortion. If the mRNA level of a gene encoding fibrinogen y as set forth in SEQ ID NO: 2 in a pregnant woman is 30% or less of that in a normal person, it can be determined that the pregnant woman is at the risk of recurrent spontaneous

abortion. If the mRNA level of a gene encoding antithrombin as set forth in SEQ ID NO: 3 in a pregnant woman is 30% or less, preferably 10% or less, more preferably 3% or less of that in a normal person, it can be determined that the pregnant woman is at the risk of recurrent spontaneous abortion. If the mRNA level of a gene encoding angiotensinogen as set forth in SEQ ID NO: 4 in a pregnant woman is 30% or less, preferably 10% or less, more preferably 5% or less of that in a normal person, it can be determined that the pregnant woman is at the risk of recurrent spontaneous abortion.

The present invention also provides a kit for diagnosing recurrent spontaneous abortion, including a molecule capable of measuring the expression level of a gene encoding at least one protein selected from the group consisting of proteins as set forth in SEQ ID NOS: 1-5, wherein the molecule is an antibody, substrate, ligand, or cofactor specifically binding to the at least one protein; or a primer having a complementary sequence specific for the gene encoding the at least one protein.

Molecules capable of measuring the expression levels of genes encoding the five proteins (i.e., the proteins as set forth in SEQ ID NOS: 1-5) identified according to the present invention may be antibodies, substrates, ligands, or cofactors specifically binding to the proteins; or primers having complementary sequences specific for the genes encoding the proteins.

Polyclonal or monoclonal antibodies for the proteins of SEQ ID NOS: 1-5 can be prepared by a method commonly known in the biotechnology field. Thus, a diagnostic kit including at least one of the antibodies can be manufactured. The functions of the proteins of SEQ ID NOS: 1-5 have been identified. Thus, a diagnostic kit including a substrate, ligand, or cofactor for each of the proteins can also be manufactured. In addition, primers having complementary sequences specific for the genes encoding the proteins of SEQ ID NOS: 1-5 can be prepared by a method commonly known in the biotechnology field. Thus, a diagnostic kit including at least one of the primers can be manufactured.

In the diagnostic kit of the present invention, the molecule capable of measuring the expression level of the gene encoding the at least one of the proteins as set forth in SEQ ID NOS: 1-5 may be labeled with a detectable marker (e.g., a chromophore). The

diagnostic kit of the present invention may be a microarray chip (e.g., DNA chip) in which the above-described primer(s) is immobilized on a substrate.

A detailed mechanism about relationships between reduced expression levels of the proteins as set forth in SEQ ID NOS: 1-5 and recurrent spontaneous abortion, which have been found by the present invention, has not yet be elucidated, but can be considered as follows.

Fibrinogen, which is a main blood glycoprotein, consists of two subunits that contain three polypeptide chains: α, β, and γ_ Fibrinogen is synthesized in hepatic parenchymal cells, and has a half-life of 3 to 4.5 days. Hypofibrinogemic or afibrinogemic mice exhibit similar symptoms to bleeding, abortion, or abnormal blood clot formation. In experiments using mouse models, when maternal fibrinogen is absent or markedly reduced, vascularization is inhibited, thereby affecting trophoblast invasion or causing hemorrhagic abortion. Thus, if fibrinogen y is reduced during pregnancy, it is considered that embryos or fetuses abnormally grow or pregnancy is aborted.

Antithrombin, which is a plasmic serine protease inhibitor, permanently inactivates thrombin by binding commonly to proteolytic fragments of thrombin. In this manner, antithrombin participates in maintaining blood dissolution. Antithrombin deficiency, which was first known in 1965, is an autosomal genetic disorder characterized by either an absence or a dysfunctional form of antithrombin. Combination of antithrombin deficiency with other thrombophilic mutation leads to a further increase in diseases associated with loss of antithrombin. It has been reported that pregnant women with antithrombin deficiency have an increased risk of thrombosis during pregnancy. Thus, it is considered that reduced expression levels of antithrombin and fibrinogen y stimulate the appearance of diseases associated with pregnancy.

