KR102143770B1 - Association of miR―27a A>G, miR―423 C>A, miR―449b A>G and miR―604 A>G single nucleotide polymorphism with the risk of recurrent implantation failure in a Korean women - Google Patents

Abstract

The present invention miR-27a A>G (rs895819), miR-423 C>A (rs6505162), miR-449b A>G (rs10061133) and miR-604 A>G (rs2368393) monobasic polymorphism and repetition of Korean women Regarding the relationship between the risk of developing implantation failure, specifically miR-27a A>G, miR-449b A>G and miR-604 from the DNA sample of the subject to provide information necessary for predicting the risk of repetitive implantation failure in Korean women. It relates to a method and a kit therefor, characterized in that the single base polymorphism selected from A>G is determined, or the miR-423 C>A single base polymorphism is further determined.
According to the present invention, repetition of Korean women by discriminating a single nucleotide polymorphism selected from miR-27a A>G, miR-449b A>G, and miR-604 A>G, or further discriminating miR-423 C>A monobasic polymorphism It can provide very useful information for predicting the risk of developing implantation failure. In particular, when the kit of the present invention using the monobasic polymorphism is used, the above information can be more easily provided.

Description

MiR-27a A>G, miR-423 C>A, miR-449b A>G, and miR-604 A>G Association of monobasic polymorphism and the risk of recurrent implantation failure in Korean women {Association of miR-27a A>G , miR-423 C＞A, miR-449b A＞G and miR-604 A＞G single nucleotide polymorphism with the risk of recurrent implantation failure in a Korean women}

The present invention miR-27a A>G (rs895819), miR-423 C>A (rs6505162), miR-449b A>G (rs10061133) and miR-604 A>G (rs2368393) monobasic polymorphism and repetition of Korean women Regarding the relationship between the risk of developing implantation failure, specifically miR-27a A>G, miR-449b A>G and miR-604 from the DNA sample of the subject to provide information necessary for predicting the risk of repetitive implantation failure in Korean women. It relates to a method and a kit for the same, characterized in that the single base polymorphism selected from A>G is determined, or miR-423 C>A single base polymorphism is further determined.

Repeated Implantation Failure (RIF) means that the embryo's intrauterine implantation failure is repeated even after IVF. Causes of repetitive implantation failure can be divided into maternal factors (anatomy, endometrial, thrombosis) and embryonic factors (genetic factors, intrauterine developmental disorders), and the delivery success rate according to IVF is 30~ It is less than 60%. Korea's number of infertility disease treatment personnel increased from 148,000 in 2006 to 184,000 in 2010, an increase of about 36,000 over five years (an average of 5.8% increase per year), and total medical expenses increased from 14.3 billion won in 2006 to 20.3 billion won in 2010. About 6 billion won increased over 5 years (an annual average increase of 9.4%). In addition, for the socioeconomic burden of infertility, it is estimated that the majority of infertile couples currently take 4 to 8 years to conceive and give birth (2 to 4 years before being diagnosed with infertility and 2 to 4 years after diagnosis of infertility). ), despite the high cost of treatment (average of 4.45 million won per treatment), as the number of women who received treatment more than 3 times, which is the number of medical expenses for assisted reproductive procedures such as in vitro fertilization, is about 1 in 3 women. ) 86.4% of infertile couples revealed their intention to continue to receive infertility treatment until their childbirth in the future, so the impact of the cost burden of infertility treatment including assisted reproductive procedures on the home economy is serious (infertile women who responded to the survey 97.8% of respondents answered that the cost burden is high).

Meanwhile, chromosomal abnormalities of parents, thrombosis of mothers, hormonal abnormalities, infectious diseases, autoimmune diseases, uterine and fallopian tubes abnormalities, chromosomal abnormalities, polycystic ovary syndrome, and early ovarian failure are suggested as risk factors for recurrent implantation failure. Although it is a risk factor, it has not been suggested as an exact cause of repetitive implantation failure, and in most cases screening information such as the risk and incidence of unexplained repetitive implantation failure cannot be presented. Therefore, accurate risk presentation and screening tests do not exist yet.

