KR102113061B1 - Association of miR-605 A>G, miR-608 G>C, miR-631 I>D, miR-938 C>T and miR-1302-3 C>T polymorphism with the risk of recurrent implantation failure in a Korean women - Google Patents

Abstract

The present invention relates to a correlation between miR-605 A > G (rs2043556), miR-608 G > C (rs4919510), miR-631 I > D (rs5745925), miR-938 C > T (rs12416605) and miR-1302-3 C > T (rs7589328) polymorphisms and a risk of recurrent implantation failures among Korean women and, more particularly, to a method and a kit for distinguishing the polymorphism from a subject′s DNA sample to provide information required for predicting a risk of developing recurrent implantation failures among Korean women. According to the present invention, it is possible to provide information very useful in predicting a risk of developing recurrent implantation failures among Korean women by distinguishing a polymorphism of the subject′s microRNA-related gene. In particular, in case of using the kit of the present invention using the polymorphism, it is possible to more easily provide the above information.

Description

miR-605 A ＞ G, miR-608 G ＞ C, miR-631 I ＞ D, miR-938 C ＞ T and miR-1302-3 C ＞ T polymorphisms and the risk of repeated implantation failure in Korean women {Association of miR-605 A ＞ G, miR-608 G ＞ C, miR-631 I ＞ D, miR-938 C ＞ T and miR-1302-3 C ＞ T polymorphism with the risk of recurrent implantation failure in a Korean women}

The present invention miR-605 A> G (rs2043556), miR-608 G> C (rs4919510), miR-631 I> D (rs5745925), miR-938 C> T (rs12416605) and miR-1302-3 C> T (rs7589328) relates to the relationship between polymorphism and the risk of repeated implantation failure in Korean women. Specifically, a method for determining the polymorphism from a DNA sample of a subject in order to provide information necessary to predict the risk of repeated implantation failure in Korean women, And kits therefor.

Repeated Implantation Failure (RIF) means that the embryo fails to implant in the womb even after an in vitro fertilization procedure. The causes of repeated implantation failure can be divided into maternal factors (anatomical, endometrial, thrombotic) and embryonic factors (genetic factors, developmental disorders in the uterus), etc., and the success rate of delivery according to in vitro fertilization is 30 ~ It is less than 60%. The number of medical treatments for infertility diseases in Korea increased from 148,000 in 2006 to 184,000 in 2010, an increase of 36,000 over the past five years (an average annual increase of 5.8%), and the total medical expenses from 13.4 billion in 2006 to 23.2 billion in 2010. About 5 billion won increased over the past 5 years (an annual increase of 9.4%). In addition, for the socio-economic burden of infertility, it is estimated that the majority of infertile couples currently take 4 to 8 years to get pregnant and give birth (2 to 4 years before receiving infertility diagnosis and 2 to 4 years after infertility diagnosis) ), In spite of the high burden of treatment costs (average of 4.45 million won per session), one out of three women received more than four treatments in excess of three times, which is the number of medical expenses supported by the government's in vitro fertilization procedure ) 86.4% of infertile couples indicated their intention to continue to receive infertility treatment until childbirth in the future, and the cost burden of infertility treatment, including assisted reproductive procedures, has a significant impact on the home economy (infertility women who responded to the survey) 97.8% of the respondents said that the cost is high).

On the other hand, chromosomal abnormalities of parents, thrombosis of mothers, hormonal abnormalities, infectious diseases, autoimmune diseases, uterine and fallopian tube malformations, chromosomal abnormalities, polycystic ovary syndrome, and early ovarian dysfunction have been suggested as risk factors for repeated implantation failure. Although it is a risk factor, it has not been suggested as an etiology of repeated repetitive failure, and in most cases, screening information, such as the risk and incidence of repeated failure, is unknown. For this reason, accurate risk presentation and screening tests have not yet existed.

Reprod Biomed Online. 2014 Jan; 28 (1): 14-38.

Therefore, the main object of the present invention is to provide a new marker useful for predicting the risk of developing repeated failure of Korean women and a method of using the same.

Another object of the present invention is to provide a kit useful for predicting the risk of recurrence of repetitive implantation failure in Korean women using the marker.