Angiotensinogen is known to be associated with a rennin-angiotensin system (RAS) that regulates a vasopressor, an electrolyte, and fluid hemostasis, and to play a critical role in embryo development. Angiotensinogen, angiotensin (AT) II, and AT Il receptors (ATi and AT ₂ ) are known to be expressed in embryos during pregnancy and to be associated with organ development. In angiotensinogen gene-deficient mouse

models, it is known that there is a higher likelihood of failure of initial embryo development. With respect to complement components, early (3 months) pregnancy failure (about 20% incidence) can be characterized by onset of hypocomplementemia, and in some cases, is associated with complement deposition in the placenta. Complement activation is required in mouse models with recurrent fetal loss characterized by increased or recurrent abortion associated with antiphospholipid antibodies. Thus, it is considered that recurrent abortion is associated with the reduced levels of the above-described blood coagulation factors, angiotensinogen, and complement components. Hereinafter, the present invention will be described more specifically with reference to the following examples. However, the following examples are provided only for illustrative purposes and are not intended to limit the scope of the present invention.

Examples

1. Test methods

Separation of follicular fluids and chorions from recurrent spontaneous abortion (hereinafter, referred to as simply "RSA") women and normal women, removal of abundant proteins through immunoaffinity column chromatography, two-dimensional electrophoresis and image analysis, protein identification, Western blot analysis, reverse-transcription PCR and real-time PCR, and data processing and statistical analysis were performed as follows.

(1) Separation of follicular fluids and chorions

(1-1) Separation of follicular fluids

Three women (average age: 34) who had experienced three or more consecutive pregnancy losses within initial three months of gestation due to indefinite causes of the pregnancy losses were chosen as a RSA group. These women had no history of abortion and thromboembolism between four and six months of gestation. In order to exclude known etiologies of recurrent spontaneous abortion, diagnostic procedures

were performed prior to tests. The diagnostic procedures included hysteroscopy, parental and maternal karyotype, cervical cultures for Clamydia, urea plasma and mycoplasma, comprehensive hormonal status, and evaluation of antiphospholipid syndrome with IgM and IgG anticardiolipin antibody assessment. Normal women (average age: 33) who had experienced at least two births and no abortion were chosen as a control group. Written consents were obtained from all of the women who had participated in the tests.

Follicular fluids were harvested according to a known method (Chi, H. J., Kim, D. H., Koo, J. J., Chang, S. S., Fertil. Steril. 1998, 70, 871-877). Human follicular fluids were obtained from three normal women and three RSA women based on a human IVF program at the CHA fertility center (Korea). Follicular fluids derived from follicles with different sizes were collected in sterilized tubes containing K-EDTA or sodium heparin. The collected follicular fluids were centrifuged at 3,000 rpm for 30 minutes. Aliquots of supernatants were heat-inactivated at 59 ⁰ C for 35 minutes, cooled on ice, and sterilized with a sterilization filter (pore size: 0.22 μm). All the samples were stored at -80 ⁰ C until used.

(1-2) Separation of chorions

Chorions were harvested from RSA women and normal women according to a known method (Choi, H. K., Choi, B. C ₁ Lee, S. H., Kim, J. W. et al., MoI. Reprod. Dev.

2003, 66, 24-31). Six fetal chorion samples were obtained from six pregnant women between six and eight weeks of gestation (i.e., three RSA women and three normal women). Chorions for a RSA group were obtained from women who had experienced three or more consecutive pregnancy losses due to indefinite causes of the pregnancy losses, and chorions for a control group were obtained from women who had not experienced abortion, abnormal pregnancy, preterm delivery, and stillbirth. Average ages of the RSA women and the normal women were 34 and 32, respectively.

Chorions were detected by immunocytochemical assay using proliferating cell nuclear antigen (PCNA) and by histological assay with haematoxylin and eosin (H/E) staining and then subjected to reverse-transcription PCR. Karyotyping of all the samples was performed using a G-banding technique, which revealed genotypes of 46,

XX chromosomes. Formal written consents were obtained from all of the women who had participated in the tests. All the samples were collected and frozen within 30 minutes and stored at -80 ⁰ C until used.

