WO2024054087A1 - Biomarker for diagnosing infertility in elderly women and use thereof - Google Patents

Biomarker for diagnosing infertility in elderly women and use thereof Download PDF

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WO2024054087A1
WO2024054087A1 PCT/KR2023/013493 KR2023013493W WO2024054087A1 WO 2024054087 A1 WO2024054087 A1 WO 2024054087A1 KR 2023013493 W KR2023013493 W KR 2023013493W WO 2024054087 A1 WO2024054087 A1 WO 2024054087A1
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protein
infertility
gene
level
mrna
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이재호
고정재
김명주
김영상
김유진
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차의과학대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/367—Infertility, e.g. sperm disorder, ovulatory dysfunction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • This relates to biomarkers for diagnosing infertility in elderly women and their uses.
  • ART In assisted reproductive technology
  • age is an important factor in the successful outcome of pregnancy.
  • IVF in vitro fertilization
  • Follicle development is a complex process regulated by several factors, either independent or dependent on gonadotropin.
  • Oocyte quality is determined by the somatic cells present in the ovary, including granulosa cells (GCs) and cumulus cells (CC), which are responsible for oocyte growth, development, and gradual acquisition of developmental capacity. It is related to functional ability.
  • GCs granulosa cells
  • CC cumulus cells
  • One aspect is to provide a composition for diagnosing infertility in elderly women.
  • Another aspect is to provide kits for diagnosing infertility in elderly women.
  • Another aspect is to provide informational methods for diagnosing infertility in elderly women.
  • Another aspect is to provide a method for screening for infertility treatments in elderly women.
  • compositions for diagnosing infertility in elderly women including an agent for measuring the level of low density lipoprotein receptor (LDLR) protein or mRNA of its gene.
  • the composition is an agent for measuring the level of mRNA of a protein or gene thereof selected from the group consisting of steroidogenic acute regulatory protein (StAR), adenylate cyclase (ADCY), and hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1). may additionally be included.
  • StAR steroidogenic acute regulatory protein
  • ADCY adenylate cyclase
  • HSD17B1 hydroxysteroid 17-beta dehydrogenase 1
  • the term "subfertility” refers to a case where it is difficult to have a child due to various reasons such as decreased uterine function and decreased ovarian function. As women age, the number and quality of eggs decreases, so women are unable to have children after the age of 35. Decreased ovarian function is considered the biggest cause of in
  • differentially expressed genes involved in the steroidogenic pathway were identified in cumulus cells from pregnant or non-pregnant women aged 33 or older or 40 or older.
  • LDLR and StAR significantly increases and the expression of ADCY and HSD17B1 significantly decreases in pregnant women regardless of age, making LDLR, StAR, ADCY, and HSD17B1 a molecular diagnosis that can diagnose and predict infertility. It was confirmed that it can be used as a marker. In addition, it was confirmed that the expression or activity of LDLR was increased by lovastatin.
  • infertility treatment promotes the development and ovulation of follicles by increasing the expression or activity of proteins involved in the steroid production pathway or their mRNA, and improves egg quality to achieve natural pregnancy, artificial insemination, or in vitro fertilization. It may be possible to successfully induce pregnancy through assisted reproductive technology such as surgery.
  • the term "elderly woman” refers to a pregnant woman who is medically over 35 years of age, such as over 35 years of age, over 36 years of age, over 37 years of age, over 38 years of age, over 39 years of age, or over 40 years of age. It may be a woman over the age of three. In one embodiment, the elderly woman may be a woman aged 35 or older but younger than 45.
  • the term "marker” refers to a substance that can be used for diagnosis by distinguishing between normal individuals and individuals with infertility. Polypeptides, proteins or nucleic acids, genes, lipids, glycolipids, etc. that are increased in individuals with infertility. It includes all organic biomolecules such as glycoproteins or sugars.
  • the grade of infertility can be confirmed by measuring the protein or mRNA level of the marker in a biological sample of an individual with infertility.
  • the subject may include a vertebrate, mammal, or human ( Homo sapiens ).
  • the human may be Korean.
  • the biological sample may be tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine.
  • mRNA level measurement refers to the process of determining the presence and expression level of mRNA of genes in a biological sample in order to diagnose vascular stenosis disease, and measures the amount of mRNA. Analysis methods for this include reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), and RNase protection assay (RPA). assay), Northern blotting, and DNA chip.
  • RT-PCR reverse transcription polymerase reaction
  • Competitive RT-PCR competitive reverse transcription polymerase reaction
  • Real-time RT-PCR real-time reverse transcription polymerase reaction
  • RNase protection assay RNase protection assay
  • the agent for measuring the mRNA level of the genes is a primer pair or probe, and since the nucleic acid information of the genes is known in GeneBank, etc., those skilled in the art can design primers or probes that specifically amplify specific regions of these genes based on the sequences. You can. Accordingly, an agent for measuring the mRNA level of the gene may include a primer pair, probe, or antisense nucleotide that specifically binds to the gene.
  • primer pair includes all combinations of primer pairs consisting of forward and reverse primers that recognize the target gene sequence, and specifically, it is a primer pair that provides analysis results with specificity and sensitivity. High specificity can be granted when the nucleic acid sequence of the primer is a sequence that is inconsistent with the non-target sequence present in the sample, so that the primer amplifies only the target gene sequence containing the complementary primer binding site and does not cause non-specific amplification. .
  • probe refers to a substance that can specifically bind to a target substance to be detected in a sample, and refers to a substance that can specifically confirm the presence of the target substance in the sample through said binding.
  • the type of probe molecule is not limited as it is a material commonly used in the art, but may preferably be PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA, or DNA. More specifically, the probe is a biomaterial that is derived from or similar to living organisms or includes those produced in vitro, such as enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, nerve cells, DNA, and RNA.
  • DNA may include cDNA, genomic DNA, and oligonucleotides
  • RNA may include genomic RNA, mRNA, and oligonucleotides
  • proteins may include antibodies, antigens, enzymes, peptides, etc.
  • antisense oligonucleotide is DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, and binds to the complementary sequence in the mRNA and inhibits the translation of the mRNA into protein. It works. Antisense oligonucleotide sequence refers to a DNA or RNA sequence that is complementary to the mRNA of the genes and is capable of binding to the mRNA. This may inhibit translation, translocation into the cytoplasm, maturation, or any other essential activity for overall biological functions of the gene mRNA.
  • the length of the antisense oligonucleotide may be 6 to 100 bases, preferably 8 to 60 bases, and more preferably 10 to 40 bases.
  • the antisense oligonucleotide can be synthesized in vitro using a conventional method and administered in vivo, or the antisense oligonucleotide can be synthesized in vivo.
  • One example of synthesizing antisense oligonucleotides in vitro is using RNA polymerase I.
  • One example of allowing antisense RNA to be synthesized in vivo is to transcribe the antisense RNA using a vector with the origin of the multiple cloning site (MCS) in the opposite direction. It is preferable that the antisense RNA has a translation stop codon within the sequence so that it is not translated into a peptide sequence.
  • protein level measurement refers to the process of confirming the presence and expression level of a marker protein for infertility diagnosis in a biological sample for the purpose of diagnosing infertility.
  • the amount of protein can be confirmed using an antibody that specifically binds to the marker protein, and also, for example, the protein expression level itself can be measured without using an antibody.
  • the protein level measurement or comparative analysis methods include protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption/Ionization Time of Flight Mass Spectrometry) analysis, and SELDI-TOF (Surface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry analysis, radioimmunoassay, radioimmunodiffusion method, Ouchteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry.
  • the agent for measuring the protein level may include an antibody that specifically binds to the estrogen receptor, MUCIN5AC, or aromatase protein.
  • the term “antibody” may refer to a specific protein molecule directed to an antigenic site.
  • the antibody binds specifically to the low density lipoprotein receptor (LDLR), steroidogenic acute regulatory protein (StAR), adenylate cyclase (ADCY), and/or hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1).
  • LDLR low density lipoprotein receptor
  • StAR steroidogenic acute regulatory protein
  • ADCY adenylate cyclase
  • HSD17B1 hydroxysteroid 17-beta dehydrogenase 1
  • Antibodies can be easily produced using techniques well known in the art.
  • Antibodies herein also include intact forms with two full-length light chains and two full-length heavy chains, as well as functional fragments of the antibody molecule.
  • Functional fragments of antibody molecules refer to fragments that possess at least an antigen-binding function, and include Fab, F(ab'), F(ab') 2, and Fv.
  • Another aspect provides a kit for diagnosing infertility in elderly women comprising the composition.
  • the specific details of the composition are as described above.
  • the kit is a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an enzyme-linked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit. ) It may be a kit.
  • RT-PCR reverse transcription polymerase chain reaction
  • DNA chip kit a DNA chip kit
  • ELISA enzyme-linked immunosorbent assay
  • MRM multiple reaction monitoring
  • the kit for diagnosing infertility in elderly women may further include one or more other component compositions, solutions, or devices suitable for the analysis method.
  • the diagnostic kit may be a diagnostic kit containing essential elements necessary to perform a reverse transcription polymerase reaction.
  • the reverse transcription polymerase reaction kit includes each primer pair specific for the marker gene.
  • the primer is a nucleotide having a sequence specific to the nucleic acid sequence of each gene, and is about 7 bp to 50 bp in length, more preferably about 10 bp to 30 bp in length. It may also include primers specific to the nucleic acid sequence of the control gene.
  • the reverse transcription polymerase reaction kit consists of a test tube or other suitable container, reaction buffer (pH and magnesium concentration varies), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, and RNAse inhibitor DEPC- It may include water (DEPC-water), sterilized water, etc.
  • a DNA chip kit may include a substrate to which a cDNA or oligonucleotide corresponding to a gene or fragment thereof is attached, and reagents, agents, enzymes, etc. for producing a fluorescent probe.
  • the substrate may also include cDNA or oligonucleotides corresponding to control genes or fragments thereof.
  • the kit may be a diagnostic kit containing essential elements necessary to perform LISA.
  • ELISA kits contain specific antibodies against these proteins. Antibodies have high specificity and affinity for each marker protein and almost no cross-reactivity to other proteins, and are either monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. Additionally, ELISA kits may include antibodies specific for control proteins. Other ELISA kits include reagents that can detect bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated with antibodies) and their substrates or those that can bind to antibodies. It may contain other substances, etc. Additionally, for example, the kit may be a rapid kit containing the essential elements necessary to perform a rapid test that can provide analysis results.
  • Rapid kits contain specific antibodies against proteins. Antibodies have high specificity and affinity for each marker protein and almost no cross-reactivity to other proteins, and are either monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. Additionally, the rapid kit may include antibodies specific for control proteins. In addition, the rapid kit includes reagents that can detect bound antibodies, such as a nitrocellulose membrane on which specific antibodies and secondary antibodies are immobilized, a membrane bound to antibody-bound beads, an absorption pad, and a sample pad. It may include substances, etc. Additionally, for example, the kit may be a multiple reaction monitoring (MRM) kit in MS/MS mode that includes the essential elements necessary to perform mass spectrometry.
  • MRM multiple reaction monitoring
  • SIM Select Ion Monitoring
  • MRM selects a specific ion from among the broken ions once more and uses another continuously connected MS source. This is a method of passing it through one more time, colliding with it, and then using the ions obtained from these.
  • Another aspect provides an informative method for diagnosing infertility in elderly women, including measuring the mRNA level of low density lipoprotein receptor (LDLR) protein or its gene.
  • the method includes measuring the mRNA level of a protein or gene thereof selected from the group consisting of steroidogenic acute regulatory protein (StAR), adenylate cyclase (ADCY), and hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1). may additionally be included.