According to one aspect of the present invention, the present invention is a method for determining miR-1302-3 C> T (rs7589328) single nucleotide polymorphism from a DNA sample of a subject in order to provide information necessary for predicting the risk of recurrence of repeated implantation failure in Korean women. Gives

In the method of the present invention, in addition to the miR-1302-3 C> T (rs7589328) single base polymorphism, miR-605 A> G (rs2043556), miR-631 I> D (rs5745925) and miR-938 C> It is preferable to further discriminate one or more polymorphisms selected from T (rs12416605), and it is more preferable to further discriminate miR-608 G> C (rs4919510) monobasic polymorphism.

In the method of the present invention, in addition to the miR-1302-3 C> T (rs7589328) single base polymorphism, it is preferable to further determine aPTT (activated partial thromboplastin time) from a blood sample of a subject.

According to another aspect of the present invention, the present invention provides a kit for predicting the risk of repeated implantation failure in Korean women, including means for detecting miR-1302-3 C> T (rs7589328) monobasic polymorphism.

In the kit of the present invention, in addition to the means for detecting the miR-1302-3 C> T (rs7589328) single base polymorphism, the means for detecting the miR-605 A> G (rs2043556) single base polymorphism, miR-631 I> D (rs5745925) It is preferable to further include at least one polymorphism detection means selected from means for detecting polymorphism and means for detecting miR-938 C> T (rs12416605) single base polymorphism, in addition to miR-608 G> C (rs4919510) It is more preferable to further include means for detecting monobasic polymorphism.

In the kit of the present invention, it is preferable that the miR-1302-3 C> T (rs7589328) monobasic polymorphism is further included in addition to the means for detecting activated partial thromboplastin time (aPTT).

According to another aspect of the present invention, the present invention discriminates miR-608 G> C (rs4919510) single nucleotide polymorphism from a subject's DNA sample in order to provide information necessary for predicting the risk of repeated implantation failure in Korean women, and the subject It provides a method for determining the activated partial thromboplastin time (aPTT) from a blood sample.

According to another aspect of the present invention, the present invention predicts the risk of repeated implantation failure in Korean women comprising miR-608 G> C (rs4919510) means for detecting monobasic polymorphisms and reagents for measuring activated partial thromboplastin time (apt). Kit.

In the kit of the present invention, the means for detecting the miR-1302-3 C> T (rs7589328) single base polymorphism is an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 14, an oligo represented by the nucleotide sequence of SEQ ID NO: 15 It is preferred to be a primer-restriction enzyme set comprising a nucleotide primer and an Nla III restriction enzyme.

In the kit of the present invention, the means for detecting the miR-605 A> G (rs2043556) single base polymorphism is an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 6, an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 7 And a Hin -I restriction enzyme.

In the kit of the present invention, the means for detecting the miR-631 I> D (rs5745925) polymorphism is an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 10, an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 11 and Nla It is preferably a primer-restriction enzyme set comprising IV restriction enzyme.

In the kit of the present invention, the means for detecting the miR-938 C> T (rs12416605) single base polymorphism is an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 12, an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 13 And a primer-restriction enzyme set comprising Hha I restriction enzyme.

In the kit of the present invention, the means for detecting the miR-608 G> C (rs4919510) single base polymorphism is an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 8, an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 9 And a Pvu II restriction enzyme.

According to the present invention, it is possible to provide very useful information for predicting the risk of repeated implantation failure in Korean women by determining the polymorphism of a micro RNA-related gene in a subject. In particular, when using the kit of the present invention using the gene polymorphism, it is possible to provide the above information more easily.

1 is a graph showing the synergistic effect of miR-608GC + CC and miR-1302-3CC and aPTT at the risk of repeated implantation failure.

The miR-605 A> G, miR-608 G> C, miR-631 I> D, miR-938 C> T and miR-1302-3 C> T polymorphisms used in the present invention may appear in human microRNA genes. The polymorphisms are registered in the United States National Center for Bioinformatics' single base polymorphism database (NCBI dbSNP, http: // http: //www.ncbi.nlm.nih.gov/snp/) as rs2043556, rs4919510, rs5745925, rs12416605, and rs7589328, respectively. It is.

miR-605 A> G, miR-608 G> C, miR-938 C> T and miR-1302-3 C> T polymorphism is single nucleotide polymorphism (SNP) (hereinafter abbreviated as 'SNP') , And miR-631 I> D polymorphism is insertion deletion polymorphism (hereinafter abbreviated as 'InDel').