(2) Removal of abundant proteins through immunoaffinity column chromatography

Six abundant proteins present in the follicular fluids obtained in Section (1-1), i.e., albumin, transferrin, IgG, IgA, haptoglobin, and antitrypsin were removed using multiple affinity removal column (MARC). A 4.6x50 mm MARC capable of binding with 20 μl of human follicular fluid was used. Chromatographic separation of the abundant proteins using MARC was performed using a mobile phase reagent kit according to a standard LC protocol provided by the manufacturer. That is, the human follicular fluid samples were five times diluted with a buffer A containing a protease inhibitor, centrifuged at room temperature (RT) for 1-2 minutes at 16,00Og, and filtered with a 0.22-μm spin filter. The samples were loaded on multiple affinity removal column (MARC), and the flow-through fractions were collected and stored at -20 "C until used. In order to analyze the quantitatively reduced follicular fluid proteins on 2-D gels, the flow-through fractions obtained from the MARC were combined, precipitated using a 10% trichloroacetic acid solution which was previously cooled at -20 ⁰ C for one hour, and washed with a cold acetone solution. The resultant pellets were re-solubilized in a 2-DE sample buffer.

(3) Two-dimensional electrophoresis and image analysis

First, for one-dimensional analysis, 1 mg of follicular fluid proteins were loaded on an immobilized pH 3-10 nonlinear gradient strip according to a known method (Cho,

Y. M., Bae, S. H., Choi, B. K., Cho, S. Y. et al., Proteomics 2003, 3, 1883-1894).

Two-dimensional separation was performed on 9-16% linear gradient polyacrylamide gels. The proteins were immobilized with a solution containing 40% methanol and 5% phosphoric acid for one hour, and the gels were stained with CBB G-250 for 12 hours. The gels were scanned using a GS-710 imaging densitometer and the images were

analyzed by lmge Master™ 2D Platinum computer software program. A relative protein volume was determined by measuring the intensity of each spot on every gel.

(4) Protein identification

(4-1) In-gel digestion and mass spectrometry (MS) analysis The gel spots were sliced into smaller cubes, and the cubes were digested with trypsin according to a known method (Cho, S. Y., Lee, E. Y., Lee, J. S., Kim, H. Y. et al., Proteomics 2005, 5, 3386-3396). For MALDI-TOF MS analysis, trypsin peptides were concentrated with Poros R2 and oligo R3 column and eluted with α-cyano-4-hydroxycinnamic acid. Spectra were obtained using a 4700 TOF/TOF spectrophotometer. Peptide mass maps of proteins were obtained by applying monoisotopic peak intensities to MASCOT and MS-Fit.

(4-2) LC-MS/MS

For Nano-LC-ESI-MS/MS analysis, the trypsin-digested peptides were concentrated with Poros R2 and oligo R3 column. All LC-MS/MS experiments were performed using an Agilent Nanoflow Proteomics Solution featuring an Agilent 1100 Series nano-LC for MS/MS coupled through an orthogonal nanospray ion source to an Agilent 1100 Series LC/MSD Trap XCT ion mass spectrometer. The Nano-LC system was operated in a sample enrichment/desalting mode using ZORBAX 300SB-C18 enrichment column (0.3 x 50 mm, 5 μm). Chromatography was performed using ZORBAX 300 SB-C18 (75 μm x 150 mm) nanocolumn. A solvent gradient initiated at 3% solvent B (formic acid in 0.1 % acetonitrile) and 97% solvent A (0.1% formic acid solution). The solvent gradient was applied as follows: from 0 to 5 minutes to 3% solvent B isocratically, from 5 to 10 minutes to 3 to 10% solvent B, from 10 to 50 minutes to 10 to 45% solvent B, from 50 to 55 minutes to 45 to 90% solvent B, from 55 to 61 minutes to 90% solvent B, and 10 minutes to 3% solvent B.

The LC/MSD Trap XCT was operated in a unique peptide scan auto-MS/MS mode. The ionization mode was positive nanoelectrospray with an Agilent orthogonal source. A drying gas flow was 5 L/min and a drying gas temperature was 300 ⁰ C .

Vcap was typically 1800-1900V (1 at 30 V, and capillary exit offset at 75V). The trap drive was set at 85 V with averages of one or two. ICC was on; maximum accumulation time of 150 ms, smart target of 125,000, and MS scan range of 300 to 2200. Automatic MS/MS was in ultra scan mode, with the number of parents of 2, averages of 2, fragmentation amplitude of 1.15 V, SmartFrag on (30-200%), active exclusion on (after 2 spectra for 1 min), prefer +2 on, MS/MS scan range of 100-1800, and ultra scan on.