  • StAR steroidogenic acute regulatory protein
  • ADCY adenylate cyclase
  • HSD17B1 hydroxysteroid 17-beta dehydrogenase 1
  • the infertility occurs because the level of gene expression or the expression level of the corresponding protein decreases in an individual with infertility compared to a normal control group (an individual capable of normal pregnancy, or a sample unrelated to infertility from the same individual), so when the level decreases, It can be diagnosed as infertility.
  • the method according to one aspect may include determining infertility when the measured protein expression level or gene expression level is lower than the protein expression level or gene expression level of the normal control sample.
  • Another aspect includes contacting a test substance with a low density lipoprotein receptor (LDLR) protein; and selecting a test substance that increases the expression or activity level of the low density lipoprotein receptor (LDLR) protein as a treatment for infertility compared to an untreated control group.
  • LDLR low density lipoprotein receptor
  • test substance for example, a drug candidate, a test compound, or a test composition may include a small molecule compound, an antibody, an antisense nucleotide, a short interfering RNA, a short hairpin RNA, a nucleic acid, a protein, a peptide, Other extracts or natural products may be included.
  • the contact may be performed in vitro. For example, transforming a cell with a vector expressing the proteins; It may include treating the transformed cells with a drug candidate.
  • the expression of the marker is decreased in infertility, so a candidate substance that increases the expression or activity of the marker can be usefully used in the treatment of infertility.
  • a composition according to one aspect can not only non-invasively diagnose infertility in elderly women, but can also be a useful new target for infertility treatment. In addition, it promotes follicular development and ovulation by increasing the expression or activity of biomarkers according to one aspect, and improves egg quality to successfully induce pregnancy through natural pregnancy or assisted reproductive technology such as artificial insemination and in vitro fertilization. can do.
  • Figure 1a shows hierarchical clustering of data using normalized values (log2) based on age and ancestry.
  • Figure 1B shows a multidimensional scaling analysis plot of the data using normalized values (log2) based on age and inseam.
  • Figure 1C shows a heatmap of the hierarchical clustering graph of pregnant (young and older) and non-pregnant (older and younger) groups.
  • Figure 2a shows the results confirming the difference in ovarian steroid hormones synthesized in the young and elderly pregnant group and the young and elderly non-pregnant group.
  • Figure 2b shows the results of KEGG pathway analysis to identify ovarian-specific signaling pathways.
  • Figure 3A shows LDLR, StAR, ADCY, and HSD17B1 gene expression levels in young adult pregnant and young adult non-pregnant groups.
  • Figure 3B shows LDLR, StAR, ADCY, and HSD17B1 gene expression levels in older pregnancy and older non-pregnant groups.
  • Example 1 Collection of cumulus cells (CC) and confirmation of oocyte characteristics
  • age-related anti-Müllerian Hormone was significantly decreased in the elderly pregnancy group compared to the young pregnancy group.
  • clinical outcomes did not show significant differences within each age group as a function of BMI, number of previous IVF cycles, or basal FSH levels.
  • Even when all IVF patients were stimulated using the GnRH antagonist protocol there was no significant difference in the dose of gonadotropin administered.
  • There were no significant differences in oocyte characteristics such as oocyte retrieval, maturation rate, and fertilization rate between groups.
  • pregnancy was associated with oocyte and embryo quality in both young and old groups. It was confirmed that the clinical pregnancy group had a significantly higher number of high-quality embryos and blastocyst formation rate compared to the non-pregnancy group.
  • Example 1 whole-transcriptome analysis was performed to confirm differences in mRNA expression profiles between the pregnant and non-pregnant groups and the young and elderly groups.
  • mRNA from the cumulus cells isolated in Example 1 was extracted, and transcriptome data was obtained for the extracted mRNA using next-generation sequencing (NGS).
  • NGS next-generation sequencing
  • mRNA was isolated from the four groups of cumulus cells selected in Example 1 using the DynaBeads mRNA DIRECT Kit (Life Technologies, Oslo, Norway), and NanoDrop ND-1000 spectrophotometer (Nyxor Biotech, Paris, France) was used. and quantified.
  • mRNA was sequenced using a MiSeq sequencer (MiSeq System, Illumina, San Diego, CA, USA), and a sample sheet was prepared to provide detailed information. Afterwards, bioinformatics analysis was performed on the whole-transcriptome raw data obtained from NGS.
  • MiSeq sequencer MiSeq System, Illumina, San Diego, CA, USA
  • Figure 1a shows hierarchical clustering of data using normalized values (log2) based on age and ancestry.
  • Figure 1B shows a multidimensional scaling analysis plot of the data using normalized values (log2) based on age and inseam.
  • Figure 1C shows a heatmap of the hierarchical clustering graph of pregnant (young and older) and non-pregnant (older and younger) groups.
  • the gene expression pattern of the pregnant group was significantly different from that of the non-pregnant group.
  • the young non-pregnant group and the old non-pregnant group showed similar genetic profile ratios.
  • similar z-scores were evident within the non-pregnant groups (both young and old).
  • gene expression profiles were found to be at similarly high levels between pregnancy groups, but there were differences in expression patterns between the young pregnancy group and the elderly pregnancy group.
  • the non-pregnant group the young and elderly groups showed similar expression patterns.
  • the elderly pregnant group showed stronger gene expression activity than the non-pregnant group, and the young and elderly non-pregnant groups had lower gene expression profiles compared to the young and elderly pregnant groups.
  • DEG Differential expression gene analysis was performed to identify genes showing expression changes of more than twofold among the 17,317 genes derived in Example 2-2. Specifically, log2(FPKM+1) values were calculated and normalized using the quantile method. Transcripts with p-value ⁇ 0.05 and fold-change value > 2 were included as differentially expressed genes (total 1366).
  • hierarchical clustering analysis was performed using complete linkage and Euclidean distance of similarity measures. The top 10 gene ontology terms are displayed in ascending order of p-value. The top ranked up- and down-regulated gene lists were then analyzed and the up- and down-regulated gene clusters were plotted as a function of age and pregnancy outcome, respectively. Each group was examined according to age and fertility.
  • SNORD159, ZNF117, ITGA2, LOC102723753, SFRP4, and NME1-NME2 were upregulated, and NGFR, ADNP, SLC44A2, LOC728715, HMGB2, PLCD1, SDSL, ACTA1, It was confirmed that DUSP15 and ZMIZ1-AS1 were commonly downregulated.
  • the upregulated genes eg, ZNF117, ITGA2 were confirmed to be genes related to ovarian functions such as cumulus cell proliferation, differentiation, and steroid production.
  • Example 3 Using the results of differential expression gene analysis in Example 3, youth vs. Old age and pregnancy vs. The impact of the normalization method was evaluated on both non-pregnant real and virtual data. Specifically, the DEG data obtained in Example 3 was uploaded to the DAVID program (http://david.abcc.ncifcrf.gov/tools.jsp, v.6.8 web software). Functional categories were clustered using a functional annotation clustering tool, and representative Gene Ontology categories were identified.
  • a database of GO/canonical pathways and feature gene sets was generated using Metascape (http://david.abcc.ncifcrf.gov/tools.jsp, v.6.8 web software) to identify significantly enriched terms in each gene set. was used to infer.
  • Metascape http://david.abcc.ncifcrf.gov/tools.jsp, v.6.8 web software
  • KEGG Korean of Genes and Genomes pathway analysis was performed to identify ovary-specific signaling pathways in the gene enrichment data of DEGs with p-value ⁇ 0.05.
  • the threshold was set at corrected Fisher Exact P-value (EASE score) ⁇ 0.05.
  • Figure 2a shows the results confirming the difference in ovarian steroid hormones synthesized in the young and elderly pregnant group and the young and elderly non-pregnant group.
  • Figure 2b shows the results of KEGG pathway analysis to identify ovarian-specific signaling pathways.
  • the up-regulated differentially expressed genes in cumulus cells were related to the ovarian steroidogenic pathway (LDLR and StAR), and the down-regulated differentially expressed genes were related to pregnancy status (ADCY and 17 ⁇ -sideroxy). It was confirmed that it was related to steroid dehydrogenase (17 beta-hydroxysteroid dehydrogenase 1, HSD17B1).
  • Real-time qPCR was performed to verify data for young and older pregnancy groups. Specifically, mRNA was purified using the DynaBeads mRNA DIRECT Kit. Total RNA was reverse transcribed into cDNA using AccuPower® CycleScript RT PreMix (Bioneer) and poly-dT. qPCR was performed using AccuPower Taq PCR PreMix (Bioneer) in a spectrofluorometric thermal cycler (SimpliAmp Thermal Cycler; Thermo Fisher Scientific). Based on the KEGG signaling pathway, up- and down-regulated target genes among genes involved in ovarian steroidogenesis as a function of age and pregnancy outcome were selected.
  • Figure 3A shows LDLR, StAR, ADCY, and HSD17B1 gene expression levels in young adult pregnant and young adult non-pregnant groups.
  • Figure 3B shows LDLR, StAR, ADCY, and HSD17B1 gene expression levels in older pregnancy and older non-pregnant groups.
  • genes involved in the steroidogenic pathway including LDLR and StAR proteins, are upregulated in cumulus cells of the pregnant group, and LDLR and StAR can be used as molecular diagnostic markers to diagnose and predict infertility in elderly women.

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Abstract

The present invention relates to a biomarker for diagnosing infertility in elderly women and a use thereof, and can not only non-invasively diagnose infertility in elderly women, but can also be a useful new target for infertility treatment.

Description

고령 여성의 난임 진단을 위한 바이오마커 및 이의 용도Biomarkers for diagnosing infertility in elderly women and their uses
본 출원은 2022년 9월 8일 출원된 대한민국 특허출원 제 10-2022-0114366호 를 우선권으로 주장하고, 상기 명세서 전체는 본 출원의 참고문헌이다.This application claims priority to Republic of Korea Patent Application No. 10-2022-0114366 filed on September 8, 2022, and the entire specification is a reference to this application.
고령 여성의 난임 진단을 위한 바이오마커 및 이의 용도에 관한 것이다.This relates to biomarkers for diagnosing infertility in elderly women and their uses.
보조생식기술(assisted reproductive technology, ART)에서, 나이는 임신의 성공적인 결과에 중요한 요소이다. 구체적으로, 40세 이상의 난임 여성의 체외수정(in vitro fertilization, IVF) 시술 성공률은 매우 낮을 뿐만 아니라, 보조생식기술에서 고령화에 따른 난모세포 및 배아의 능력 감소로 인한 불임의 특별한 치료방법이 없다. 난포 발달은 생식샘자극호르몬(gonadotropin)에 독립적이거나 의존하는 등 여러 요인에 의해 복잡한 과정으로 조절된다. 난모세포(oocyte)의 질은 난모세포의 성장, 발달 및 발달 능력의 점진적인 획득을 담당하는 과립막세포(granulosa cells, GCs) 및 난구세포(cumulus cell, CC)를 포함하여 난소에 존재하는 체세포의 기능적 능력과 관련이 있다. In assisted reproductive technology (ART), age is an important factor in the successful outcome of pregnancy. Specifically, not only is the success rate of in vitro fertilization (IVF) treatment for infertile women over 40 years old very low, but there is no special treatment method for infertility due to the decline in oocyte and embryo capacity due to aging in assisted reproductive technology. Follicle development is a complex process regulated by several factors, either independent or dependent on gonadotropin. Oocyte quality is determined by the somatic cells present in the ovary, including granulosa cells (GCs) and cumulus cells (CC), which are responsible for oocyte growth, development, and gradual acquisition of developmental capacity. It is related to functional ability.