miR-605 A> G SNP can be identified as the 101st base in the human DNA region represented by SEQ ID NO: 1, which may be adenine or guanine.

miR-608 G> C SNP can be identified as the 110th base in the human DNA region represented by SEQ ID NO: 2, which may be guanine or cytosine.

miR-631 I> D InDel can be distinguished from the 85th to 86th base (CT) in the human DNA region represented by SEQ ID NO: 3, but the sequence is present (insertion, insertion) or deletion (deletion). Can be.

miR-938 C> T SNP can be identified as the 114th base in the human DNA region represented by SEQ ID NO: 4, which can be cytosine or thymine.

miR-1302-3 C> T SNP can be identified as the 139th base in the human DNA region represented by SEQ ID NO: 5, which may be cytosine or thymine.

The determination of SNP and InDel as described above may be performed by performing a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method or a sequencing method.

The polymerase chain reaction-restriction enzyme fragment length formation method performs a polymerase chain reaction to amplify a target gene or DNA region, and when treated with a specific restriction enzyme, the restriction enzyme recognizes whether digestion is achieved or the restriction enzyme does not recognize It is a method of distinguishing polymorphism, deletion, addition or substitution of bases by distinguishing whether or not digestion is not possible. At this time, whether or not DNA is amplified and digested by restriction enzymes can be confirmed through electrophoresis using agarose gel.

As the sequencing method, a known Maxam-Gilbert or Sanger method can be used.

When polymorphism is determined by the PCR-RFLP method, primers for amplifying consecutive 50 to 10,000 bp sites including each SNP or InDel site, and each SNP site is a specific base or InDel site is inserted or deleted Restriction enzymes capable of cleaving DNA can be used.

More preferably, the following primers for amplification and restriction enzymes can be used.

miR-605 A> G SNP discrimination: forward primer of SEQ ID NO: 6 and reverse primer of SEQ ID NO: 7, Hin fI restriction enzyme

miR-608 G> C SNP discrimination: forward primer of SEQ ID NO: 8 and reverse primer of SEQ ID NO: 9, Pvu II restriction enzyme

miR-631 I> D InDel discrimination: forward primer of SEQ ID NO: 10 and reverse primer of SEQ ID NO: 11, Nla IV restriction enzyme

miR-938 C> T SNP discrimination: forward primer of SEQ ID NO: 12 and reverse primer of SEQ ID NO: 13, Hha I restriction enzyme

miR-1302-3 C> T SNP discrimination: forward primer of SEQ ID NO: 14 and reverse primer of SEQ ID NO: 15, Nla III restriction enzyme

When the miR-605 A> G SNP discriminant primer and restriction enzyme are used, a DNA fragment of 185bp is amplified through a polymerase chain reaction, and when the restriction enzyme is processed, the DNA fragment of 140bp and 45bp for the AA genotype and the AG genotype In the case of 185bp, 140bp and 45bp DNA fragments, in the case of GG genotype DNA fragments of 185bp are generated.

When the miR-608 G> C SNP discriminant primer and restriction enzyme are used, a DNA fragment of 348 bp is amplified through a polymerase chain reaction, and when the restriction enzyme is processed, DNA fragments of 178 bp, 121 bp, and 49 bp for the GG genotype, GC DNA fragments of 299bp, 178bp, 121bp, and 49bp for genotype, and DNA fragments of 299bp and 49bp for CC genotype are generated.

When the miR-631 I> D InDel discriminant primer and restriction enzyme are used, a DNA fragment of 194 bp or 196 bp is amplified through a polymerase chain reaction, and when treated with a restriction enzyme, DNA fragments of 55 bp, 56 bp, and 85 bp for the II genotype , DNA fragments of 55 bp, 56 bp, 85 bp and 139 bp for ID genotype, and 55 bp and 139 bp DNA fragments for DD genotype are generated.

When the miR-938 C> T SNP discriminant primer and restriction enzyme are used, a DNA fragment of 137 bp is amplified through a polymerase chain reaction, and when processing the restriction enzyme, a DNA fragment of 112 bp and 25 bp in the CC genotype and CT genotype In the case of 137bp, 112bp and 25bp DNA fragments, in the case of the TT genotype DNA fragments of 137bp are generated.