(4-3) Bioinformatics Each MS/MS spectrum was searched against the nonredundant protein sequence database using Spectrum Mill software to identify the proteins. Sequences of uninterpreted CID spectra were identified by correlation with the peptide sequences present in the protein sequence database (NCBInr 2005,10) using the Spectrum Mill MS Proteomics Workbench. Results for the Spectrum Mill search were initially assessed by "score" and "scored peak intensity (SPI)". The software creates theoretical peptides for all or a limited group of database proteins, calculates corresponding MS/MS spectra, and compares them to experimental spectra to find the match. Score means points to the matched (Bonus) or unmatched (Penalty) peaks. Bonus points are awarded for each matched peak, at one point per peak regardless of peak height. Penalty points for unmatched peaks are based on peak height/height of the tallest peak. Scored peak intensities are calculated as follows: from peaks remaining after peak detection, this is the percentage of total intensity in the query set spectrum, which is matched to peaks in the library spectrum. Scored peak intensities lower than 50% suggest a poor match, or absence of corresponding fragment isotypes in the query set spectrum. Adjusting the value of minimum matched peak intensity to less than 50% (default value) will enable reporting of poorer quality matches. As a general rule, for declaring a protein hit, protein score >13, peptide score > 10, and SPI (%) > 70 were applied throughout the data analysis procedures as suggested by the manufacturer. All the proteins identified in the present invention are based on assignment of at least two peptides.

(5) Western blot analysis

40 μg of human follicular fluid proteins were diluted to 1/10 with a PBS buffer and subjected to SDS-PAGE. In order to identify three fibrinogen chains (α, β, and Y) and antithrombin, albumin was removed from the follicular fluids using an Aurum serum protein mini kit. The follicular fluid proteins were blotted on nitrocellulose membranes. The membranes were blocked in a blocking solution containing anti-fibrinogen antibodies (for each of α, β, and Y) (1 :500), and then incubated in a blocking solution containing horseradish peroxidase-conjugated anti-goat immunogloblin G (IgG) secondary polyclonal antibodies (1 :5,000). In addition, a blocking solution containing an antithrombin antibody (1 :1 ,000) and an anti-mouse secondary monoclonal antibody were used. Signals were detected by an ECL system. Bands on the Western blot were scanned using Fluor-S™ M ulti Imager, and band densities were measured. The band densities were expressed as mean ± SEM.

(6) Reverse-transcription PCR and real-time PCR

(6-1) Total RNA preparation and cDNA synthesis

Total RNA was isolated from human chorions stored at -80 ⁰ C using a TRIzol reagent. Synthesis of cDNAs was performed using a Superscript™ Frist-Strand Synthesis System. 3 μg of total RNA and 0.5 μg of oligo (dT)i2-is primers were incubated at 70 ^° C for 10 minutes, and 20 μl of a mixture containing a 5 x first strand buffer (250 mM Tris-HCI, 375 mM KCI, and 15 mM MgCI ₂ ), 10 mM DTT, and 0.5 mM dNTP was added thereto. A tube containing the mixture was incubated at 42 "C for two minutes, and Superscript™ Il reverse transcriptase (50 units/μl) was added thereto. The resultant mixture was incubated at 42 ⁰ C for 50 minutes, and the enzymes were denatured at 70 ^° C for 15 minutes.

(6-2) Semi-quantitative reverse-transcription PCR analysis

The expression levels of genes encoding complement component C3c chain E, fibrinogen y, antithrombin, angiotensinogen, and hemopexin precursor in the chorions obtained in Section (1-2) were determined based on the expression level of the GAPDH gene used as control. Primers of these genes were designed based on the gene

sequences deposited in National Center of Biotechnology Information (NCBI) GenBank. The primer sequences and the estimate sizes of PCR products are listed in Table 2 below.