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

Therefore, the main object of the present invention is to provide a novel marker useful for predicting the risk of repetitive implantation failure in 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 repetitive implantation failure in Korean women using the marker.

According to one aspect of the present invention, the present invention provides information necessary for predicting the risk of repetitive implantation failure in Korean women, miR-27a A>G(rs895819), miR-449b A>G(rs10061133) from a DNA sample of a subject. ) And miR-604 A>G (rs2368393).

In the method of the present invention, it is preferable to further discriminate the miR-423 C>A(rs6505162) monobasic polymorphism.

According to another aspect of the present invention, the present invention provides a means for detecting a monobasic polymorphism selected from miR-27a A>G (rs895819), miR-449b A>G (rs10061133) and miR-604 A>G (rs2368393). It provides a kit for predicting the risk of repetitive implantation failure in Korean women, including.

In the kit of the present invention, it is preferable to further include a means for detecting miR-423 C>A (rs6505162) monobasic polymorphism.

In the kit of the present invention, the means for detecting the miR-27a A>G mononucleotide polymorphism is an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 5, an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 6, and Dra III It is a primer-restriction enzyme set comprising a restriction enzyme, and the means for detecting miR-449b A>G mononucleotide polymorphism is an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 9, and the nucleotide sequence of SEQ ID NO: 10. It is a primer-restriction enzyme set comprising an oligonucleotide primer and a Bsm AI restriction enzyme, and the means for detecting the miR-604 A>G mononucleotide polymorphism is an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 11, SEQ ID NO: 12 It is preferably a primer-restriction enzyme set comprising an oligonucleotide primer and a Bss SI restriction enzyme represented by the nucleotide sequence of.

In the kit of the present invention, the means for detecting the miR-423 C>A mononucleotide polymorphism is an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 7, an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 8, and Aci I It is preferable that it is a primer-restriction enzyme set comprising a restriction enzyme.

According to the present invention, repetition of Korean women by discriminating a single nucleotide polymorphism selected from miR-27a A>G, miR-449b A>G, and miR-604 A>G, or further discriminating miR-423 C>A monobasic polymorphism It can provide very useful information for predicting the risk of developing implantation failure. In particular, when the kit of the present invention using the monobasic polymorphism is used, the above information can be more easily provided.

The miR-27a A>G, miR-423 C>A, miR-449b A>G and miR-604 A>G mononucleotide polymorphisms used in the present invention (hereinafter, abbreviated as'SNP') appear in human microRNA genes. Registered as rs895819, rs6505162, rs10061133, and rs2368393 in the U.S. National Bioinformatics Center's monobasic polymorphism database (NCBI dbSNP, http://http://www.ncbi.nlm.nih.gov/snp/), respectively. Has been.

The miR-27a A>G SNP can be identified by the 181th base in the human DNA site represented by SEQ ID NO: 1, and this base may be adenine or guanine.

The miR-423 C>A SNP can be identified by the 141st base in the human DNA region represented by SEQ ID NO: 2, and this base may be cytosine or adenine.

The miR-449b A>G SNP can be identified by the 141st base in the human DNA site represented by SEQ ID NO: 3, and this base may be adenine or guanine.

The miR-604 A>G SNP can be identified by the 181th base in the human DNA site represented by SEQ ID NO: 4, and this base may be adenine or guanine.

Discrimination of the SNP as described above can be performed by performing a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, a nucleotide sequence analysis method, and the like.

Polymerase chain reaction-restriction enzyme fragmentation method is to amplify a target gene or DNA site by performing a polymerase chain reaction, and when treated with a specific restriction enzyme, the restriction enzyme recognizes and digests it or the restriction enzyme does not recognize it. This is a method of distinguishing polymorphism, deletion, addition or substitution of bases by distinguishing whether digestion is not possible due to failure. At this time, whether or not DNA is amplified and digested by restriction enzymes can be confirmed through electrophoresis using an agarose gel.

As a method for sequencing, a known method of Maxam-Gilbert or Sanger may be used.

