KR20200058832A - 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 relates to a correlation between miR-27a A>G (rs895819), miR-423 C > A (rs6505162), miR-449b A > G (rs10061133) and miR-604 A > G (rs2368393) single nucleotide polymorphisms and a risk of recurrent implantation failures among Korean women and, more particularly, to a method and kit for distinguishing a single nucleotide polymorphism selected from miR-27a A > G, miR-449b A > G and miR-604 A > G from a subject′s DNA sample or further distinguishing a single nucleotide polymorphism of miR-423 C > A 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 single nucleotide polymorphism selected from miR-27a A > G, miR-449b A > G and miR-604 A > G from a subject′s DNA sample or further distinguishing a single nucleotide polymorphism of miR-423 C > A to provide information required for predicting a risk of developing recurrent implantation failures among Korean women. In particular, in case of using the kit of the present invention using the single nucleotide polymorphism, it is possible to more easily provide the above information.

Description

miR-27a A> G, miR-423 C> A, miR-449b A> G and miR-604 A> G Association between monobasic polymorphism and risk of repeated 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 Concerning the association of the risk of implantation failure, specifically miR-27a A> G, miR-449b A> G and miR-604 from DNA samples of subjects to provide information necessary to predict the risk of repeated implantation failure in Korean women It relates to a method and a kit for determining a single base polymorphism selected from A> G or further discriminating miR-423 C> A single base polymorphism.

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 miR-27a A> G (rs895819), miR-449b A> G (rs10061133) from a subject's DNA sample to provide the information necessary to predict the risk of repeated implantation failure in Korean women ) And miR-604 A> G (rs2368393).

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

According to another aspect of the present invention, the present invention provides a means for detecting a single base 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 repeated implantation failure in Korean women.

In the kit of the present invention, it is preferable to further include 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 single base 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 the miR-449b A> G single base polymorphism is an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 9, represented by 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 single base polymorphism is an oligonucleotide primer represented by the nucleotide sequence of SEQ ID NO: 11, SEQ ID NO: 12 It is preferred that it is a primer-restriction enzyme set comprising an oligonucleotide primer represented by the base sequence of and a Bss SI restriction enzyme.

In the kit of the present invention, the means for detecting the miR-423 C> A single base 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 preferably a primer-restriction enzyme set comprising a restriction enzyme.

According to the present invention, the repetition of Korean women by discriminating single base polymorphism selected from miR-27a A> G, miR-449b A> G and miR-604 A> G or further discriminating miR-423 C> A single base polymorphism It can provide very useful information to predict the risk of implantation failure. In particular, when using the kit of the present invention using the single-basic polymorphism, it is possible to provide the above information more easily.

The miR-27a A> G, miR-423 C> A, miR-449b A> G and miR-604 A> G single base polymorphisms (hereinafter abbreviated as 'SNP') used in the present invention may appear in the human microRNA gene. SNPs can be registered in the National Biological Information Center's single base polymorphism database (NCBI dbSNP, http://www.ncbi.nlm.nih.gov/snp/) as rs895819, rs6505162, rs10061133, and rs2368393, respectively. It is.

miR-27a A> G SNP can be classified as the 181th base in the human DNA region represented by SEQ ID NO: 1, which may be adenine or guanine.

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

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

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

The SNP can be determined by performing a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, a sequencing method, and the like.

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 site and a restriction enzyme capable of cleaving DNA when each SNP site is a specific base are used. Can be.

More preferably, the following primers for amplification and restriction enzymes can 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 discriminant primer and restriction enzyme are used, a DNA fragment of 182 bp is amplified through a polymerase chain reaction, and when restriction enzyme is processed, 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, and 182 bp DNA fragments in case of GG genotype are generated.

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

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

When the miR-604 A> G SNP discriminant primer and restriction enzyme are used, a DNA fragment of 243 bp is amplified through a polymerase chain reaction, and when processing the restriction enzyme, DNA fragments of 169 bp and 74 bp in the case of AA genotype, AG genotype In this case, DNA fragments of 243 bp, 169 bp, and 74 bp, and in the case of the GG genotype, DNA fragments of 243 bp 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 based on this, repeated implantation of Korean women It can provide information to predict the risk of failure. This is attributable to a significant difference ( p <0.05) confirmed through comparative analysis of the repeat failure 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 results of each SNP discrimination.

1) When the miR-27a A> G genotype of the subject is the AG genotype, RIF is less likely to occur compared to the AA genotype (predominant genotype).

2) When the miR-449b A> G genotype of the test subject is the AG genotype, it is more likely that RIF occurs three or more times or more than four times compared to the AA genotype.