한편, 체외수정시술을 경험한 고령의 여성 중에서 임신에 성공하여 정상 출산을 한 이력이 있는 여성은 임신 실패를 경험한 여성과 비교하여 난포 발달 동안 과립막세포 및 난구세포의 반응이 다르게 나타날 수 있다. 40세 이상 여성의 경우, 난모세포의 발달 능력이 점차 감소하나, 현재까지 고령 여성의 임신 실패를 완화할 수 있는 중요한 요소가 확인된 바 없다. Meanwhile, among older women who have experienced in vitro fertilization, those who have successfully become pregnant and had a live birth may show different responses of granulosa cells and cumulus cells during follicular development compared to women who have experienced a failed pregnancy. . In women over 40 years of age, the ability to develop oocytes gradually decreases, but to date, no important factors that can alleviate pregnancy failure in elderly women have been identified.
따라서, 노령 난소의 저하된 기능을 개선하고, 이를 통하여 임신 성공률 및 유전자 변형이 낮은 양질의 수정란을 획득하기 위하여 치료법을 개발할 필요가 있다.Therefore, there is a need to develop treatments to improve the deteriorated function of aged ovaries and thereby obtain high-quality fertilized eggs with low pregnancy success rate and low genetic modification.
일 양상은 고령 여성의 난임 진단용 조성물을 제공하는 것이다. One aspect is to provide a composition for diagnosing infertility in elderly women.
다른 양상은 고령 여성의 난임 진단용 키트를 제공하는 것이다. Another aspect is to provide kits for diagnosing infertility in elderly women.
다른 양상은 고령 여성의 난임 진단을 위한 정보제공방법을 제공하는 것이다. Another aspect is to provide informational methods for diagnosing infertility in elderly women.
다른 양상은 고령 여성의 난임 치료제를 스크리닝하는 방법을 제공하는 것이다.Another aspect is to provide a method for screening for infertility treatments in elderly women.
일 양상은 LDLR(low density lipoprotein receptor) 단백질 또는 이의 유전자의 mRNA의 수준을 측정하는 제제를 포함하는 고령 여성의 난임 진단용 조성물을 제공한다. 일 구체예에 있어서, 상기 조성물은 StAR(steroidogenic acute regulatory protein), ADCY(adenylate cyclase), HSD17B1(hydroxysteroid 17-beta dehydrogenase 1)로 구성된 군에서 선택되는 단백질 또는 이의 유전자의 mRNA의 수준을 측정하는 제제를 추가로 포함할 수 있다. 본 명세서에서 용어, "난임(subfertility)"이란 자궁 기능 저하, 난소 기능 저하 등 다양한 원인으로 아이를 가지기 어려운 경우로서, 여성은 나이가 들수록 난자의 수가 감소하고 질이 나빠지므로 만 35세 이후에는 여성의 난소 기능 저하가 난임의 가장 큰 원인으로 지목된다. One aspect provides a composition for diagnosing infertility in elderly women, including an agent for measuring the level of low density lipoprotein receptor (LDLR) protein or mRNA of its gene. In one embodiment, the composition is an agent for measuring the level of mRNA of a protein or gene thereof selected from the group consisting of steroidogenic acute regulatory protein (StAR), adenylate cyclase (ADCY), and hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1). may additionally be included. In this specification, the term "subfertility" refers to a case where it is difficult to have a child due to various reasons such as decreased uterine function and decreased ovarian function. As women age, the number and quality of eggs decreases, so women are unable to have children after the age of 35. Decreased ovarian function is considered the biggest cause of infertility.
일 실시예에서는 33세 이상 또는 40세 이상의 임신 또는 비-임신 여성의 난구세포에서 스테로이드 생성 경로에 관여하는 차등 발현 유전자(LDLR, StAR, ADCY, HSD17B1)를 확인하였다. 구체적으로, 연령에 관계 없이 임신 여성에서 LDLR, StAR의 발현이 유의하게 증가하고, ADCY, HSD17B1의 발현이 유의하게 감소함으로써, LDLR, StAR, ADCY, HSD17B1를 난임 여부를 진단 및 예측할 수 있는 분자 진단 마커로 활용할 수 있음을 확인하였다. 또한, 로바스타틴에 의하여 LDLR의 발현 또는 활성이 증가하는 것을 확인하였다. In one example, differentially expressed genes (LDLR, StAR, ADCY, HSD17B1) involved in the steroidogenic pathway were identified in cumulus cells from pregnant or non-pregnant women aged 33 or older or 40 or older. Specifically, the expression of LDLR and StAR significantly increases and the expression of ADCY and HSD17B1 significantly decreases in pregnant women regardless of age, making LDLR, StAR, ADCY, and HSD17B1 a molecular diagnosis that can diagnose and predict infertility. It was confirmed that it can be used as a marker. In addition, it was confirmed that the expression or activity of LDLR was increased by lovastatin.
따라서, 일 구체예에 있어서, 난임 치료란 스테로이드 생성 경로에 관여하는 단백질 또는 이의 mRNA의 발현 또는 활성을 증가시킴으로써 난포의 발달 및 배란을 촉진하고, 난자의 질을 개선하여 자연 임신 또는 인공수정, 시험관 시술과 같은 보조생식기술에 의한 임신을 성공적으로 유도하는 것일 수 있다. Therefore, in one embodiment, infertility treatment promotes the development and ovulation of follicles by increasing the expression or activity of proteins involved in the steroid production pathway or their mRNA, and improves egg quality to achieve natural pregnancy, artificial insemination, or in vitro fertilization. It may be possible to successfully induce pregnancy through assisted reproductive technology such as surgery.
본 명세서에서 용어, "고령 여성"이란 의학적으로 임산부가 만 35세 이상인 경우를 의미하므로, 만 35세 이상, 만 36세 이상, 만 37세 이상, 만 38세 이상, 만 39세 이상 또는 만 40세 이상의 여성인 것일 수 있다. 일 구체예에 있어서, 상기 고령 여성은 만 35세 이상 내지 만 45세 미만인 여성일 수 있다. In this specification, the term "elderly woman" refers to a pregnant woman who is medically over 35 years of age, such as over 35 years of age, over 36 years of age, over 37 years of age, over 38 years of age, over 39 years of age, or over 40 years of age. It may be a woman over the age of three. In one embodiment, the elderly woman may be a woman aged 35 or older but younger than 45.
본 명세서에서 용어, "마커(marker)"란 정상군 개체와 난임을 가진 개체를 구분하여 진단할 수 있는 물질로, 난임을 가지는 개체에서 증가를 보이는 폴리펩티드, 단백질 또는 핵산, 유전자, 지질, 당지질, 당단백질 또는 당 등과 같은 유기 생체 분자들을 모두 포함한다. 특히 상기 난임을 가지는 개체의 생물학적 시료에서 상기 마커의 단백질 또는 이의 mRNA 수준을 측정함으로써 난임의 등급을 확인할 수 있다. 구체적으로, 상기 개체는 척추동물, 포유동물, 또는 인간 (Homo sapiens)을 포함할 수 있다. 예를 들면, 상기 인간은 한국인일 수 있다. 또한, 상기 생물학적 시료는 조직, 세포, 전혈, 혈청, 혈장, 타액, 객담, 뇌척수액 또는 뇨일 수 있다.As used herein, the term "marker" refers to a substance that can be used for diagnosis by distinguishing between normal individuals and individuals with infertility. Polypeptides, proteins or nucleic acids, genes, lipids, glycolipids, etc. that are increased in individuals with infertility. It includes all organic biomolecules such as glycoproteins or sugars. In particular, the grade of infertility can be confirmed by measuring the protein or mRNA level of the marker in a biological sample of an individual with infertility. Specifically, the subject may include a vertebrate, mammal, or human ( Homo sapiens ). For example, the human may be Korean. Additionally, the biological sample may be tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine.
본 명세서에 용어, "mRNA 수준 측정"이란 혈관 협착 질환을 진단하기 위하여 생물학적 시료에서 유전자들의 mRNA 존재 여부와 발현 정도를 확인하는 과정으로 mRNA의 양을 측정한다. 이를 위한 분석 방법으로는 역전사 중합효소반응(RT-PCR), 경쟁적 역전사 중합효소반응(Competitive RT-PCR), 실시간 역전사 중합효소반응(Real-time RT-PCR), RNase 보호 분석법(RPA; RNase protection assay), 노던 블랏팅(Northern blotting), DNA 칩 등이 있다. As used herein, the term “mRNA level measurement” refers to the process of determining the presence and expression level of mRNA of genes in a biological sample in order to diagnose vascular stenosis disease, and measures the amount of mRNA. Analysis methods for this include reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), and RNase protection assay (RPA). assay), Northern blotting, and DNA chip.
상기 유전자의 mRNA 수준을 측정하는 제제는 프라이머 쌍 또는 프로브이며, 상기 유전자들의 핵산 정보가 GeneBank 등에 알려져 있으므로 당업자는 상기 서열을 바탕으로 이들 유전자의 특정 영역을 특이적으로 증폭하는 프라이머 또는 프로브를 디자인할 수 있다. 따라서, 상기 유전자의 mRNA 수준을 측정하는 제제는 상기 유전자에 특이적으로 결합하는 프라이머 쌍, 프로브 또는 안티센스 뉴클레오티드를 포함할 수 있다.The agent for measuring the mRNA level of the genes is a primer pair or probe, and since the nucleic acid information of the genes is known in GeneBank, etc., those skilled in the art can design primers or probes that specifically amplify specific regions of these genes based on the sequences. You can. Accordingly, an agent for measuring the mRNA level of the gene may include a primer pair, probe, or antisense nucleotide that specifically binds to the gene.
본 명세서에서 용어, "프라이머 쌍"은 표적 유전자 서열을 인지하는 정방향 및 역방향의 프라이머로 이루어진 모든 조합의 프라이머쌍을 포함하고, 상세하게는 특이성 및 민감성을 가지는 분석 결과를 제공하는 프라이머 쌍이다. 프라이머의 핵산 서열이 시료내 존재하는 비-표적 서열과 불일치하는 서열이어서, 상보적인 프라이머 결합 부위를 함유하는 표적 유전자 서열만 증폭하고 비특이적 증폭을 유발하지 않는 프라이머일 때, 높은 특이성을 부여할 수 있다.As used herein, the term “primer pair” includes all combinations of primer pairs consisting of forward and reverse primers that recognize the target gene sequence, and specifically, it is a primer pair that provides analysis results with specificity and sensitivity. High specificity can be granted when the nucleic acid sequence of the primer is a sequence that is inconsistent with the non-target sequence present in the sample, so that the primer amplifies only the target gene sequence containing the complementary primer binding site and does not cause non-specific amplification. .
본 명세서에서 용어, "프로브"란 시료 내의 검출하고자 하는 표적 물질과 특이적으로 결합할 수 있는 물질을 의미하며, 상기 결합을 통하여 특이적으로 시료 내의 표적 물질의 존재를 확인할 수 있는 물질을 의미한다. 프로브 분자의 종류는 당업계에서 통상적으로 사용되는 물질로서 제한은 없으나, 바람직하게는 PNA (peptide nucleic acid), LNA (locked nucleic acid), 펩타이드, 폴리펩타이드, 단백질, RNA 또는 DNA 일 수 있다. 보다 구체적으로, 상기 프로브는 바이오 물질로서 생물에서 유래되거나 이와 유사한 것 또는 생체외에서 제조된 것을 포함하는 것으로 예를 들어, 효소, 단백질, 항체, 미생물, 동식물 세포 및 기관, 신경세포, DNA, 및 RNA일 수 있으며, DNA는 cDNA, 게놈 DNA, 올리고뉴클레오타이드를 포함하며, RNA는 게놈 RNA, mRNA, 올리고뉴클레오타이드를 포함하며, 단백질의 예로는 항체, 항원, 효소, 펩타이드 등을 포함할 수 있다.As used herein, the term "probe" refers to a substance that can specifically bind to a target substance to be detected in a sample, and refers to a substance that can specifically confirm the presence of the target substance in the sample through said binding. . The type of probe molecule is not limited as it is a material commonly used in the art, but may preferably be PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA, or DNA. More specifically, the probe is a biomaterial that is derived from or similar to living organisms or includes those produced in vitro, such as enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, nerve cells, DNA, and RNA. DNA may include cDNA, genomic DNA, and oligonucleotides, RNA may include genomic RNA, mRNA, and oligonucleotides, and examples of proteins may include antibodies, antigens, enzymes, peptides, etc.