When the miR-1302-3 C> T SNP discriminant primer and restriction enzyme are used, a DNA fragment of 177 bp is amplified through a polymerase chain reaction, and when the restriction enzyme is processed, the DNA fragment of 177 bp in the CC genotype or CT genotype DNA fragments of 177 bp, 148 bp and 29 bp in case, and DNA fragments of 148 bp and 29 bp in case of TT genotype are generated.

According to the present invention, it is possible to provide information for discriminating each SNP or InDel in the same manner as described above and predicting the risk of repeated implantation failure of Korean women based on the same. This is attributable to a significant difference ( FDR-p <0.05) identified through comparative analysis of repeat failure patients and control groups.

Existing academia defines recurring implantation failure (hereinafter abbreviated as 'RIF') when embryos fail to implant in the womb even after in vitro fertilization, but these implantation failures have occurred twice recently. There is an opinion that even if it occurs continuously, it should also be viewed as RIF. Accordingly, in the present invention, a case in which the occurrence of an implantation failure occurs twice consecutively or the total number of implantation failures is 3 or more is defined as RIF.

According to the present invention, it is possible to predict the risk of developing RIF in Korean women as follows based on the result of each SNP or InDel discrimination.

1) When the miR-1302-3 C> T genotype of the subject is a CT genotype, RIF is less likely to occur compared to a CC genotype (dominant genotype).

2) When the subject's miR-605 A> G / miR-938 C> T / miR-1302-3 C> T haplotype is A-T-C haplotype, it is more likely to develop RIF than the A-C-C haplotype.

3) When the miR-938 C> T / miR-1302-3 C> T haplotype of the subject is C-T haplotype, it is less likely to develop RIF than the C-C haplotype.

4) If the subject's miR-608 G> C genotype is GC or CC genotype and the activated partial thromboplastin time (apTTT) is 22.6 seconds or less, miR-608 G> C genotype is GG genotype and aPTT exceeds 22.6 seconds. In comparison, RIF is likely to occur.

5) If the subject's miR-1302-3 C> T genotype is a CC genotype and aPTT is 22.6 seconds or less, compared to a person whose miR-1302-3 C> T genotype is CT or TT genotype and aPTT exceeds 22.6 seconds, RIF Is likely to occur.

6) Subject's miR-605 A> G / miR-608 G> C / miR-631 I> D / miR-938 C> T / miR-1302-3 C> T If the haplotype is AGITC haplotype, AGICC People with haplotypes are more likely to develop RIF.

7) Subject's miR-605 A> G / miR-608 G> C / miR-631 I> D / miR-938 C> T / miR-1302-3 C> T If the haplotype is GGICT haplotype, AGICC People with a haplotype are less likely to develop RIF.

8) Subject's miR-605 A> G / miR-631 I> D / miR-938 C> T / miR-1302-3 C> T When the haplotype is GICT haplotype, it is RIF compared to the AICC haplotype. Is unlikely to occur.

9) When the subject's miR-605 A> G / miR-608 G> C / miR-938 C> T / miR-1302-3 C> T haplotype is AGTC haplotype, it is RIF compared to the person who is AGCC haplotype. Is likely to occur.

10) When the subject's miR-605 A> G / miR-631 I> D / miR-1302-3 C> T haplotype is G-I-T haplotype, it is less likely to develop RIF than the A-I-C haplotype.

11) When the subject's miR-631 I> D / miR-938 C> T / miR-1302-3 C> T haplotype is I-C-T haplotype, it is less likely to develop RIF than the I-C-C haplotype.

12) When the subject's miR-605 A> G / miR-1302-3 C> T haplotype is G-T haplotype, it is less likely to develop RIF than the A-C haplotype.

13) When the subject's miR-631 I> D / miR-1302-3 C> T haplotype is an I-T haplotype, it is less likely to develop RIF than a person who is an I-C haplotype.

14) When the miR-631 I> D / miR-1302-3 C> T combination type of the subject is the II / CT combination type, it is less likely to develop RIF than the person who is the II / CC combination type.

15) When the miR-938 C> T / miR-1302-3 C> T combination type of the subject is the CC / CT combination type, it is less likely to develop RIF than the CC / CC combination type person.