When the polymorphism is determined by the PCR-RFLP method, a primer for amplifying a continuous 50 to 10,000 bp site including each SNP site and a restriction enzyme capable of cleaving DNA when each SNP site is a specific base are used. I can.

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

miR-27a A>G SNP discrimination: forward primer of SEQ ID NO: 5 and reverse primer of SEQ ID NO: 6, Dra III restriction enzyme

miR-423 C>A SNP discrimination: forward primer of SEQ ID NO: 7 and reverse primer of SEQ ID NO: 8, Aci I restriction enzyme

miR-449b A>G SNP discrimination: forward primer of SEQ ID NO: 9 and reverse primer of SEQ ID NO: 10, Bsm AI restriction enzyme

miR-604 A>G SNP discrimination: forward primer of SEQ ID NO: 11 and reverse primer of SEQ ID NO: 12, Bss SI restriction enzyme

When the miR-27a A>G SNP identification primer and restriction enzyme are used, a DNA fragment of 182 bp is amplified through polymerase chain reaction, and when the restriction enzyme is treated, DNA fragments of 155 bp and 27 bp in the case of AA genotype, AG genotype DNA fragments of 182 bp, 155 bp, and 27 bp in case of GG genotype are generated.

When the miR-423 C>A SNP identification primer and restriction enzyme are used, a 144 bp DNA fragment is amplified through polymerase chain reaction, and when the restriction enzyme is treated, a DNA fragment of 122 bp and 22 bp in the case of CC genotype, CA genotype DNA fragments of 144 bp, 122 bp, and 22 bp in the case of the AA genotype are generated.

When the miR-449b A>G SNP identification primer and restriction enzyme are used, a 119 bp DNA fragment is amplified through polymerase chain reaction, and when the restriction enzyme is treated, a DNA fragment of 119 bp in the case of AA genotype and 119 bp in the case of AG genotype. , 97bp and 22bp DNA fragments, in the case of the GG genotype, 97bp and 22bp DNA fragments are generated.

When the miR-604 A>G SNP identification primer and restriction enzyme are used, a 243 bp DNA fragment is amplified through polymerase chain reaction, and when the restriction enzyme is treated, DNA fragments of 169 bp and 74 bp in the case of AA genotype, AG genotype DNA fragments of 243 bp, 169 bp and 74 bp in the case of GG genotype and 243 bp of DNA fragments are generated.

According to the present invention, each SNP of miR-27a A>G, miR-423 C>A, miR-449b A>G and miR-604 A>G is determined in the same manner as described above, and repetitive implantation of Korean women based on this It can provide information to predict the risk of developing failure. This is due to the significant difference ( p <0.05) confirmed through comparative analysis of the repeat implantation failure patient and control group.

In the existing academic world, the case where the embryo fails to implant in the uterus at least three times after IVF is defined as repeated implantation failure (hereinafter abbreviated as'RIF'), but recently, such implantation failure has occurred twice. Even if it occurs continuously, there is an opinion that it should also be viewed as RIF. Accordingly, in the present invention, a case in which two successive implantation failures occur or the total number of implantation failures is three or more is defined as RIF.

According to the present invention, the risk of developing RIF in Korean women can be predicted based on the result of each SNP determination as follows.

1) If the subject's miR-27a A>G genotype is AG genotype, the likelihood of RIF development is lower than those with AA genotype (predominant genotype).

2) If the subject's miR-449b A>G genotype is AG genotype, it is more likely to develop 3 or more or 4 or more RIFs than those with AA genotype.

3) If the subject's miR-604 A>G genotype is GG genotype, the likelihood of RIF occurrence is lower than that of AA genotype.

4) When the subject's miR-27a A>G / miR-423 C>A combination type is the AG/CC combination type, the possibility of RIF occurrence is lower than that of the AA/CC combination type.

5) If the subject's miR-27a A>G / miR-604 A>G combination type is AA/GG combination type, AG/AG combination type, or AG/GG combination type, RIF is less likely than those of AA/AA combination type. .