본 명세서에서 용어, "안티센스 올리고뉴클레오티드"는 특정 mRNA의 서열에 상보적인 핵산 서열을 함유하고 있는 DNA 또는 RNA 또는 이들의 유도체로서, mRNA 내의 상보적인 서열에 결합하여 mRNA의 단백질로의 번역을 저해하는 작용을 한다. 안티센스 올리고뉴클레오티드 서열은 상기 유전자들의 mRNA에 상보적이고 상기 mRNA에 결합할 수 있는 DNA 또는 RNA 서열을 의미한다. 이는 상기 유전자 mRNA의 번역, 세포질 내로의 전위(translocation), 성숙(maturation) 또는 다른 모든 전체적인 생물학적 기능에 대한 필수적인 활성을 저해할 수 있다. 안티센스 올리고뉴클레오티드의 길이는 6 내지 100 염기, 바람직하게는 8 내지 60 염기, 보다 바람직하게는 10 내지 40 염기일 수 있다. 상기 안티센스 올리고뉴클레오티드는 통상의 방법으로 시험관 내에서 합성되어 생체 내로 투여하거나 생체 내에서 안티센스 올리고뉴클레오티드가 합성되도록 할 수 있다. 시험관 내에서 안티센스 올리고뉴클레오티드를 합성하는 한가지 예는 RNA 중합효소 I를 이용하는 것이다. 생체 내에서 안티센스 RNA가 합성되도록 하는 한 가지 예는 다중클로닝부위(MCS)의 기원이 반대 방향에 있는 벡터를 사용하여 안티센스 RNA가 전사되도록 하는 것이다. 상기 안티센스 RNA는 서열 내에 번역 중지 코돈이 존재하도록 하여 펩타이드 서열로 번역되지 않도록 하는 것이 바람직하다.As used herein, the term "antisense oligonucleotide" is DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, and binds to the complementary sequence in the mRNA and inhibits the translation of the mRNA into protein. It works. Antisense oligonucleotide sequence refers to a DNA or RNA sequence that is complementary to the mRNA of the genes and is capable of binding to the mRNA. This may inhibit translation, translocation into the cytoplasm, maturation, or any other essential activity for overall biological functions of the gene mRNA. The length of the antisense oligonucleotide may be 6 to 100 bases, preferably 8 to 60 bases, and more preferably 10 to 40 bases. The antisense oligonucleotide can be synthesized in vitro using a conventional method and administered in vivo, or the antisense oligonucleotide can be synthesized in vivo. One example of synthesizing antisense oligonucleotides in vitro is using RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to transcribe the antisense RNA using a vector with the origin of the multiple cloning site (MCS) in the opposite direction. It is preferable that the antisense RNA has a translation stop codon within the sequence so that it is not translated into a peptide sequence.
본 명세서에 용어, "단백질 수준 측정"이란 난임 진단을 위하여 생물학적 시료에서 난임 진단용 마커 단백질의 존재 여부와 발현 정도를 확인하는 과정이다. 상기 마커 단백질에 대하여 특이적으로 결합하는 항체를 이용하여 단백질의 양을 확인할 수 있으며, 또한, 예를 들면, 항체를 이용하지 않고 단백질 발현 수준 자체를 측정할 수 있다. As used herein, the term “protein level measurement” refers to the process of confirming the presence and expression level of a marker protein for infertility diagnosis in a biological sample for the purpose of diagnosing infertility. The amount of protein can be confirmed using an antibody that specifically binds to the marker protein, and also, for example, the protein expression level itself can be measured without using an antibody.
상기 단백질 수준 측정 또는 비교 분석 방법으로는 단백질 칩 분석, 면역측정법, 리간드 바인딩 어세이, MALDI-TOF(Matrix Desorption/Ionization Time of Flight Mass Spectrometry)분석, SELDI-TOF(Surface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry)분석, 방사선 면역분석, 방사 면역 확산법, 오우크테로니 면역 확산법, 로케트 면역전기영동, 조직면역 염색, 보체 고정 분석법, 2차원 전기영동 분석, 액상 크로마토그래피-질량분석(liquid chromatography-Mass Spectrometry, LC-MS), LC-MS/MS(liquid chromatography-Mass Spectrometry/ Mass Spectrometry), 웨스턴 블랏, 및 ELISA(enzyme linked immunosorbentassay) 등이 있다. 따라서, 상기 단백질 수준을 측정하는 제제는 에스트로겐 수용체, MUCIN5AC, 또는 아로마타제 단백질에 특이적으로 결합하는 항체를 포함할 수 있다.The protein level measurement or comparative analysis methods include protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption/Ionization Time of Flight Mass Spectrometry) analysis, and SELDI-TOF (Surface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry analysis, radioimmunoassay, radioimmunodiffusion method, Ouchteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry. Mass Spectrometry, LC-MS), LC-MS/MS (liquid chromatography-Mass Spectrometry/Mass Spectrometry), Western blot, and ELISA (enzyme linked immunosorbent assay). Accordingly, the agent for measuring the protein level may include an antibody that specifically binds to the estrogen receptor, MUCIN5AC, or aromatase protein.
본 명세서에서 용어, "항체"는 항원성 부위에 대해서 지시되는 특이적인 단백질 분자를 의미할 수 있다. 본 발명의 목적상, 항체는 상기 LDLR(low density lipoprotein receptor), StAR(steroidogenic acute regulatory protein), ADCY(adenylate cyclase), 및/또는 HSD17B1(hydroxysteroid 17-beta dehydrogenase 1)에 대해 특이적으로 결합하는 항체를 의미하며, 다클론 항체, 단클론 항체 및 재조합 항체를 모두 포함한다. 항체를 생성하는 것은 당업계에 널리 공지된 기술을 이용하여 용이하게 제조할 수 있다. 또한 본 명세서의 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 완전한 형태뿐만 아니라, 항체 분자의 기능적인 단편을 포함한다. 항체분자의 기능적인 단편이란 적어도 항원 결합 기능을 보유하고 있는 단편을 뜻하며, Fab, F(ab'), F(ab') 2 및 Fv 등이 있다.As used herein, the term “antibody” may refer to a specific protein molecule directed to an antigenic site. For the purpose of the present invention, the antibody binds specifically to the low density lipoprotein receptor (LDLR), steroidogenic acute regulatory protein (StAR), adenylate cyclase (ADCY), and/or hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1). It refers to antibodies and includes polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. Antibodies can be easily produced using techniques well known in the art. Antibodies herein also include intact forms with two full-length light chains and two full-length heavy chains, as well as functional fragments of the antibody molecule. Functional fragments of antibody molecules refer to fragments that possess at least an antigen-binding function, and include Fab, F(ab'), F(ab') 2, and Fv.
다른 양상은 상기 조성물을 포함하는 고령 여성의 난임 진단용 키트를 제공한다. 상기 조성물의 구체적인 내용은 전술한 바와 같다. Another aspect provides a kit for diagnosing infertility in elderly women comprising the composition. The specific details of the composition are as described above.
일 구체예에 있어서, 상기 키트는 RT-PCR(Reverse transcription polymerase chain reaction) 키트, DNA 칩 키트, ELISA(Enzyme-linked immunosorbent assay) 키트, 단백질 칩 키트, 래피드(rapid) 키트 또는 MRM(Multiple reaction monitoring) 키트일 수 있다.In one embodiment, the kit is a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an enzyme-linked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit. ) It may be a kit.
또한, 상기 고령 여성의 난임 진단용 키트는 분석 방법에 적합한 한 종류 또는 그 이상의 다른 구성성분 조성물, 용액 또는 장치를 더 포함하여 구성될 수 있다. 예를 들면, 상기 진단용 키트는 역전사 중합효소반응을 수행하기 위해 필요한 필수 요소를 포함하는 진단용 키트일 수 있다. 역전사 중합효소반응 키트는 마커 유전자에 대한 특이적인 각각의 프라이머 쌍을 포함한다. 프라이머는 상기 각 유전자의 핵산서열에 특이적인 서열을 가지는 뉴클레오타이드로서, 약 7 bp 내지 50 bp의 길이, 보다 바람직하게는 약 10 bp 내지 30 bp 의 길이이다. 또한 대조군 유전자의 핵산 서열에 특이적인 프라이머를 포함할 수 있다. 그외 역전사 중합효소반응 키트는 테스트 튜브 또는 다른 적절한 컨테이너, 반응 완충액(pH 및 마그네슘 농도는 다양), 데옥시뉴클레오타이드(dNTPs), Taq-폴리머라아제 및 역전사효소와 같은 효소, DNAse, RNAse 억제제 DEPC-수(DEPC-water), 멸균수등을 포함할 수 있다. 또한, 예를 들면, DNA 칩 키트는 유전자 또는 그의 단편에 해당하는 cDNA 또는 올리고뉴클레오티드(oligonucleotide)가 부착되어 있는 기판, 및 형광표식 프로브를 제작하기 위한 시약, 제제, 효소 등을 포함할 수 있다. 또한 기판은 대조군 유전자 또는 그의 단편에 해당하는 cDNA 또는 올리고뉴클레오티드를 포함할 수 있다. 또한, 예를 들면, 상기 키트는 LISA를 수행하기 위해 필요한 필수 요소를 포함하는 진단 키트일 수 있다. ELISA 키트는 상기 단백질에 대한 특이적인 항체를 포함한다. 항체는 각 마커 단백질에 대한 특이성 및 친화성이 높고 다른 단백질에 대한 교차 반응성이 거의 없는 항체로, 단클론 항체, 다클론 항체 또는 재조합 항체이다. 또한 ELISA 키트는 대조군 단백질에 특이적인 항체를 포함할 수 있다. 그 외 ELISA 키트는 결합된 항체를 검출할 수 있는 시약, 예를 들면, 표지된 2차 항체, 발색단(chromophores), 효소(예: 항체와 컨주게이트됨) 및 그의 기질 또는 항체와 결합할 수 있는 다른 물질 등을 포함할 수 있다. 또한, 예를 들면, 상기 키트는 분석결과를 알 수 있는 신속한 테스트를 수행하기 위해 필요한 필수 요소를 포함하는 래피드(rapid) 키트 일 수 있다. 래피드 키트는 단백질에 대한 특이적인 항체를 포함한다. 항체는 각 마커 단백질에 대한 특이성 및 친화성이 높고 다른 단백질에 대한 교차 반응성이 거의 없는 항체로, 단클론 항체, 다클론 항체 또는 재조합 항체이다. 또한 래피드 키트는 대조군 단백질에 특이적인 항체를 포함할 수 있다. 그 외 래피드 키트는 결합된 항체를 검출할 수 있는 시약, 예를 들면, 특이항체와 2차 항체가 고정된 나이트로 셀룰로오스 멤브레인, 항체가 결합된 비드에 결합된 멤브레인, 흡수패드와 샘플 패드 등 다른 물질 등을 포함할 수 있다. 또한, 예를 들면, 상기 키트는 질량 분석을 수행하기 위해 필요한 필수 요소를 포함하는 MS/MS 모드인 MRM(Multiple reaction monitoring) 키트일 수 있다. SIM(Selected Ion Monitoring)이 질량분석기의 소스 부분에서 한 번 충돌하여 생긴 이온을 이용하는 방법인 반면, MRM은 상기 한 번 깨진 이온 중에서 특정 이온을 한 번 더 선택하여 연속적으로 연결된 또 다른 MS의 소스를 한 번 더 통과시켜 충돌시킨 후 이 중에서 얻은 이온들을 이용하는 방법이다. 상기 MRM(Multiple reaction monitoring) 분석 방법들을 통하여, 정상 대조군, 또는 동일 개체의 단백질 발현 수준과 난임이 있는 개체에서의 단백질 발현 수준을 비교할 수 있고, 또한, 난임 관련 질환 마커 유전자에서 단백질로의 유의한 발현량 증감여부를 판단하여, 난임의 발병 여부를 진단할 수 있다.In addition, the kit for diagnosing infertility in elderly women may further include one or more other component compositions, solutions, or devices suitable for the analysis method. For example, the diagnostic kit may be a diagnostic kit containing essential elements necessary to perform a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit includes each primer pair specific for the marker gene. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each gene, and is about 7 bp to 50 bp in length, more preferably about 10 bp to 30 bp in length. It may also include primers specific to the nucleic acid sequence of the control gene. In addition, the reverse transcription polymerase reaction kit consists of a test tube or other suitable container, reaction buffer (pH and magnesium concentration varies), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, and RNAse inhibitor DEPC- It may include water (DEPC-water), sterilized water, etc. Additionally, for example, a DNA chip kit may include a substrate to which a cDNA or oligonucleotide corresponding to a gene or fragment thereof is attached, and reagents, agents, enzymes, etc. for producing a fluorescent probe. The substrate may also include cDNA or oligonucleotides corresponding to control genes or fragments thereof. Additionally, for example, the kit may be a diagnostic kit containing essential elements necessary to perform LISA. ELISA kits contain specific antibodies against these proteins. Antibodies have high specificity and affinity for each marker protein and almost no cross-reactivity to other proteins, and are either monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. Additionally, ELISA kits may include antibodies specific for control proteins. Other ELISA kits include reagents that can detect bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated with antibodies) and their substrates or those that can bind to antibodies. It may contain other substances, etc. Additionally, for example, the kit may be a rapid kit containing the essential elements necessary to perform a rapid test that can provide analysis results. Rapid kits contain specific antibodies against proteins. Antibodies have high specificity and affinity for each marker protein and almost no cross-reactivity to other proteins, and are either monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. Additionally, the rapid kit may include antibodies specific for control proteins. In addition, the rapid kit includes reagents that can detect bound antibodies, such as a nitrocellulose membrane on which specific antibodies and secondary antibodies are immobilized, a membrane bound to antibody-bound beads, an absorption pad, and a sample pad. It may include substances, etc. Additionally, for example, the kit may be a multiple reaction monitoring (MRM) kit in MS/MS mode that includes the essential elements necessary to perform mass spectrometry. While SIM (Selected Ion Monitoring) is a method that uses ions created by a single collision in the source part of a mass spectrometer, MRM selects a specific ion from among the broken ions once more and uses another continuously connected MS source. This is a method of passing it through one more time, colliding with it, and then using the ions obtained from these. Through the above MRM (multiple reaction monitoring) analysis methods, it is possible to compare the protein expression level in a normal control group or the same individual with the protein expression level in an individual with infertility, and also to achieve significant conversion from infertility-related disease marker genes to proteins. By determining whether the expression level increases or decreases, the onset of infertility can be diagnosed.
다른 양상은 LDLR(low density lipoprotein receptor) 단백질 또는 이의 유전자의 mRNA 수준을 측정하는 단계를 포함하는 고령 여성의 난임 진단을 위한 정보제공방법을 제공한다. 일 구체예에 있어서, 상기 방법은 StAR(steroidogenic acute regulatory protein), ADCY(adenylate cyclase), 및 HSD17B1(hydroxysteroid 17-beta dehydrogenase 1)로 구성된 군에서 선택되는 단백질 또는 이의 유전자의 mRNA 수준을 측정하는 단계를 추가로 포함할 수 있다. Another aspect provides an informative method for diagnosing infertility in elderly women, including measuring the mRNA level of low density lipoprotein receptor (LDLR) protein or its gene. In one embodiment, the method includes measuring the mRNA level of a protein or gene thereof selected from the group consisting of steroidogenic acute regulatory protein (StAR), adenylate cyclase (ADCY), and hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1). may additionally be included.
상기 난임은 정상 대조군(정상 임신이 가능한 개체, 또는 동일 개체에서 난임과 관련이 없는 시료)과 비교하여, 난임을 갖는 개체에서 유전자 발현 수준 또는 해당 단백질의 발현 수준이 감소하므로, 상기 수준이 감소하면 난임으로 진단할 수 있다. The infertility occurs because the level of gene expression or the expression level of the corresponding protein decreases in an individual with infertility compared to a normal control group (an individual capable of normal pregnancy, or a sample unrelated to infertility from the same individual), so when the level decreases, It can be diagnosed as infertility.
따라서, 일 양상에 따른 방법은 상기 측정된 단백질의 발현 수준 또는 유전자의 발현 수준이 정상 대조군 시료의 단백질의 발현 수준 또는 유전자의 발현 수준보다 낮은 경우 난임으로 판단하는 것을 포함할 수 있다. Accordingly, the method according to one aspect may include determining infertility when the measured protein expression level or gene expression level is lower than the protein expression level or gene expression level of the normal control sample.
또 다른 양상은 LDLR(low density lipoprotein receptor) 단백질과 피검 물질을 접촉시키는 단계; 및 무처리 대조군에 비하여 상기 LDLR(low density lipoprotein receptor) 단백질의 발현 또는 활성 수준을 증가시킨 피검 물질을 난임의 치료제로 선별하는 단계를 포함하는 고령 여성의 난임 치료제를 스크리닝 하는 방법을 제공한다. Another aspect includes contacting a test substance with a low density lipoprotein receptor (LDLR) protein; and selecting a test substance that increases the expression or activity level of the low density lipoprotein receptor (LDLR) protein as a treatment for infertility compared to an untreated control group.
상기 피검 물질, 예를 들면, 약물 후보 물질, 피검 화합물 또는 피검 조성물은 저분자 화합물, 항체, 안티센스 뉴클레오티드, 작은 간섭 RNA(short interfering RNA), 짧은 헤어핀 RNA(short hairpin RNA), 핵산, 단백질, 펩티드, 기타 추출물 또는 천연물을 포함할 수 있다. The test substance, for example, a drug candidate, a test compound, or a test composition may include a small molecule compound, an antibody, an antisense nucleotide, a short interfering RNA, a short hairpin RNA, a nucleic acid, a protein, a peptide, Other extracts or natural products may be included.
상기 접촉은 시험관 내(in vitro)에서 수행되는 것일 수 있다. 예를 들면, 상기 단백질들을 발현하는 벡터를 세포에 형질전환 하는 단계; 상기 형질전환된 세포에 약물 후보물질을 처리하는 단계를 포함할 수 있다. The contact may be performed in vitro. For example, transforming a cell with a vector expressing the proteins; It may include treating the transformed cells with a drug candidate.
일 구체예에 따른 상기 마커는 난임에서 발현이 감소하므로, 상기 마커의 발현 또는 활성을 증가시키는 후보 물질은 난임의 치료에 유용하게 사용될 수 있다.According to one embodiment, the expression of the marker is decreased in infertility, so a candidate substance that increases the expression or activity of the marker can be usefully used in the treatment of infertility.
일 양상에 따른 조성물은 고령 여성의 난임 여부를 비침습적으로 진단할 수 있을 뿐만 아니라, 난임 치료에 유용한 새로운 타겟이 될 수 있다. 또한, 일 양상에 따른 바이오마커의 발현 또는 활성을 증가시킴으로써 난포의 발달 및 배란을 촉진하고, 난자의 질을 개선하여 자연 임신 또는 인공수정, 시험관 시술과 같은 보조생식기술에 의한 임신을 성공적으로 유도할 수 있다.A composition according to one aspect can not only non-invasively diagnose infertility in elderly women, but can also be a useful new target for infertility treatment. In addition, it promotes follicular development and ovulation by increasing the expression or activity of biomarkers according to one aspect, and improves egg quality to successfully induce pregnancy through natural pregnancy or assisted reproductive technology such as artificial insemination and in vitro fertilization. can do.
도 1a는 연령 및 인심을 기반으로 한 정규화된 값(log2)을 사용한 데이터의 계층적 클러스터링을 나타낸다. Figure 1a shows hierarchical clustering of data using normalized values (log2) based on age and ancestry.
도 1b는 연령 및 인심을 기반으로 한 정규화된 값(log2)을 사용한 데이터의 다차원 스케일링 분석 플롯을 나타낸다. Figure 1B shows a multidimensional scaling analysis plot of the data using normalized values (log2) based on age and inseam.
도 1c는 임신(청년 및 고령) 및 비-임신(고령 및 청년) 그룹의 계층적 클러스터링 그래프의 히트맵을 나타낸다.Figure 1C shows a heatmap of the hierarchical clustering graph of pregnant (young and older) and non-pregnant (older and younger) groups.
도 2a는 청년 및 고령 임신 그룹과 청년 및 고령 비-임신 그룹에서 합성된 난소 스테로이드 호르몬의 차이를 확인한 결과이다. Figure 2a shows the results confirming the difference in ovarian steroid hormones synthesized in the young and elderly pregnant group and the young and elderly non-pregnant group.
도 2b는 난소 특이적 신호전달 경로를 식별하기 위하여 KEGG 경로 분석을 수행한 결과이다. Figure 2b shows the results of KEGG pathway analysis to identify ovarian-specific signaling pathways.
도 3a는 청년 임신 및 청년 비-임신 그룹의 LDLR, StAR, ADCY, 및 HSD17B1 유전자 발현 수준을 나타낸다.Figure 3A shows LDLR, StAR, ADCY, and HSD17B1 gene expression levels in young adult pregnant and young adult non-pregnant groups.
도 3b는 고령 임신 및 고령 비-임신 그룹의 LDLR, StAR, ADCY, 및 HSD17B1 유전자 발현 수준을 나타낸다.Figure 3B shows LDLR, StAR, ADCY, and HSD17B1 gene expression levels in older pregnancy and older non-pregnant groups.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.
[실시예][Example]
실시예 1. 난구세포(cumulus cell, CC)의 수집 및 난모세포(oocyte)의 특성 확인Example 1. Collection of cumulus cells (CC) and confirmation of oocyte characteristics
본 실험은 차의과학대학교의 기관연구윤리위원회(승인번호: 1044308-201611-BR-027-04)의 승인을 받았으며, 환자의 서면 동의 하에 실시하였다. 남성 요인, 난관(tubal) 또는 자궁 요인으로 인한 불임 진단을 받은 환자를 대상자로 선정하였고, 다낭성난소증후군 등 내분비질환으로 인한 불임 여성이나 유전질환이 있는 여성, 불임 인자를 알 수 없는 여성은 조사대상자에서 제외하였다. 최종 선정된 환자를 33세 이하(n=16) 또는 40세 이상(n=16)의 청년 임신(Young pregnancy, Young P, n=8); 청년 비-임신(Young NP, n=8); 고령 임신(Old pregnancy, Old P, n=8); 및 고령 비-임신(Old NP, n=8) 그룹으로 분류하였다.This experiment was approved by the Institutional Research Ethics Committee of CHA University of Science and Technology (approval number: 1044308-201611-BR-027-04) and was conducted with written consent from the patients. Patients diagnosed with infertility due to male, tubal, or uterine factors were selected as subjects, while women with infertility due to endocrine diseases such as polycystic ovary syndrome, women with genetic diseases, or women whose infertility factors were unknown were selected as subjects. excluded from. The final selected patients were young pregnancy (Young P, n=8) aged under 33 (n=16) or over 40 (n=16); Young Non-Pregnant (Young NP, n=8); Old pregnancy (Old P, n=8); and older non-pregnant (Old NP, n=8) groups.