The kit for predicting the risk of repeated implantation failure of Korean women of the present invention is to enable easy identification of the SNP or InDel as described above, thereby to predict the risk of repeated implantation failure of Korean women, SNP Alternatively, as a means for detecting InDel, materials commonly used for the detection of SNP or InDel, for example, primers for PCR, primers and restriction enzymes for PCR-RFLP, and probes for DNA-DNA hybridization may be used. In the present invention, it is preferable to use PCR-RFLP primers and restriction enzymes as detection means, particularly, the primers and restriction enzymes for SNP or InDel discrimination mentioned above.

In addition to the detection means as described above, the kit of the present invention may further include reagents, instruments, and the like used for SNP or InDel detection.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

[Example]

1. Method

1-1. Study group

119 RIF patients diagnosed as having failed implantation after 2 complete IVF-ET procedures using 10 or more embryos (mean ± standard deviation [SD] age, 33.90 ± 5.64 years, mean body mass index [BMI], 21.63 ± 3.40 ), A blood sample was taken. Samples were collected from the Obstetrics and Gynecology Center of Infertility Treatment Center in Bundang Cha Hospital between March 2010 and December 2012. Serum human chorionic gonadotropin (HCG) concentrations were <5 mIU / mL 14 days after embryo transfer in all patients, and all embryos were examined by an embryonician prior to transplantation and considered good.

Both male and female partners of couples undergoing RIF were evaluated, and participants diagnosed with RIF due to anatomy, chromosome, hormone, infection, autoimmunity or thrombosis were excluded from the study group. Anatomical abnormalities were assessed using several imaging techniques, including ultrasonography, tubal angiography, hysteroscopy, computed tomography, and magnetic resonance imaging. Karyotype analysis was performed using standard protocols. Participants who have experienced hormonal causes of RIF, including hyperprolactinemia, luteal deficiency, and thyroid disease, have prolactin in the peripheral blood, thyroid-stimulating hormone (TSH), free T4, follicle-stimulating hormone (FSH), and luteinizing hormone LH ), And progesterone levels were measured and excluded from the study. In addition, erythematous anticoagulants and anticardiolipin antibodies were additionally measured to exclude autoimmune diseases such as lupus and antiphospholipid antibody syndrome. The thrombophilia was confirmed by protein C and protein S deficiency and the presence of anti-α2 glycoprotein antibodies. Male partners were evaluated by performing semen analysis, karyotype analysis, and hormone analysis, including estradiol (E2), testosterone, FSH, and LH measurements. Of the 167 patients selected for the study, 48 patients with intrauterine adhesions, hypothyroidism, trisomy, or chromosomal translocation (patient or spouse) or antiphospholipid antibody syndrome were excluded from the patient group.

The control group included 212 participants (age range, 27-45 years; mean ± SD age, 34.24 ± 3.35 years; BMI, normal normal karyotype (46XX), normal menstrual cycle, at least one normal pregnancy experience, and no abortion experience). 21.01 ± 2.75). This study was reviewed and approved by the Deliberation Committee of Bundang Cha Hospital on February 23, 2010 (reference no.PBC09-120). Prior consent was obtained from all participants, and data were collected equally from all groups.

1-2. Genotyping

Genomic DNA was extracted from anticoagulated blood samples of all patients and controls using G-DEX Genomic DNA Extraction Kit For Blood (iNtRON Biotechnology Inc, Seongnam, Korea). The following five miRNA monobasic polymorphisms (SNPs) were selected using the human genome SNP database (dbSNP, http://www.ncbi.nlm.nih.gov/snp): miR-605 A> G (rs2043556) , miR-608 G> C (rs4919510), miR-631 I> D (rs5745925), miR-938 C> T (rs12416605) and miR-1302-3 C> T (rs7589328). Genotyping was performed by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) analysis. Table 1 shows the PCR primers and temperatures used to detect each polymorphism.