6) If the subject's miR-423 C>A / miR-449b A>G combination type is CC/AG combination type or CA/GG combination type, RIF is more likely to occur than those of CC/AA combination type.

7) If the subject's miR-423 C>A / miR-604 A>G combination type is the CC/AG combination type, the possibility of RIF occurrence is lower than those of the CC/AA combination type.

8) If the subject's miR-449b A>G / miR-604 A>G combination type is the AG/AA combination type, RIF is more likely to occur than those of the AA/AA combination type.

9) If the subject's miR-27a A>G / miR-423 C>A / miR-449b A>G / miR-604 A>G haplotype is ACGA haplotype, AAGG haplotype or GAAA haplotype, ACAA haplotype RIF is more likely to occur than people with body type.

10) When the subject's miR-27a A>G / miR-423 C>A / miR-449b A>G / miR-604 A>G haplotype is GCAG haplotype, RIF occurs compared to those of ACAA haplotype It is unlikely.

11) If the subject's miR-27a A>G / miR-423 C>A / miR-449b A>G haplotype is the G-C-A haplotype, the possibility of RIF occurrence is lower than that of the A-C-A haplotype.

12) If the subject's miR-27a A>G / miR-423 C>A / miR-604 A>G haplotype is GCG haplotype or GAG haplotype, the possibility of RIF occurrence is lower than those of ACA haplotype. .

13) When the subject's miR-27a A>G / miR-449b A>G / miR-604 A>G haplotype is A-G-A haplotype, RIF is more likely to occur than those of A-A-A haplotype.

14) If the subject's miR-27a A>G / miR-449b A>G / miR-604 A>G haplotype is G-A-G haplotype, the possibility of RIF occurrence is lower than those of A-A-A haplotype.

15) If the subject's miR-423 C>A / miR-449b A>G / miR-604 A>G haplotype is an A-G-G haplotype, the possibility of RIF occurrence is lower than that of a C-A-A haplotype.

16) If the subject's miR-27a A>G / miR-423 C>A haplotype is a G-C haplotype, the possibility of RIF occurrence is lower than that of the A-C haplotype.

17) If the subject's miR-27a A>G / miR-604 A>G haplotype is a G-G haplotype, the possibility of RIF occurrence is lower than that of the A-A haplotype.

18) If the subject's miR-423 C>A / miR-449b A>G haplotype is a C-G haplotype, RIF is more likely than a C-A haplotype.

19) If the subject's miR-449b A>G / miR-604 A>G haplotype is an A-G haplotype, the possibility of RIF occurrence is lower than that of the A-A haplotype.

20) If the subject's miR-449b A>G genotype is AG or GG genotype and the activated partial thromboplastin time (aPTT) is 29.4 seconds or less, the miR-449b A>G genotype is AA genotype and the aPTT exceeds 29.4 seconds. In comparison, RIF is more likely to occur.

The kit for predicting the risk of repetitive implantation failure in Korean women of the present invention makes it possible to easily perform SNP identification as described above, and based on this, the risk of repetitive implantation failure in Korean women can be predicted, which detects SNP. As a means, substances commonly used for detection of SNPs, for example, primers for PCR, primers and restriction enzymes for PCR-RFLP, probes for DNA-DNA hybridization, and the like may be used. In the present invention, it is preferable to use PCR-RFLP primers and restriction enzymes as detection means, and in particular, the SNP identification primers and restriction enzymes mentioned above are preferable.

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

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

[Example]

1. Method

1-1. Research group

Between March 2010 and December 2012, RIF patients were recruited at the Obstetrics and Gynecology of the Infertility Treatment Center at Bundang Cha Hospital (Seongnam, Korea), and blood samples were collected from 120 RIF patients and 219 control participants. All patients and control participants were Koreans. This study was approved by the Review Committee of Bundang Cha Hospital, and informed consent was obtained from all patients (reference no. PBC09-120).