모든 환자는 체외수정(in vitro fertilization, IVF) 절차 동안 생식샘자극호르몬분비호르몬(gonadotropin-releasing hormone,　GnRH) 길항제 프로토콜을 사용하여 기존의 COS 또는 최소 자극 프로토콜을 받았다. 난자를 수득한 후, 세포질 정자 주입(intracytoplasmic sperm injection)을 위하여 0.1% 히알루로니다제(ORIGIO CooperSurgical, Mεløv, Denmark)를 이용하여 난모세포를 제거하고, 난구세포(cumulus cell, CC)를 분리하였다. 이후, 분리된 난구세포를 PBS로 세척한 후, mRNA를 정제할 때까지 -80℃에서 보관하였다. 분리된 난구세포의 특성을 하기 표 1에 나타내었다.All patients received a conventional COS or minimal stimulation protocol using a gonadotropin-releasing hormone (GnRH) antagonist protocol during in vitro fertilization (IVF) procedures. After obtaining the oocytes, oocytes were removed using 0.1% hyaluronidase (ORIGIO CooperSurgical, Mεløv, Denmark) for intracytoplasmic sperm injection, and cumulus cells (CC) were isolated. . Afterwards, the isolated cumulus cells were washed with PBS and stored at -80°C until mRNA purification. The characteristics of the isolated cumulus cells are shown in Table 1 below.
청년 임신(n=8)Young adult pregnancy (n=8) 청년 비-임신(n=8)Young Adult Non-Pregnant (n=8) 고령 임신(n=8)Older pregnancy (n=8) 고령 비-임신(n=8)Older non-pregnant (n=8)
연령age 31.16 ± 1.1631.16 ± 1.16 33.8 ± 1.7833.8 ± 1.78 39.83 ± 0.7539.83 ± 0.75 40.83 ± 1.8340.83 ± 1.83
혈청 AMH(ng/mL)Serum AMH (ng/mL) 4.49 ± 2.794.49 ± 2.79 2.80 ± 1.272.80 ± 1.27 1.95 ± 1.011.95 ± 1.01 1.99 ± 0.971.99 ± 0.97
BMI(㎏/m2)BMI (kg/m 2 ) 23.30 ± 3.4123.30 ± 3.41 21.49 ± 2.5621.49 ± 2.56 24.20 ± 3.1524.20 ± 3.15 22.33 ± 2.5622.33 ± 2.56
기초 FSH (mIU/mL)Basal FSH (mIU/mL) 7.96 ± 2.997.96 ± 2.99 8.36 ± 2.208.36 ± 2.20 8.23 ± 2.168.23 ± 2.16 7.89 ± 1.477.89 ± 1.47
이전 IVF 주기Previous IVF cycle 2.6 ± 1.672.6 ± 1.67 2.0 ± 1.222.0 ± 1.22 1.0 ± 0.811.0 ± 0.81 2.16 ± 1.602.16 ± 1.60
총 고나도트로핀(Gonadotropin) 용량Total Gonadotropin Dose 3030 ± 497 3030 ± 497 2308 ± 8202308 ± 820 2525 ± 3182525 ± 318 2995 ± 3592995 ± 359
항뭄 난포(antral follicle) 수Number of antral follicles 11.25 ± 5.31 11.25 ± 5.31 8.88 ± 7.648.88 ± 7.64 8.80 ± 3.638.80 ± 3.63 9.33 ± 4.969.33 ± 4.96
수거된 난모세포의 수Number of Oocytes Collected 13.16 ± 6.43 13.16 ± 6.43 10.4 ± 2.3010.4 ± 2.30 8.66 ± 5.888.66 ± 5.88 10.16 ± 4.9910.16 ± 4.99
성숙률(%)Maturity rate (%) 77.01 ± 15.31* 77.01 ± 15.31 * 64.77 ± 7.0864.77 ± 7.08 87.12 ± 16.11* 87.12 ± 16.11 * 81.22 ± 17.7881.22 ± 17.78
MII 난모세포 수MII oocyte count 9.5 ± 3.939.5 ± 3.93 6.8 ± 2.046.8 ± 2.04 5.0 ± 3.375.0 ± 3.37 6.66 ± 4.846.66 ± 4.84
수정률(%)fertility(%) 91.05 ± 8.6191.05 ± 8.61 68.85 ± 32.2368.85 ± 32.23 83.33 ± 23.5783.33 ± 23.57 75.82 ± 17.9375.82 ± 17.93
전이 배아의 수 Number of transfer embryos 1.16 ± 0.40 1.16 ± 0.40 1.63 ± 0.921.63 ± 0.92 2.33 ± 0.812.33 ± 0.81 2.00 ± 0.002.00 ± 0.00
전이 배아 BL(%)/CL(%) 단계Transfer embryo BL(%)/CL(%) stage 100 */0100 */0 62.5/37.562.5/37.5 83.33 */16.6783.33 */16.67 66.66/33.3366.66/33.33
* p<0.05*p<0.05
그 결과, 표 1에 나타낸 바와 같이, 연령과 관련된 항 뮬러관호르몬(Anti Mullerian Hormone, AMH)은 청년 임신 그룹과 비교하여 고령 임신 그룹에서 유의하게 감소하였다. 그러나, 임상 결과는 BMI, 이전 IVF 주기수 또는 기초 FSH 수준의 함수로 각 연력 그룹 내에서 유의한 차이를 나타내지 않았다. GnRH 길항제 프로토콜을 사용하여 모든 IVF 환자를 자극한 결과에서도, 투여된 생식샘자극호르몬(gonadotropin)의 용량에 유의한 차이는 없었다. 난자 채취, 성숙율, 수정율과 같은 난자이 특성은 그룹 간 유의한 차이가 없었다. 임상 결과에 따르면, 임신은 젊은 그룹과 고령 그룹 모두에서 난모세포 및 배아의 질과 관련이 있었다. 임상 임신군은 비-임신군과 비교하여 고품질 배아 수 및 배반포 형성율이 유의하게 높은 것을 확인할 수 있었다.As a result, as shown in Table 1, age-related anti-Müllerian Hormone (AMH) was significantly decreased in the elderly pregnancy group compared to the young pregnancy group. However, clinical outcomes did not show significant differences within each age group as a function of BMI, number of previous IVF cycles, or basal FSH levels. Even when all IVF patients were stimulated using the GnRH antagonist protocol, there was no significant difference in the dose of gonadotropin administered. There were no significant differences in oocyte characteristics such as oocyte retrieval, maturation rate, and fertilization rate between groups. According to clinical results, pregnancy was associated with oocyte and embryo quality in both young and old groups. It was confirmed that the clinical pregnancy group had a significantly higher number of high-quality embryos and blastocyst formation rate compared to the non-pregnancy group.
실시예 2. 난구세포의 전사체 데이터 분석Example 2. Analysis of transcriptome data of cumulus cells
2-1. mRNA 추출 및 mRNA-seqNGS 분석2-1. mRNA extraction and mRNA-seqNGS analysis
상기 실시예 1의 결과를 바탕으로 임신 및 비-임신 그룹과 청년 및 고령 그룹 사이의 mRNA 발현 프로파일의 차이를 확인하기 위하여 전체-전사체(whole-transcriptome) 분석을 수행하였다. 먼저, 상기 실시예 1에서 분리한 난구세포 난구세포의 mRNA를 추출하고, 추출된 mRNA에 대하여 차세대 유전체 분석(New Generation Sequencing, NGS)를 이용하여 전사체데이터를 획득하였다. 구체적으로, DynaBeads mRNA DIRECT Kit (Life Technologies, Oslo, Norway)를 사용하여 실시예 1에서 선정한 4개 그룹의 난구세포로부터 mRNA를 분리하고, NanoDrop ND-1000 spectrophotometer (Nyxor Biotech, Paris, France)를 사용하여 정량화하였다. 정제된 mRNA를 MiSeq sequencer(MiSeq System, Illumina, San Diego, CA, USA)를 사용하여 시퀀싱하고, 세부 정보를 제공하기 위하여 샘플 시트를 준비하였다. 이후, NGS에서 얻은 전체-전사체(whole-transcriptome) 원시데이터에 대하여 생물정보학 분석을 수행하였다.Based on the results of Example 1, whole-transcriptome analysis was performed to confirm differences in mRNA expression profiles between the pregnant and non-pregnant groups and the young and elderly groups. First, mRNA from the cumulus cells isolated in Example 1 was extracted, and transcriptome data was obtained for the extracted mRNA using next-generation sequencing (NGS). Specifically, mRNA was isolated from the four groups of cumulus cells selected in Example 1 using the DynaBeads mRNA DIRECT Kit (Life Technologies, Oslo, Norway), and NanoDrop ND-1000 spectrophotometer (Nyxor Biotech, Paris, France) was used. and quantified. Purified mRNA was sequenced using a MiSeq sequencer (MiSeq System, Illumina, San Diego, CA, USA), and a sample sheet was prepared to provide detailed information. Afterwards, bioinformatics analysis was performed on the whole-transcriptome raw data obtained from NGS.
2-2. mRNA-seq NGS 데이터의 생물정보학 분석2-2. Bioinformatics analysis of mRNA-seq NGS data
인간 게놈 서열 (UCSC hg19, annotation RefSeq_2017_06_12)을 참조로 사용하여 전체-전사체 데이터를 분석하였다. 초기 NGS 데이터에서 stringTie-e 옵션을 사용하여 실시예 1에서 선정한 4개 그룹 조건에서 27,685개의 유전자를 확인하였다. 이후, Cufflinks를 사용하여 상기 데이터를 매핑된 백만 킬로베이스 당 단편들(kilobase of exon per million fragments mapped, FPKMs)로 정규화하였다. 유의미한 유전자 발현을 필터링하기 위하여 2개 이상의 FPKM 값이 0인 유전자를 제외하였다. 샘플 간의 관계를 명확하게 하기 위하여, 17,317개의 유전자에 대한 발현 데이터에 대해 감독되지 않은 계층적 클러스터링 분석(unsupervised hierarchical clustering analysis) 및 다차원 스케일링 multidimensional scaling, MDS) 분석을 수행하였다. 이후, 생물학적 복제물(replicates) 사이에 이상치(outlier) 샘플 및 유사한 발현 패턴의 존재를 확인하였다. Whole-transcriptome data were analyzed using the human genome sequence (UCSC hg19, annotation RefSeq_2017_06_12) as reference. In the initial NGS data, 27,685 genes were identified in the four group conditions selected in Example 1 using the stringTie-e option. The data was then normalized to kilobase of exon per million fragments mapped (FPKMs) using Cufflinks. To filter out significant gene expression, genes with two or more FPKM values of 0 were excluded. To clarify relationships between samples, unsupervised hierarchical clustering analysis and multidimensional scaling (MDS) analysis were performed on expression data for 17,317 genes. Afterwards, the presence of outlier samples and similar expression patterns among biological replicates was confirmed.