PCR for detecting miR-605 A> G (rs2043556) and miR-608 G> C (rs4919510) polymorphisms was performed after initial degeneration at 95 ° C for 15 minutes, then denaturation at 95 ° C for 20 seconds, and 60 ° C for 40 seconds. Annealing and extension at 72 ° C for 30 seconds were performed as 40 cycles, and final extension was performed at 72 ° C for 5 minutes. Hin fI for miR-605 and Pvu II for miR-608 (New England BioLabs, Bevery, Massachusetts) were treated at 37 ° C. for 16 hours. PCR for detecting miR-631 I> D (rs5745925), miR-938 C> T (rs12416605) and miR-1302-3 C> T (rs7589328) polymorphisms was performed at 94 ° C for 5 minutes and then at 94 ° C 30-second denaturation, 30-second annealing (61 ° C for miR-631, 64 ° C for miR-938, 58 ° C for miR-1302-3) and an extension of 30 seconds at 72 ° C to 35 cycles, 5 at 72 ° C It was carried out in a manner of final extension for a minute. Nla IV for miR-631, Hha I for miR-938, Nla III for miR-1302-3 (New England BioLabs, Bevery, Massachusetts) were treated at 37 ° C. for 16 hours.

About 20% of the PCR analysis for each polymorphism was randomly selected and repeated. In order to verify PCR-RFLP analysis, DNA sequencing of approximately 20% of the samples randomized was performed using an ABI 3730XL DNA Analyzer (Applied Biosystems, Foster City, California). The quality control sample was 100% consistent.

1-3. Blood coagulation status analysis

Platelet count (PLT), white blood cell (WBC) and hemoglobin (Hgb) were measured using a Sysmex XE 2100 Automated Hematology System (Sysmex Corporation, Kobe, Japan). Prothrombin time (PT) and activated partial thromboplastin time (apTT) were measured with an ACL TOP automated photo-optical coagulometer (Mitsubishi Chemical Medience, Tokyo, Japan).

1-4. Hormone analysis

To measure FSH, LH, E2, TSH and prolactin levels, blood samples were collected by venipuncture on days 3 to 5 of the menstrual cycle. Serum was prepared and hormone levels were determined using radioimmunoassay (E2, TSH and prolactin; Beckman Coulter) or enzymatic immunoassay (FSH and LH; Siemens, Munich, Germany) according to the manufacturer's instructions.

1-5. Statistical analysis

Fisher exact test and logistic regression were used to analyze the difference in miRNA gene polymorphism frequency between control and RIF patients. Odds ratio, AORs, 95% CI were calculated, and the association between miRNA polymorphism and RIF risk was investigated. Data are expressed as mean ± standard deviation for continuous variables or as a percentage for categorical variables. Statistical analysis was performed using MedCalc, version 12.1.4 (MedCalc Software bvba, Mariakerke, Belgium) or GraphPad Prism 4.0 (GraphPad Software Inc., San Diego, CA). In addition, a correlation analysis between blood coagulation factor and miRNA polymorphism was attempted; Analysis was performed based on clinical indicators of PLT, PT and aPTT coagulation factors. In order to confirm the association between miRNA polymorphism and blood coagulation factor in a high coagulation environment, stratification analysis was performed by grouping values of related factors into lower 15% and upper 15% and miRNA polymorphism. The frequency of polymorphic haplotypes was estimated using the HAPSTAT program (v.3.0, http://www.bios.unc.edu/~lin/hapstat/) with a strong synergistic effect. P values <0.05 were considered statistically significant. FDR (false discovery rate) was also used to coordinate multiple comparisons; Associations with FDR-corrected P values <0.05 were considered statistically significant. Statistical verification ability of positive association was calculated using G * POWER 3.0 (Institut fur Psychologie, Christian-Albrechts-Universitat Kiel).

2. Results

The demographic characteristics and clinical profiles of RIF patients and controls are shown in Table 2. Table 3 shows the genotyping frequency of miRNA polymorphisms in control participants and RIF patients. All of the genes analyzed showed polymorphism frequencies similar to the Hardy-Weinberg equilibrium (HWE) estimates for each group.

Analysis showed that the miR-1302-3 polymorphism was significantly associated with reduced RIF risk (CT: AOR = 0.234; 95% CI, 0.089-0.618, P = 0.003; CT + TT: AOR = 0.227; 95 % CI, 0.086-0.598, P = 0.003). In addition, three or more times (CT: AOR = 0.261; 95% CI, 0.099-0.690; P = 0.007, CT + TT: AOR = 0.253; 95% CI, 0.096-0.668; P = 0.006) and four or more times (CT : AOR = 0.301; 95% CI, 0.103-0.882; P = 0.029, CT + TT: AOR = 0.292; 95% CI, 0.100-0.853; P = 0.024) between this allele and RIF Showed a statistically significant negative correlation (see Table 4). Therefore, miR-1302-3 polymorphism in both groups was associated with a decrease in the incidence of RIF.