3) When the miR-604 A> G genotype of the test subject is the GG genotype, the likelihood of RIF generation is lower than that of the AA genotype.

4) When the subject's miR-27a A> G / miR-423 C> A combination type is the AG / CC combination type, it is less likely to develop RIF than the AA / CC combination type person.

5) When the miR-27a A> G / miR-604 A> G combination of subjects is AA / GG combination, AG / AG combination or AG / GG combination, RIF is less likely to occur compared to AA / AA combination. .

6) When 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 the CC / AA combination type.

7) When the miR-423 C> A / miR-604 A> G combination type of the subject is the CC / AG combination type, it is less likely to develop RIF than the CC / AA combination type person.

8) When 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 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 fold RIFs are more likely to occur than people with body types.

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

11) When the subject's miR-27a A> G / miR-423 C> A / miR-449b A> G haplotype is G-C-A haplotype, it is less likely to develop RIF than the A-C-A haplotype.

12) When the subject's miR-27a A> G / miR-423 C> A / miR-604 A> G haplotype is GCG haplotype or GAG haplotype, it is less likely to develop RIF than a person who is 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, it is more likely to develop RIF than the A-A-A haplotype.

14) When the subject's miR-27a A> G / miR-449b A> G / miR-604 A> G haplotype is G-A-G haplotype, it is less likely to develop RIF than the A-A-A haplotype.

15) When the subject's miR-423 C> A / miR-449b A> G / miR-604 A> G haplotype is A-G-G haplotype, it is less likely to develop RIF than the C-A-A haplotype.

16) When the subject's miR-27a A> G / miR-423 C> A haplotype is G-C haplotype, it is less likely to develop RIF than the A-C haplotype.

17) If the subject's miR-27a A> G / miR-604 A> G haplotype is G-G haplotype, it is less likely to develop RIF than the A-A haplotype.

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

19) When the subject's miR-449b A> G / miR-604 A> G haplotype is A-G haplotype, it is less likely to develop RIF than 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 (apPTT) is 29.4 seconds or less, miR-449b A> G genotype is AA genotype and aPTT exceeds 29.4 seconds. In comparison, RIF is more likely to occur.

The kit for predicting the risk of repeated implantation failure of a Korean woman of the present invention is to enable easy identification of the SNP as described above, thereby predicting the risk of repeated implantation failure of a Korean woman. As a means, materials commonly used for the detection of SNPs, for example, primers for PCR, primers for PCR-RFLP and restriction enzymes, probes for DNA-DNA hybridization, and the like can be used. In the present invention, it is preferable to use PCR-RFLP primers and restriction enzymes as detection means, and particularly, the above-mentioned primers and restriction enzymes for SNP discrimination are preferred.

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 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

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

Patients who experienced abortion (less than 20 weeks of pregnancy) were identified by level or ultrasound and / or physical examination of chorionic gonadotropin. Anatomical, chromosomal, hormonal, infectious, autoimmune, or thrombotic implantation failures were excluded from the study group because they were the exclusion criteria generally applied when diagnosing RIF. Anatomical abnormalities of RIF patients were confirmed by ultrasonography, 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 peripheral blood. To confirm the autoimmune disease lupus and antiphospholipid syndrome, lupus anticoagulant and anticardiolipin antibodies were identified, respectively. The thrombotic cause was defined as thrombocytopenia, and was evaluated by the lack 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 conditions were excluded. In addition, an embryonician assessed the quality of the embryo for IVF. Women in the control group were recruited from the Bundang Cha Hospital and met the following registration criteria: regular menstrual cycle, at least one natural pregnancy experience, no experience of miscarriage, karyotype 46, XX.