도 1a는 연령 및 인심을 기반으로 한 정규화된 값(log2)을 사용한 데이터의 계층적 클러스터링을 나타낸다.Figure 1a shows hierarchical clustering of data using normalized values (log2) based on age and ancestry.
도 1b는 연령 및 인심을 기반으로 한 정규화된 값(log2)을 사용한 데이터의 다차원 스케일링 분석 플롯을 나타낸다. Figure 1B shows a multidimensional scaling analysis plot of the data using normalized values (log2) based on age and inseam.
도 1c는 임신(청년 및 고령) 및 비-임신(고령 및 청년) 그룹의 계층적 클러스터링 그래프의 히트맵을 나타낸다. Figure 1C shows a heatmap of the hierarchical clustering graph of pregnant (young and older) and non-pregnant (older and younger) groups.
그 결과, 도 1a에 나타낸 바와 같이, 임신 그룹의 유전자 발현 양상은 비-임신 그룹의 유전자 발현 양상과 유의하게 차이를 나타내었다. 또한, 도 1b에 나타낸 바와 같이, 청년 비-임신 그룹과 고령 비-임신 그룹은 유사한 유전자 프로파일 비율을 나타내었다. 또한, 도 1c에 나타낸 바와 같이, 비-임신 그룹(청년 및 고령 모두) 내에서 유사한 z-score를 명확히 나타내었다. 또한, 임신 그룹 간에 유전자 발현 프로파일은 유사하게 높은 수준을 나타내었으나, 청년 임신 그룹과 고령 임신 그룹 사이의 발현 양상의 차이를 나타내었다. 또한, 비-임신 그룹에서 청년 그룹과 고령 그룹은 유사한 발현 양상을 나타내었다. 또한, 고령 임신 그룹은 비-임신 그룹에 비해 유전자 발현 활성이 더 강하게 나타났고, 청년 및 고령 비-임신 그룹은 청년 및 고령 임신 그룹에 비해 유전자 발현 프로필이 더 낮은 것을 확인할 수 있었다.As a result, as shown in Figure 1a, the gene expression pattern of the pregnant group was significantly different from that of the non-pregnant group. Additionally, as shown in Figure 1B, the young non-pregnant group and the old non-pregnant group showed similar genetic profile ratios. Additionally, as shown in Figure 1C, similar z-scores were evident within the non-pregnant groups (both young and old). In addition, gene expression profiles were found to be at similarly high levels between pregnancy groups, but there were differences in expression patterns between the young pregnancy group and the elderly pregnancy group. Additionally, in the non-pregnant group, the young and elderly groups showed similar expression patterns. In addition, the elderly pregnant group showed stronger gene expression activity than the non-pregnant group, and the young and elderly non-pregnant groups had lower gene expression profiles compared to the young and elderly pregnant groups.
실시예 3. 유전자 온톨로지(gene ontology, GO) 분석Example 3. Gene ontology (GO) analysis
상기 실시예 2-2에서 도출된 17,317개의 유전자 중에서 2배 이상의 발현 변화를 나타내는 유전자를 확인하기 위하여 차등 발현 유전자(differential expression gene, DEG) 분석을 수행하였다. 구체적으로, log2(FPKM+1) 값을 계산하고 분위수법(quantile method)으로 정규화하였다. p-값≤0.05인 배수-변화(fold-change)값>2인 전사(Transcript)를 차등 발현 유전자로 포함하였다(총 1366개). DEG 발현 패턴을 표시하기 위하여, 유사성 척도의 완전한 연결과 유클리디안(Euclidean) 거리를 사용하여 계층적 클러스터링 분석을 수행하였다. 상위 10개 유전자 온톨로지(gene ontology) 용어를 p-값의 오름차순순으로 표시하였다. 이후, 상위 랭크된 상향 및 하향 조절된 유전자 목록을 분석하고, 연령 및 임신 결과의 함수로써 각각 상향 및 하향 유전자 클러스터를 플로팅(plot)하였다. 연령 및 임신 능력에 따라 각 그룹을 조사하였다. 임신 능력과 관련된 주요 DEG를 식별하기 위하여, 다음의 세 쌍을 비교하였다: (1) 고령 임신 vs. 고령 비-임신; (2) 청년 임신 vs. 청년 비-임신; (3) 연령에 관계 없이 임신 vs. 비-임신. 각 그룹에 하나의 샘플만이 포함되어 있으므로, 배수-변화 값(>2 또는 <0.5)을 사용하여 DEG를 식별하였다. T-test를 사용하여 데이터를 분석하고, Benjamini-Hochberg 절차에 의해 p-값을 조정하였다. 그러나, 모든 조정 p-값이 통계적으로 유의하지 않았기 때문에, 조정된 p<0.05 대신 p<0.05를 사용하여 DEG를 식별하고, 청년 및 고령 임신 그룹의 상위 6개의 상향 및 상위 10개의 하향 조절 유전자를 하기 표 2에 나타내었다.Differential expression gene (DEG) analysis was performed to identify genes showing expression changes of more than twofold among the 17,317 genes derived in Example 2-2. Specifically, log2(FPKM+1) values were calculated and normalized using the quantile method. Transcripts with p-value ≤ 0.05 and fold-change value > 2 were included as differentially expressed genes (total 1366). To display DEG expression patterns, hierarchical clustering analysis was performed using complete linkage and Euclidean distance of similarity measures. The top 10 gene ontology terms are displayed in ascending order of p-value. The top ranked up- and down-regulated gene lists were then analyzed and the up- and down-regulated gene clusters were plotted as a function of age and pregnancy outcome, respectively. Each group was examined according to age and fertility. To identify key DEGs associated with fertility, the following three pairs were compared: (1) older pregnancy vs. Elderly, non-pregnant; (2) Young adult pregnancy vs. Young Adult Non-Pregnant; (3) Pregnancy at any age vs. Non-Pregnant. Since each group included only one sample, fold-change values (>2 or <0.5) were used to identify DEGs. Data were analyzed using T-test, and p-value was adjusted by Benjamini-Hochberg procedure. However, since all adjusted p-values were not statistically significant, we used adjusted p<0.05 instead of p<0.05 to identify DEGs and ranked the top 6 up- and top 10 down-regulated genes in the young and older pregnancy groups. It is shown in Table 2 below.
유전자gene 청년 비-임신 vs. 임신 Fold changeYoung Adult Non-Pregnant vs. Pregnancy Fold change 고령 비-임신 vs. 임신 Fold changeOlder non-pregnant vs. Pregnancy Fold change
상향 조절upregulation SNORD159SNORD159 small nucleolar RNA, C/D box 159small nucleolar RNA, C/D box 159 2.0184692.018469 2.1604062.160406
ZNF117ZNF117 zinc finger protein 117zinc finger protein 117 3.9041573.904157 2.6357052.635705
ITGA2ITGA2 integrin subunit alpha 2 integrin subunit alpha 2 2.4149542.414954 3.0198053.019805
LOC102723753LOC102723753 HECT and RLD domain containing E3 ubiqitin protein ligase 2 pseudogeneHECT and RLD domain containing E3 ubiquitin protein ligase 2 pseudogene 2.8249692.824969 3.5337613.533761
SFRP4SFRP4 secreted frizzled related protein 4Secreted frizzled related protein 4 2.6492692.649269 3.6174783.617478
NME1-NME2NME1-NME2 NME1-NME2 readthroughNME1-NME2 readthrough 3.5320603.532060 17.91897417.918974
하향 조절down regulation NGFRNGFR nerve growth factor receptornerve growth factor receptor -13.181860-13.181860 -7.632480-7.632480
ADNPADNP activity dependent neuroprotector homeoboxactivity dependent neuroprotector homeobox -6.182802-6.182802 -3.508153-3.508153
SLC44A2SLC44A2 solute carrier family 44 member 2single carrier family 44 member 2 -5.652775-5.652775 -4.948898-4.948898
LOC728715LOC728715 ovostatin homolog 2ovostatin homolog 2 -5.646341-5.646341 -2.095229-2.095229
HMGB2HMGB2 high mobility group box 2high mobility group box 2 -4.669963-4.669963 -3.403419-3.403419
PLCD1PLCD1 phospholipase C delta 1phospholipase C delta 1 -4.559903-4.559903 -4.480157-4.480157
SDSLSDSL serine dehydratase likeserine dehydratase like -4.485008-4.485008 -2.728038-2.728038
ACTA1ACTA1 actin, alpha 1, skeletal muscleactin, alpha 1, skeletal muscle -3.889844-3.889844 -2.643790-2.643790
DUSP15DUSP15 dual specificity phosphatase 15dual specificity phosphatase 15 -3.829421-3.829421 -2.535768-2.535768
ZMIZ1-AS1ZMIZ1-AS1 ZMIZ1 antisense RNA 1ZMIZ1 antisense RNA 1 -3.759088-3.759088 -2.906539-2.906539
그 결과, 표 2에 나타낸 바와 같이, 청년 및 고령 임신 그룹에서 SNORD159, ZNF117, ITGA2, LOC102723753, SFRP4, 및 NME1-NME2가 상향 조절되었으며 NGFR, ADNP, SLC44A2, LOC728715, HMGB2, PLCD1, SDSL, ACTA1, DUSP15 및 ZMIZ1-AS1가 공통적으로 하향 조절된 것을 확인할 수 있엇다. 구체적으로, 상향 조절된 유전자(예를 들어, ZNF117, ITGA2)는 난구세포의 증식, 분화 및 스테로이드 생성과 같은 난소 기능과 관련된 유전자인 것을 확인할 수 있었다. As a result, as shown in Table 2, in the young and old pregnancy groups, SNORD159, ZNF117, ITGA2, LOC102723753, SFRP4, and NME1-NME2 were upregulated, and NGFR, ADNP, SLC44A2, LOC728715, HMGB2, PLCD1, SDSL, ACTA1, It was confirmed that DUSP15 and ZMIZ1-AS1 were commonly downregulated. Specifically, the upregulated genes (eg, ZNF117, ITGA2) were confirmed to be genes related to ovarian functions such as cumulus cell proliferation, differentiation, and steroid production.
실시예 4. KEGG 경로 분석을 통한 주요 신호 맵 확인Example 4. Confirmation of main signal map through KEGG pathway analysis
상기 실시예 3의 차등 발현 유전자 분석 결과를 이용하여, 청년 vs. 고령 및 임신 vs. 비-임신의 실제 데이터와 가상 데이터 모두에서 정규화 방법의 영향을 평가하였다. 구체적으로, 상기 실시예 3에서 획득한 DEG 데이터를 DAVID 프로그램 (http://david.abcc.ncifcrf.gov/tools.jsp, v.6.8 web software)에 업로드 하였다. 기능적 주석 클러스터링 도구를 사용하여 기능적 범주를 클러스터링 하였으며, 대표적인 유전자 온톨로지(Gene Ontology) 범주를 식별하였다. GO/정규 경로 (canonical pathway) 및 특징 유전자 세트의 데이터베이스는 메타스케이프(http://david.abcc.ncifcrf.gov/tools.jsp, v.6.8 웹 소프트웨어)를 사용하여 각 유전자 세트에서 상당히 풍부한 용어를 추론하는데 사용되었다. GO 분석 후, KEGG(Kyoto Encyclopedia of Genes and Genomes) 경로 분석을 수행하여 p-값<0.05인 DEG의 유전자 농축 데이터에서 난소 특이적 신호전달 경로를 식별하였다. 임계값은 보정된 Fisher Exact P-value (EASE score)≤0.05로 설정하였다.Using the results of differential expression gene analysis in Example 3, youth vs. Old age and pregnancy vs. The impact of the normalization method was evaluated on both non-pregnant real and virtual data. Specifically, the DEG data obtained in Example 3 was uploaded to the DAVID program (http://david.abcc.ncifcrf.gov/tools.jsp, v.6.8 web software). Functional categories were clustered using a functional annotation clustering tool, and representative Gene Ontology categories were identified. A database of GO/canonical pathways and feature gene sets was generated using Metascape (http://david.abcc.ncifcrf.gov/tools.jsp, v.6.8 web software) to identify significantly enriched terms in each gene set. was used to infer. After GO analysis, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis was performed to identify ovary-specific signaling pathways in the gene enrichment data of DEGs with p-value <0.05. The threshold was set at corrected Fisher Exact P-value (EASE score)≤0.05.