To identify allele combinations associated with RIF prevalence, allele combination analysis was performed with 5, 4, 3 and 2 SNP combinations (see Tables 5-10). Of all the correlations investigated, the miR-605A / 938T / 1302-3C (OR = 31.670; 95% CI, 1.802-556.500; P = 0.0003) allele combination was associated with increased RIF incidence. Conversely, the miR-938C / 1302-3T (OR = 0.259; 95% CI, 0.100-0.674; P = 0.003) allele combination was associated with reduced RIF development.

As a result of geno-gene binding genotyping analysis of Tables 11 and 12, miR-631II / miR-1302-3CT (OR = 0.244; 95% CI, 0.092-0.650; P = 0.005) binding genotype and miR-938CC / miR-1302- The 3CT (OR = 0.270; 95% CI, 0.101-0.718; P = 0.009) binding genotype was found to be associated with reduced RIF prevalence.

In addition, as a result of performing an interaction analysis to evaluate the possible synergistic effect between the binding polymorphism and the environment, in particular, in the case of miR-608GC + CC and miR-1302-3CC, the prevalence of RIF significantly increased in relation to the blood coagulation factor level. (Table 13 and Figure 1). Table 14 shows the statistical validity of the positive genetic association measured in this study.

<Table description>

Table 2: SD, standard deviation; BMI, body mass index; aPTT, activated partial thromboplastin time; PT, prothrombin time; WBC, white blood cell; Hgb, hemoglobin; PLT, platelet count; BUN, blood urea nitrogen; NA = not applicable; RIF = recurrent implantation failure; ^a Fisher's exact test; ^b Mann-Whitney test.

Table 3: AOR adjusted to participant age; COR = crude odds ratio; AOR = adjusted odds ratio; CI = confidence interval; HWE = Hardy-Weinberg equilibrium; ^a FDR (false discovery rates)-adjusted P values.

Table 4: AOR = adjusted odds ratio; CI = confidence interval; ^a FDR-adjusted P value.

Tables 5 to 10: OR and 95% CI of each allele combination calculated using Fisher's exact test to reference the frequencies of all other alleles; P value according to Fisher's exact test; OR = odds ratio; CI = confidence interval; ^a Fisher's exact test; ^b FDR-adjusted P value.

Tables 11 and 12: AOR = adjusted odds ratio; CI = confidence interval; ^a Fisher's exact test; ^b FDR-adjusted P value.

Table 13: aPTT, activated partial thromboplastin time; PT, prothrombin time; PLT, platelet count; NA = not applicable; RIF = recurrent implantation failure; ^* aPTT 22.6 sec and PLT 201 10 ³ / μl are the lower 25% cut off level in patients and controls and PT 11.6 sec are the upper 25% cut off level in patients and controls.

Table 14: AOR = adjusted odds ratio; CI = confidence interval.