Table 2

GenBank Annealing RT-PCR

Genes accession SEQ ID Primer sequences temperature Base pairs number CC) cycle number

Complement

6 5'-attgcagaagagaacatcgt-3' component C3c, AH513239 54 35 259 chain E 7 5'-ttcgaacaac agagtagggt- 3 '

8 5'-caagacattgcc aataaggg- 3 '

Fibrinogen y BT007081 56 30 281

9 5 ' - cttaatgc at atgggatggc- 3 '

10 5'-cctgtaatgacaccctccag-3'

Antithrombm X68793 57 35 264 11 5'-attgctctgcattttccttg-3'

12 5' -cctctc tctatctgggagcc-3'

Angiotensinogen M69110 58 30 297 13 5'-ctgtgaagtccagagagcgt-3'

Hemapexw 14 5' -taaaggggagtttgtgtgga-3'

J03048 55 35 253 precursor 15 5'-acattctccacggtgacatt-3'

PCR conditions were as follows: initial denaturation at 94 ⁰ C for 3 minutes, 30-35 cycles of denaturation at 94 ⁰ C for 1 minute, annealing at 54-58 ⁰ C for 1 minute, and polymerization for 1 minute and 30 seconds, with incubation at 72 ⁰ C for 10 minutes for a final step. The PCR products were stored at 4°C . The sequences of the PCR products were determined by an ABI prism automated DNA sequencer. In order to determine the sizes of the PCR products, the PCR products were subjected to electrophoresis on 1.5% agarose gel, the gels were stained with ethidium bromide, and DNA bands were observed under UV. The densities of the DNA bands were measured by Fluor-S™ Multilmage and compared with the density of GAPDH used as control.

(6-3) Real-time PCR analysis

Real-time PCR analysis was performed using DNA engine Opticon 2 fluorescence detection system according to the protocol of DyNAmo SYBR green qPCR kit containing modified Taq DNA polymerase, SYBR green, optimum PCR buffer, 5 mM MgCl2, and dNTP mix containing dUTP. PCR was performed as follows: denaturation at 95 "C for 10 minutes and 30-35 cycles of 95 ⁰ C for 30 seconds, 55 °C /59 ⁰ C for 30 seconds, and 72 °C for 30 seconds. At this time, a melting curve program (65°C~95°C with a heating rate of 0.2 ^° C/sec) was applied with continuous fluorescence measurement. The PCR products were cooled to 4°C . Fluorescence data were acquired after extension during PCR using SYBR Green 1. The PCR products were analyzed by the melting curve. The melting curve analysis of PCR products can be used in discriminating nonspecific or specific PCR products because of the sequence specificity of the melting curve. Relative gene expression levels were determined using a 2ddCt method. In order to determine the sizes of the PCR products, the PCR products were subjected to electrophoresis on 1.5% agarose gel, the gels were stained with ethidium bromide, and DNA bands were observed under UV.

(7) Data processing and statistical analysis

For statistical analysis for the test results, the Student's t-test was used to determine whether significant differences were present in two test groups. The results were expressed as mean ± standard deviation (SD). P < 0.005 indicates that there is a statistically significant difference between the two groups.

2. Test results

The analysis results obtained according to the above-described tests are as follows.

(1) Two-dimensional analysis for proteins present in follicular fluids of RSA women

FIG. 1 shows two-dimensional electrophoretic analysis (pH 3-10) results for proteins extracted from three RSA women (as a RSA group) and three normal women

(as a control group). As shown in FIG. 1 , five proteins were abnormally expressed in the RSA group.

The five proteins were identified by MALDI-TOF-MS and Nano LC MS/MS. As a result, the proteins were identified as complement component C3c chain E (SEQ ID NO: 1), fibrinogen Y (SEQ ID NO: 2), antithrombin (SEQ ID NO: 3), angiotensinogen (SEQ ID NO: 4), and hemopexin precursor (SEQ ID NO: 5).

Table 3

Matched Sequence

Spot Accession Theoretical

Protein Name Score ¹ ' Peptides Coverage Matching Sequence ID Number MW/ pi

Number (%)

SNLDEDIIAEENIVSRS EDLKEPPK, Complement component C3c, . „ I ₄ M ₀ Z ₃ W N ₆ \ LMNlFLK, LPYSVVRNEQVEIRAVLYNYRQNQELK, 21482/584 P ^l x c*h-ai™n " E- *' RHQαrVTIPPKSSLSVPYVIPLKTOLQEVVKAAVYHHFISDOVRKSL