Patients experiencing miscarriage (less than 20 weeks of pregnancy) were confirmed by levels of human chorionic gonadotropin or by ultrasound and/or physical examination. Anatomical, chromosomal, hormonal, infectious, autoimmune, or thrombotic implantation failure is a generally applied exclusion criterion when diagnosing RIF, so the diagnosed subjects were excluded from the study group. Anatomical abnormalities in RIF patients were confirmed by ultrasound, hysterosalpingography, hysteroscopy, computed tomography, or magnetic resonance imaging. Karyotyping was performed using standard protocols. Hormonal causes including hyperprolactinemia, luteal deficiency, and thyroid disease were evaluated by measuring the levels of prolactin, thyroid-stimulating hormone, free T4, follicle-stimulating hormone, luteinizing hormone and progesterone in the peripheral blood. In order to identify the autoimmune diseases lupus and antiphospholipid syndrome, lupus anticoagulant and anticardiolipin antibodies were identified, respectively. The cause of thrombosis was defined as thrombocytopenia, and evaluated by the deficiency of protein C and protein S and the presence of anti-β2 glycoprotein antibody. In this study, subjects diagnosed with uterine malformations or underlying medical diseases were excluded. In addition, an embryologist evaluated the quality of embryos for IVF. Women in the control group were recruited from Bundang CHA Hospital and met the following registration criteria: regular menstrual cycle, at least one natural pregnancy experience, no miscarriage, karyotype 46, XX.

1-2. Evaluation of homocysteine, folic acid, total cholesterol, uric acid, blood urea nitrogen, creatinine and blood coagulation status

Blood samples were collected from RIF patients after a 12 hour fast. Homocysteine was measured by fluorescence polarization immunoassay using an Abbott IMx analyzer (Abbott Laboratories, Abbott Park, IL, USA). Folic acid was measured by a competitive immunoassay using an ACS 180 Plus automated chemiluminescence system (Bayer Diagnostics, Tarrytown, NY, USA). Total cholesterol, uric acid, blood urea nitrogen and creatinine were measured using a commercially available enzyme colorimetric test (Roche Diagnostics, GmbH, Mannheim, Germany). Platelet, leukocyte and hemoglobin levels were obtained using a Sysmex XE 2100 automated blood analysis system (Sysmex Corporation, Kobe, Japan). PT (prothrombin time) and aPTT (activated partial thromboplastin time) were measured with an ACL TOP automatic photo-optical coagulometer (LSI Medience, Tokyo, Japan).

1-3. Hormone analysis

On the second or third day of the menstrual cycle, a female blood sample was collected by venipuncture, and a serum sample was collected from the blood before measurement. Estradiol, thyroid-stimulating hormone and prolactin levels were measured by radioimmunoassay (Beckman Coulter), and follicle-stimulating hormone and luteinizing hormone levels were measured according to the manufacturer's instructions using enzyme immunoassay (Siemens, Munich, Germany). .

1-4. Genotyping

Genomic DNA was extracted from white blood cells using a G-DEX blood extraction kit (Intron, Seongnam, Korea). All genetic polymorphisms were determined by PCR-RFLP analysis using the isolated genomic DNA as a template (35 cycles of denaturation at 95°C for 30 seconds, annealing at 56 to 59°C for 30 seconds, and extension for 30 seconds at 72°C). For miR-27a(rs895819) A>G, 5'-GAA CTT AGC CAC TGT GAA CAC CAC TTG G-3' forward primer and 5'-TTG CTT CCT GTC ACA AAT CAC ATT G-3' reverse primer Used (annealed at 58° C.), and a 182 bp PCR product was treated with Dra III restriction enzyme. For miR-423(rs6505162) C>A, a forward primer of 5'-GAA GCC CGA AGT TTG AGG GAG AAA C-3' and a reverse primer of 5'-CGG GGA GAA ACT CAA GCG CGC G-3' were used. (Annealing 59° C.), 144 bp PCR product was treated with Aci I restriction enzyme. For miR-449b(rs10061133) A>G, 5'-GGT ATC CAG AGC ACT TCA TTG ACA-3' positive??anti primer and 5'-ACC TGA ATC AGG TAG GCA GTG TCT-3' reverse primer Used (annealed at 56° C.), and a 119 bp PCR product was treated with Bsm AI restriction enzyme. For miR-604 (rs2368393) A>G, 5'-CTT GGC TCA GTG GTC TGT TT-3' forward primer and 5'-GTA CAG GGA CTG AAA GGT GAA G-3' reverse primer were used (annealed 59° C.), 243bp PCR product was treated with Bss SI restriction enzyme. Each restriction enzyme (New England BioLabs, Beverly, MA, USA) treatment was performed at 37°C for 16 hours.