<110> CHA University Industry-Academic Cooperation Foundation <120> Association of miR-605 A> G, miR-608 G> C, miR-631 I> D, miR-938 C> T          and miR-1302-3 C> T polymorphism with the risk of recurrent          implantation failure in a Korean women <130> PA-D18218 <160> 15 <170> KoPatentIn 3.0 <210> 1 <211> 201 <212> DNA <213> Homo sapiens <400> 1 atgtcattga gaggccagga aaaaacacat gtgcagagag caatatacct gtggctgtca 60 gcctgtaaca taggtaacct gtatctgcag tcctaacttg rttctaaatc tcatagtgcc 120 ttctctgttt ttcccaagga gaaggcacca tgggatttag aaccaagcta gggctagaga 180 catatgttac aggctgtgaa a 201 <210> 2 <211> 254 <212> DNA <213> Homo sapiens <400> 2 ggccagcctg gacaatataa tgagactcta tctctaataa aaaataatta aaaaaaaatt 60 cccaagatcc actgggccaa ggtgggccag gggtggtgtt gggacagcts cgtttaaaaa 120 ggcatctcca agagcttcca tcaaaggctg cctcttggtg cagcacaggt agaaaatggg 180 gctggggagg cagatggaac caggtcatgg aaggccacag atgcccagct gggagtttgg 240 gttttattct gtga 254 <210> 3 <211> 209 <212> DNA <213> Homo sapiens <400> 3 atcccactcc aggatgggaa acccatggcc gagtgggaag aaaccagctg aggtcacatc 60 accagaggag ggagagtgtg gcccctgact cagtccatca gcttgtgtag ctgaggtctg 120 ggccaggtct aaccaggctc cccactcctc ccaacctgag cctgccctct gatctctgcc 180 tgttcctctg tcccacaggg ggcaaaggc 209 <210> 4 <211> 239 <212> DNA <213> Homo sapiens <400> 4 cttaatgatg tttcatgaag tgccccctgg tactttaagt gttcatgaag gcataccaag 60 ttcttaaagg tgtaccacgg ttcatgaagg tgcactgggt tcacctttaa gggyacatgg 120 tacaccttcc aacatgccgt acctgcttaa cctgccttgt ctcaccctcc cttctctgct 180 gtgggacaac ctggatccca ggccccaaag gctcagaggt attacataca ctcttccat 239 <210> 5 <211> 262 <212> DNA <213> Homo sapiens <400> 5 gagtgttagg aagagagtag ggtggccaga ggcagcaaat aaaatataaa atgcttaatt 60 ttgaatctca gataaacaac caataatgtt ttttagcata agtatgtccc aaactaagct 120 tgggaaatat ttatgctayg aaattattcg ttgtttatct aaaattcaaa ctagctgggc 180 atcctgtctt ttaatctggc aaccctaaaa ggcaagagcc aaaaatgccg gagggaagcc 240 aacggattcc aggagggaca ac 262 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for miR-605 A> G (rs2043556) <400> 6 agagcagtta cgccacatga 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for miR-605 A> G (rs2043556) <400> 7 gccttctcct tgggaaaaac 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for miR-608 G> C (rs4919510) <400> 8 gtgggtcaca cttgtaatct 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for miR-608 G> C (rs4919510) <400> 9 aattctgagg gtgttcactg 20 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for miR-631 I> D (rs5745925) <400> 10 aatcccactc caggatggga aa 22 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for miR-631 I> D (rs5745925) <400> 11 tgacagagga acaggcagag at 22 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for miR-938 C> T (rs12416605) <400> 12 ggtgcactgg gttcaccttt aagcg 25 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for miR-938 C> T (rs12416605) <400> 13 gtaatacctc tgagcctttg gggcc 25 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for miR-1302-3 C> T (rs7589328) <400> 14 aactaagctt gggaaatatt tatgcca 27 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for miR-1302-3 C> T (rs7589328) <400> 15 gagcatcatc agtccaaagt cc 22

Claims (10)

To provide the information necessary to predict the risk of repeated implantation failure in Korean women, miR-1302-3 C> T (rs7589328) single base polymorphism was determined from a subject's DNA sample, and activated partial thromboplastin time aPTT from the blood sample of the subject. ). According to claim 1,
A method characterized by further determining one or more polymorphisms selected from miR-605 A> G (rs2043556), miR-631 I> D (rs5745925) and miR-938 C> T (rs12416605). delete According to claim 2,
miR-608 G> C (rs4919510) A method characterized by further discriminating single base polymorphism. miR-1302-3 C> T (rs7589328) Kit for predicting the risk of repeat failure in Korean women, including means for detecting monobasic polymorphism and activated partial thromboplastin time (aPTT) measurement reagent. The method of claim 5,
selected from miR-605 A> G (rs2043556) monobasic polymorphism, miR-631 I> D (rs5745925) polymorphism and miR-938 C> T (rs12416605) monobasic polymorphism Kit further comprising one or more polymorphism detection means. delete The method of claim 6,
miR-608 G> C (rs4919510) Kit further comprises a means for detecting monobasic polymorphism. To provide the information necessary to predict the risk of repeated implantation failure in Korean women, miR-608 G> C (rs4919510) single base polymorphism was determined from a subject's DNA sample and activated partial thromboplastin time (aPTT) from the subject's blood sample. How to determine. miR-608 G> C (rs4919510) Kit for predicting the risk of repeat failure in Korean women, including means for detecting monobasic polymorphisms and activated partial thromboplastin time (aPTT) measurement reagent.

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