KVVPEGIR

AIQLTYNPDESSKPNMIDAATLKSRKMLEEIMKYEASILTHDSSIRY

6797 gi|30583001 Fibrinogen y 97 1105 (31%) 32 LQEIY NSNNQK, 49465/5 7

' '"^ W ^V ^ EGFGHLSPTGTTEFWLGNEKIHUSTQSAIPYALRVELEDWNORTST ADYAMFK, ASTPNGYDNGIIWATWK, LTIGEGQQHHLGGAK

PEATNRRVWELSK, TSDQIHFFFAK, LVSANRLFGDKSLTFNETYQDISELVYGAKLQPLDFKENAEQSR,

6567 gi|999514 Antithrombm 136 17/51 (33'/ ) 44 UO « RIEDGFSLKEQLQDMGLVDLFSPEK, 48916/595

LPGIVAEGRDDLYVSDAFHKAFLEVNEEGSEAAASTAVVIAGRSLN

PNRVTFKANRPFLVFIREVPLNTI IFMGR

ANAGKPKDPTFIPAPIQAKTSPVDEKALQDQLVLV AAK.VQGLALY

6518 gi|553181 68 11/61 (18%) 23 36550/655 TPVVLPRSLDFTELDVAAEK IDRFMQAVTGWK

6406 S053181 Angiotensinogen 103 11/37 (29%) 27 ANFLGFR, 36550/655

FVQGLALYTPVVLPRSLDFTELDVAAEKIDRFMQAVTGWK WKNFPSPVDAAFR,

6375 gi|386789 Hemopejαn precursor 64 8/33 (24%) 15 51512/643 ATWTELPWPHEK

6370 3053181 Angiotensmogen 11/48 (22%) 23 36550/6 55 YTPVVLPRSLDFTELDVAAEKIDRFMQAVTGWK

1) Score is -10xLog(P), where P is the probability that the observed match is a random event.

(2) Western blot analysis of blood coagulation factors, fibrinogen and antithrombin

In order to identify the detected spots, the expression levels of fibrinogen and antithrombin in the follicular fluids were analyzed by Western blotting using respective antibodies. As shown in FIG. 2, fibrinogen α- , β-, and γ-chains were detected in the follicular fluids of the RSA group and the control group. The expression levels of fibrinogen α (95 kDa) and β (56 kDa) were not different between the RSA group and the control group (107% to 100% ±19% and 98% to 100%±17%, see FIG. 3). However,

the expression level of fibrinogen Y in the RSA group was significantly lower than that in the control group (37% to 100%±11 %, *p < 0.005, see FIG. 5). FIG. 4 shows the Western blot results of the expression of antithrombin (48 kDa) detected in the follicular fluids of the RSA group and the control group. The expression level of antithrombin in the RSA group was about three times lower than that of the control group (33% to 100%±1%, ^* p < 0.005, see FIG. 5).

(3) Expression of genes specific for chorions derived from RSA women

In order to investigate whether genes encoding the above-identified five proteins, i.e., complement component C3c chain E (SEQ ID NO: 1), fibrinogen Y (SEQ ID NO: 2), antithrombin (SEQ ID N: 3), angiotensinogen (SEQ ID NO: 4), and hemopexin precursor (SEQ ID NO: 5) are abnormally expressed in the chorions of RSA women, semi-quantitative reverse-transcription PCR was performed and the results are shown in FIG. 7. As shown in FIG. 7, the expression levels of complement component C3c chain

E, fibrinogen Y, antithrombin, and angiotensinogen in the RSA group were lower than those of the normal group (3% to 100%±1.6%, 26% to 100%±6.6%, 1% to 100%±0.4%, and 4% to 100%±2.0%, respectively, *p < 0.005). On the other hand, the mRNA expression level of hemopexin precursor in the RSA group was not significantly different from that of the normal control. This result suggests that mRNA expression level and protein expression level of the hemopexin precursor are different unlike the other proteins.

In order to quantitatively analyze the expression levels of fibrinogen y and antithrombin, real-time PCT was performed. In order to normalize the efficiency of PCR, GAPDH was used as control. Based on the normalized GAPDH mRNA levels, the mRNA expression levels of fibrinogen Y and antithrombin in the follicular fluids of the RSA group were lower than those in the follicular fluids of the normal control (*p < 0.005, see FIG. 8).

Industrial Applicability

A diagnostic method and kit according to the present invention can be effectively used in early diagnosis of recurrent spontaneous abortion.