1-5. Statistical analysis

The genotype frequencies of RIF patients and controls were compared using Fisher exact test and logistic regression. All polymorphisms were calculated to investigate deviations from the Hardy-Weinberg equilibrium. OR (odds ratio) and 95% CI (confidence interval) were used to measure the degree of association between miRNA polymorphism and RIF. Polymorphisms associated with the incidence of RIF were calculated using adjusted OR (AOR) and 95% CI in age-corrected logistic regression. OR and 95% CI were also used to evaluate the relationship between each specific polymorphism and allele combination. Gene-gene interactions between four miRNA loci were analyzed using a multifactor-dimensionality reduction (MDR) method and MDR software version 2.0 (www.epistasis.org). All possible combinations of polymorphisms were studied using MDR analysis to determine combinations with strong synergistic effects. Statistical analysis was performed using GraphPad Prism 4.0 (GraphPad Software Inc., San Diego, CA) and StatsDirect software version 2.4.4 (StatsDirect Ltd., Altrincham, UK). The significant association between the inferred genotype and RIF was evaluated by changing case and control conditions and recalculating 1,000 test statistics for each sample set.

2. Results

The clinical characteristics of the RIF patient group and the control group are shown in Table 1. RIF patients had significantly lower aPTT and higher estradiol and luteinizing hormone (both P <0.05). Regarding miRNA genotypes (all Hardy-Weinberg equilibriums), the miR-27aA>G polymorphism (AOR, 0.480; 95% CI, 0.296-0.781; P =0.003 and AOR, 0.499; 95% CI, 0.316-0.789; P =0.003) showed a protective effect against the risk of RIF (see Table 2). miR-604A>G (AOR, 0.415; 95% CI, 0.196-0.878; P =0.021 and AOR, 0.596; 95% CI, 0.377-0.945; P =0.028) polymorphism also showed a protective effect against RIF risk, The miR-449bA>G polymorphism has been shown to be associated with an increased risk of RIF. When RIF patients were stratified according to the number of repetitive implantation failures, there was an association between miR-27aA>G polymorphism and RIF risk in patients with 3 or more failures.

As a result of combination analysis, patients with miR-27aAG/miR-423CC genotype (AG/CC: AOR, 0.417; 95% CI, 0.223-0.780; P =0.006) and miR-27aAG/miR-604AG or GG genotype (AG/ AG: AOR, 0.329; 95% CI, 0.153-0.706; P =0.004 and AG/GG: AOR, 0.193; 95% CI, 0.054-0.693; P =0.012). (See Table 3). These results are consistent with the association between RIF risk and each microRNA genotype.

The results of the allele-allele combination by MDR analysis of the four miRNA polymorphisms are shown in Table 4, and only the association between the allele combination type and the prevalence of RIF is shown. Three haplotypes for miR-27a/miR-423/miR-449b/miR-604 have been shown to be associated with an increased risk of RIF: ACGA (OR, 2.352; 95% CI, 1.260-4.390; P) =0.007), AAGG (OR, 8.818; 95% CI, 1.004-77.460; P =0.030), and GAAA (OR, 5.291; 95% CI, 1.627-17.210; P =0.006); The GCAG (OR, 0.248; 95% CI, 0.115-0.537; P =0.0001) haplotype has been shown to be associated with a reduced risk. A high risk of RIF was also found to be associated with the AGA haplotype of miR-27a/miR-449b/miR-604 (OR, 1.933; 95% CI, 1.146-3.261; P =0.016). In contrast, GAG of miR-27a/miR-449b/miR-604 and AGG of miR-423/miR-449b/miR-604 were found to be associated with a reduced risk of RIF.