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

Blood samples were collected from RIF patients after 12 hours of fasting. Homocysteine was measured by fluorescence polarization immunoassay using an Abbott IMx analyzer (Abbott Laboratories, Abbott Park, IL, USA). Folic acid was measured by 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 commercially available enzyme colorimetric tests (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). The prothrombin time (PT) and activated partial thromboplastin time (apTT) were measured with an ACL TOP automatic optical-optical coagulator (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 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 gene polymorphisms were determined by PCR-RFLP analysis using isolated genomic DNA as a template (denaturation at 95 ° C for 30 seconds, annealing at 56 to 59 ° C for 30 seconds, and extension at 30 ° C for 30 seconds for 35 cycles). 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 (annealing 58 ° C.) and the 182 bp PCR product was treated with Dra III restriction enzyme. For miR-423 (rs6505162) C> A, a 5'-GAA GCC CGA AGT TTG AGG GAG AAA C-3 'forward primer and a 5'-CGG GGA GAA ACT CAA GCG CGC G-3' reverse primer were used. (Annealing 59 ° C.), a 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 'constant primer and 5'-ACC TGA ATC AGG TAG GCA GTG TCT-3' reverse primer Used (annealed 56 ° C), and the 119bp 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 (annealing 59 ° C), 243 bp 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 genotyping frequency of RIF patients and controls was 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% confidence interval (CI) were used to measure the degree of association between miRNA polymorphism and RIF. Polymorphism associated with RIF incidence was calculated using 95% CI in adjusted OR (AOR) and logistic regression corrected for age. OR and 95% CI were also used to evaluate the relationship between each specific polymorphism and allele combination. The gene-gene interaction between four miRNA loci was analyzed using the multifactor-dimensionality reduction (MDR) method and MDR software version 2.0 (www.epistasis.org). All possible combinations of polymorphism were studied using MDR analysis to determine combinations with strong synergies. 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). Significant association between the inferred genotype and RIF was evaluated by changing the case and control status, and recalculating 1,000 test statistics for each sample set.

2. Results

Table 1 shows the clinical characteristics of the RIF patient group and the control group. In RIF patients, aPTT was significantly lower and estradiol and luteinizing hormone were higher (all P <0.05). With respect to the miRNA genotype (all Hardy-Weinberg equilibrium established), 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 RIF risk (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 protective effects against RIF risk, The miR-449bA> G polymorphism has been shown to be associated with increased RIF risk. When stratified RIF patients according to the number of repeated implantation failures, there was a correlation between miR-27aA> G polymorphism and RIF risk in patients who failed more than 3 times.

As a result of the 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 / A decrease in RIF risk was found in patients with 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. Here, only the association between the allele combination and RIF prevalence was shown. Three haplotypes for miR-27a / miR-423 / miR-449b / miR-604 have been shown to be associated with increased RIF risk: 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); GCAG (OR, 0.248; 95% CI, 0.115-0.537; P = 0.0001) haplotype was shown to be associated with reduced risk. The high risk of RIF has also been shown to be correlated 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, the GAG of miR-27a / miR-449b / miR-604 and the AGG of miR-423 / miR-449b / miR-604 have been shown to be associated with reduced RIF risk.

When analyzing the prevalence of RIF in the interaction relationship between four miRNA polymorphisms and environmental factors, the miR-27aAG + GG genotype was found to be associated with RIF in patients with PTs less than 10.45 seconds (AOR, 0.058; 95%) CI, 0.015-0.251; see Table 5). In addition, miR-423CA + AA genotype has 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 is 276x10 ³ It was found to be related to RIF in patients with / µl or more (AOR, 0.041; 95% CI, 0.012-0.0140). For miR-449bAG or GG genotype, PT is 10.45 seconds or less (AOR, 0.166; 95% CI, 0.042-0.653), aPTT is 29.4 seconds or less (AOR, 7.980; 95% CI, 1.998-31.870), and platelet count is 276x10 ³ It was found to be related to RIF in patients with / µl or higher (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 PTs less than 10.45 seconds (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 age of participants; 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; OR and 95% CI of each allele combination was 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.

Table 5: RIF = recurrent implantation failure; * PT 10.45 sec, aPTT 29.4 sec in the 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)

MiR-27a A> G (rs895819), miR-449b A> G (rs10061133) and miR-604 A> G (rs2368393) from DNA samples of subjects to provide the information necessary to predict the risk of repeated implantation failure in Korean women A method of determining a single base polymorphism selected from among. According to claim 1,
miR-423 C> A (rs6505162) A method characterized by further discriminating monobasic polymorphism. Predicting the risk of repeated implantation failure in Korean women, including means for detecting single-base polymorphism selected from miR-27a A> G (rs895819), miR-449b A> G (rs10061133) and miR-604 A> G (rs2368393) Dragon kit. According to claim 3,
kit further comprising means for detecting miR-423 C> A (rs6505162) monobasic polymorphism. According to claim 3,
The means for detecting the miR-27a A> G single base 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 a primer comprising a Dra III restriction enzyme -Restriction enzyme set,
The means for detecting the miR-449b A> G single base polymorphism is 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 primer comprising Bsm AI restriction enzyme. -Restriction enzyme set,
The means for detecting the miR-604 A> G single base polymorphism is a 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 primer comprising Bss SI restriction enzyme -A kit characterized by being a restriction enzyme set. The method of claim 4,
The means for detecting the miR-423 C> A single base polymorphism is a 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 a primer comprising an Aci I restriction enzyme -A kit characterized by being a restriction enzyme set.