When analyzing the prevalence of RIF in the association of the interactions between the four miRNA polymorphisms and environmental factors, the miR-27aAG+GG genotype was found to be associated with RIF in patients with PT less than 10.45 seconds (AOR, 0.058; 95%). CI, 0.015-0.251; see Table 5). In addition, the miR-423CA+AA genotype has a PT of 10.45 seconds or less (AOR, 0.003; 95% CI, 0.000-0.047), aPTT of 29.4 seconds or less (AOR, 0.188; 95% CI, 0.039-0.904), and platelet count 276x10 ³ It was found to be associated with RIF in patients with /µl or more (AOR, 0.041; 95% CI, 0.012-0.0140). miR-449bAG or GG genotype has a PT of 10.45 seconds or less (AOR, 0.166; 95% CI, 0.042-0.653), aPTT of 29.4 seconds or less (AOR, 7.980; 95% CI, 1.998-31.870), and a platelet count of 276x10 ³ It was found to be associated with RIF in patients with /µl or more (AOR, 2.831; 95% CI, 1.123-7.138). The miR-604AG or GG genotype has been shown to be associated with RIF in patients with a PT of 10.45 seconds or less (AOR, 0.080; 95% CI, 0.018-0.351). Additionally, statistical analysis was performed to confirm the statistical validity of each meaningful genotype and haplotype combination group (see Table 6).

<Table description>

Table 1: SD, standard deviation; BMI = body mass index; PT, prothrombin; aPTT, Activated Partial Thromboplastin Time; Hcy, Homocysteine; Hgb, hemoglobin; PLT, platelet count; BUN, blood urea nitrogen; TSH, thyroid-stimulating hormone; FSH, follicle stimulating hormone; LH, luteinizing hormone; NA = not applicable; RIF = recurrent implantation failure.

Table 2: AOR adjusted to participant's age; RIF = recurrent implantation failure; AOR = adjusted odds ratio; CI = confidence interval.

Table 3: RIF = recurrent implantation failure; AOR = adjusted odds ratio; CI = confidence interval.

Table 4: RIF = recurrent implantation failure; The OR and 95% CI of each allele combination was calculated using Fisher's exact test with reference to the frequencies of all other alleles; P value according to Fisher's exact test; OR = odds ratio; CI = confidence interval.

Table 5: RIF = recurrent implantation failure; *PT 10.45 sec, aPTT 29.4 sec, lower 25% cut off level in RIF patients and controls; †Platelet 276 103/µl is the top 25% cut off level in RIF patients and controls.

Table 6: AOR = adjusted odds ratio; CI = confidence interval.

<110> CHA University Industry-Academic Cooperation Foundation <120> Association of miR-27a A>G, miR-423 C>A, miR-449b A>G and miR-604 A>G single nucleotide polymorphism with the risk of recurrent implantation failure in a Korean women <130> PA-D18256 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 321 <212> DNA <213> Homo sapiens <400> 1 gccggctggg gttcctgggg atgggatttg cttcctgtca caaatcacat tgccagggat 60 ttccaaccga ccctgagctc tgccaccgag gatgctgccc ggggacgggg tggcagagag 120 gccccgaagc ctgtgcctgg cctgaggagc agggcttagc tgcttgtgag cagggtccac 180 rccaagtcgt gttcacagtg gctaagttcc gccccccagg ccctcacctc ctctggcctt 240 gccgcctgtc ccctgctgcc gcctgtctgc ctgccatcct gctgcctggc ctccctgggc 300 tctgcctccc gtgcctactg a 321 <210> 2 <211> 321 <212> DNA <213> Homo sapiens <400> 2 ccgtacattt tcccggatgg aagcccgaag tttgagggag aaacttgtga ggaaataaag 60 gaagttaggc tgaggggcag agagcgagac ttttctattt tccaaaagct cggtctgagg 120 cccctcagtc ttgcttccta mcccgcgctt gagtttctcc ccgcttggat gctctcaggg 180 gcagtgtgaa gagagacagt tcctggcttc cttagagggc cttgttctcc ttggcagaag 240 ctcaagtcct gattcccgcc cttccttctc gttttaggaa tattgttctg agaagccaca 300 gaaaaagggt aggcatatag g 321 <210> 3 <211> 321 <212> DNA <213> Homo sapiens <400> 3 cacatattca accagctaac aatacactgc cagctcatca cacacaggta tccagagcac 60 ttcattgaca agaagaattt atccagaagc aagtggcagg gtagttgtgg ctgctgactt 120 gacccaagca gccagctaac ratacactgc ctacctgatt caggtcactg ccgattctcg 180 tggcccaggc agtcactgag gctaaatttg taacatgaaa catagtggca gaactctaat 240 cttaatagtc acattaaaga aaacttatac acaaaatgta ttacttcaag actataagaa 300 ataaaatcaa tatttgcttg a 321 <210> 4 <211> 321 <212> DNA <213> Homo sapiens <400> 4 aggtgtttgg gggtacaggg actgaaaggt gaagccaatt ccattccagt aatagaataa 60 gctgtgagga ggacccagat gtctgttctt tagaatggcc cccaagatgt atgtttctcc 120 atgatgcact gtcctgaatt ccgcagcctg tgtcagaaaa cctgcctgct agtggacacg 180 rgagcgtgga aggtcaagca cgatgctctc ctgcagacac ctctctcagt gcatggaaac 240 agaccactga gccaagagag cttgaggctc tggagacggt ttttagacta aaatctctca 300 tcaggagatg agcaaatgca a 321 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for miR-27a A> G <400> 5 gaacttagcc actgtgaaca ccacttgg 28 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for miR-27a A> G <400> 6 ttgcttcctg tcacaaatca cattg 25 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for miR-423 C>A <400> 7 gaagcccgaa gtttgaggga gaaac 25 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for miR-423 C>A <400> 8 cggggagaaa ctcaagcgcg cg 22 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for miR-449b A> G <400> 9 ggtatccaga gcacttcatt gaca 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for miR-449b A>G <400> 10 acctgaatca ggtaggcagt gtct 24 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for miR-604 A>G <400> 11 cttggctcag tggtctgttt 20 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for miR-604 A>G <400> 12 gtacagggac tgaaaggtga ag 22

Claims (6)

Method for discriminating miR-27a A>G(rs895819) single nucleotide polymorphism from a DNA sample of a subject to provide information necessary for predicting the risk of repetitive implantation failure in Korean women. The method of claim 1,
A method, characterized in that further discriminating a monobasic polymorphism selected from miR-449b A>G(rs10061133), miR-604 A>G(rs2368393) and miR-423 C>A(rs6505162). miR-27a A>G (rs895819) kit for predicting the risk of repetitive implantation failure in Korean women comprising a means for detecting monobasic polymorphism. The method of claim 3,
A kit, characterized in that it further comprises a means for detecting a monobasic polymorphism selected from miR-449b A>G (rs10061133), miR-604 A>G (rs2368393) and miR-423 C>A (rs6505162). The method of claim 3,
The means for detecting the miR-27a A>G single nucleotide polymorphism is a primer comprising an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 5, an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 6, and a Dra III restriction enzyme. -Kit, characterized in that the restriction enzyme set. The method of claim 4,
The means for detecting the miR-449b A>G single nucleotide polymorphism is a primer comprising an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 9, an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 10, and a Bsm AI restriction enzyme. -Is a set of restriction enzymes,
The means for detecting the miR-604 A>G single nucleotide polymorphism is a primer comprising an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 11, an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 12, and a Bss SI restriction enzyme. -Is a set of restriction enzymes,
The means for detecting the miR-423 C>A single nucleotide polymorphism is a primer comprising an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 7, an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 8, and an Aci I restriction enzyme. -Kit, characterized in that the restriction enzyme set.