도 2a는 청년 및 고령 임신 그룹과 청년 및 고령 비-임신 그룹에서 합성된 난소 스테로이드 호르몬의 차이를 확인한 결과이다.Figure 2a shows the results confirming the difference in ovarian steroid hormones synthesized in the young and elderly pregnant group and the young and elderly non-pregnant group.
도 2b는 난소 특이적 신호전달 경로를 식별하기 위하여 KEGG 경로 분석을 수행한 결과이다. Figure 2b shows the results of KEGG pathway analysis to identify ovarian-specific signaling pathways.
그 결과, 도 2a에 나타낸 바와 같이, 샘플 상태 및 임상 결과에 따라 스테로이드 생성 경로가 유의하게 차등 상향 조절되는 것을 확인하였다. 특히, 난소 스테로이드 생성 관련 유전자는 연령 및 임신 결과에 따라 유의하게 차등 조절되는 것을 확인하였다. As a result, as shown in Figure 2a, it was confirmed that the steroid production pathway was significantly differentially upregulated depending on sample status and clinical results. In particular, genes related to ovarian steroid production were confirmed to be significantly differentially regulated depending on age and pregnancy outcome.
또한, 도 2b에 나타낸 바와 같이, 난구세포에서 상향 조절된 차등 발현 유전자는 난소 스테로이드 생성 경로(LDLR 및 StAR)와 관련이 있고, 하향 조절된 차등 발현 유전자는 임신 상태(ADCY 및 17 β-사이드록시스테로이드 탈수소효소(17 beta-hydroxysteroid dehydrogenase 1, HSD17B1)와 관련이 있는 것을 확인할 수 있었다.Additionally, as shown in Figure 2b, the up-regulated differentially expressed genes in cumulus cells were related to the ovarian steroidogenic pathway (LDLR and StAR), and the down-regulated differentially expressed genes were related to pregnancy status (ADCY and 17 β-sideroxy). It was confirmed that it was related to steroid dehydrogenase (17 beta-hydroxysteroid dehydrogenase 1, HSD17B1).
실시예 5. 실시간 qPCR에 의한 mRNA-seq 데이터 검증Example 5. Validation of mRNA-seq data by real-time qPCR
청년 및 고령 임신 그룹에 대한 데이터를 검증하기 위하여 실시간 qPCR을 수행하였다. 구체적으로, DynaBeads mRNA DIRECT Kit를 이용하여 mRNA를 정제하였다. AccuPower® CycleScript RT PreMix (Bioneer)와 poly-dT를 사용하여 총 RNA를 cDNA로 역전사 하였다. 분광형광 열 순환기(spectrofluorometric thermal cycler) (SimpliAmp Thermal Cycler; Thermo Fisher Scientific)에서 AccuPower Taq PCR PreMix (Bioneer)를 사용하여 qPCR를 수행하였다. KEGG 신호 경로를 기반으로 연령 및 임신 결과의 함수로 난소 스테로이드 생성에 관여하는 유전자 중 상향 및 하향 조절된 표적 유전자를 선택하였다. LDLR(low density lipoprotein receptor), StAR(steroidogenic acute regulatory protein), ADCY(adenylate cyclase), HSD17B1(hydroxysteroid 17-beta dehydrogenase 1), 및 β-actin에 대한 프라이머와 함께 SsoAdvanced Universal SYBR Green Supermix (Bio-Rad)를 사용하여 실시간-qPCR를 수행하였다. 각 유전자에 대한 PCR 산물의 수준을 CT(comparative threshold cycle) 방법을 사용하여 β-actin에 대한 PCR 산물의 상응하는 수준에 대해 정규화하였다. 샘플을 3회 평가하였다. PCR 산물을 2% 아가로스 겔에서 전기영동하고 SafeView (Applied Biological Materials, Richmond, Canada)를 사용하여 시각화 하였다. 겔 문서화 시스템 (WSE-6100 LuminoGraph; ATTO, Tokyo, Japan)을 사용하여 UV 조명 하에서 겔을 이미지화 하였으며, ImageJ 소프트웨어를 사용하여 PCR 사물의 밴드를 분석하였다. Real-time qPCR was performed to verify data for young and older pregnancy groups. Specifically, mRNA was purified using the DynaBeads mRNA DIRECT Kit. Total RNA was reverse transcribed into cDNA using AccuPower® CycleScript RT PreMix (Bioneer) and poly-dT. qPCR was performed using AccuPower Taq PCR PreMix (Bioneer) in a spectrofluorometric thermal cycler (SimpliAmp Thermal Cycler; Thermo Fisher Scientific). Based on the KEGG signaling pathway, up- and down-regulated target genes among genes involved in ovarian steroidogenesis as a function of age and pregnancy outcome were selected. SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) with primers for low density lipoprotein receptor (LDLR), steroidogenic acute regulatory protein (StAR), adenylate cyclase (ADCY), hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1), and β-actin. ) was used to perform real-time-qPCR. The level of the PCR product for each gene was normalized to the corresponding level of the PCR product for β-actin using the comparative threshold cycle (CT) method. Samples were evaluated in triplicate. PCR products were electrophoresed on a 2% agarose gel and visualized using SafeView (Applied Biological Materials, Richmond, Canada). Gels were imaged under UV illumination using a gel documentation system (WSE-6100 LuminoGraph; ATTO, Tokyo, Japan), and bands of PCR artifacts were analyzed using ImageJ software.
도 3a는 청년 임신 및 청년 비-임신 그룹의 LDLR, StAR, ADCY, 및 HSD17B1 유전자 발현 수준을 나타낸다.Figure 3A shows LDLR, StAR, ADCY, and HSD17B1 gene expression levels in young adult pregnant and young adult non-pregnant groups.
도 3b는 고령 임신 및 고령 비-임신 그룹의 LDLR, StAR, ADCY, 및 HSD17B1 유전자 발현 수준을 나타낸다.Figure 3B shows LDLR, StAR, ADCY, and HSD17B1 gene expression levels in older pregnancy and older non-pregnant groups.
그 결과, 도 3a 및 도 3b에 나타낸 바와 같이, 연령에 관계 없이 임신 그룹은 비-임신 그룹과 비교하여 LDLR 및 StAR의 발현 수준이 유의하게 더 높았으나, ADCY 및 HSD17B1의 발현 수준은 유의하게 낮은 것을 확인할 수 있었다. As a result, as shown in Figures 3a and 3b, regardless of age, the pregnant group had significantly higher expression levels of LDLR and StAR compared to the non-pregnant group, but the expression levels of ADCY and HSD17B1 were significantly lower. could be confirmed.
즉, 임신 그룹의 난구세포에서 LDLR 및 StAR 단백질을 포함한 스테로이드 생성 경로 관여 유전자가 상향 조절되는 바, 상기 LDLR 및 StAR는 고령 여성의 난임 여부를 진단 및 예측할 수 있는 분자 진단 마커로 사용될 수 있다. That is, genes involved in the steroidogenic pathway, including LDLR and StAR proteins, are upregulated in cumulus cells of the pregnant group, and LDLR and StAR can be used as molecular diagnostic markers to diagnose and predict infertility in elderly women.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.

Claims (9)

  1. LDLR(low density lipoprotein receptor) 단백질 또는 이의 유전자의 mRNA의 수준을 측정하는 제제를 포함하는 고령 여성의 난임 진단용 조성물.A composition for diagnosing infertility in elderly women, comprising an agent for measuring the level of LDLR (low density lipoprotein receptor) protein or mRNA of its gene.
  2. 청구항 1에 있어서, StAR(steroidogenic acute regulatory protein), ADCY(adenylate cyclase), HSD17B1(hydroxysteroid 17-beta dehydrogenase 1)로 구성된 군에서 선택되는 단백질 또는 이의 유전자의 mRNA의 수준을 측정하는 제제를 추가로 포함하는 것인 고령 여성의 난임 진단용 조성물. The method of claim 1, further comprising an agent for measuring the level of mRNA of a protein or gene thereof selected from the group consisting of StAR (steroidogenic acute regulatory protein), ADCY (adenylate cyclase), and HSD17B1 (hydroxysteroid 17-beta dehydrogenase 1). A composition for diagnosing infertility in elderly women.
  3. 청구항 1에 있어서, 상기 단백질 수준을 측정하는 제제는 상기 단백질에 특이적으로 결합하는 항체를 포함하는 것인 고령 여성의 난임 진단용 조성물.The composition for diagnosing infertility in elderly women according to claim 1, wherein the agent for measuring the protein level includes an antibody that specifically binds to the protein.
  4. 청구항 1에 있어서, 상기 유전자의 mRNA 수준을 측정하는 제제는 상기 유전자에 특이적으로 결합하는 프라이머 쌍, 프로브 또는 안티센스 올리고뉴클레오티드인 것인 고령 여성의 난임 진단용 조성물. The composition for diagnosing infertility in elderly women according to claim 1, wherein the agent for measuring the mRNA level of the gene is a primer pair, probe, or antisense oligonucleotide that specifically binds to the gene.
  5. 청구항 1의 조성물을 포함하는 고령 여성의 난임 진단용 키트.A kit for diagnosing infertility in elderly women, comprising the composition of claim 1.
  6. LDLR(low density lipoprotein receptor) 단백질 또는 이의 유전자의 mRNA 수준을 측정하는 단계를 포함하는 고령 여성의 난임 진단을 위한 정보제공방법.A method of providing information for diagnosing infertility in elderly women, including measuring the mRNA level of LDLR (low density lipoprotein receptor) protein or its gene.
  7. 청구항 6에 있어서, StAR(steroidogenic acute regulatory protein), ADCY(adenylate cyclase), HSD17B1(hydroxysteroid 17-beta dehydrogenase 1)로 구성된 군에서 선택되는 단백질 또는 이의 유전자의 mRNA 수준을 측정하는 단계를 추가로 포함하는 것인, 고령 여성의 난임 진단을 위한 정보제공방법. The method of claim 6, further comprising measuring the mRNA level of a protein or gene thereof selected from the group consisting of steroidogenic acute regulatory protein (StAR), adenylate cyclase (ADCY), and hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1). A method of providing information for diagnosing infertility in elderly women.
  8. 청구항 6에 있어서, 상기 측정된 단백질 또는 유전자의 발현 수준이 정상 대조군 시료의 단백질 또는 유전자의 발현 수준보다 낮은 경우, 난임의 위험도가 높은 것으로 판단하는 것인 고령 여성의 난임 진단을 위한 정보제공방법.The method of claim 6, wherein if the measured expression level of the protein or gene is lower than the expression level of the protein or gene in the normal control sample, the risk of infertility is determined to be high.
  9. LDLR(low density lipoprotein receptor) 단백질과 피검 물질을 접촉시키는 단계; 및 무처리 대조군에 비하여 상기 LDLR의 발현 또는 활성 수준을 증가시킨 피검 물질을 난임 치료제로 선별하는 단계를 포함하는 고령 여성의 난임 치료제를 스크리닝하는 방법.Contacting a test substance with a low density lipoprotein receptor (LDLR) protein; And a method of screening for an infertility treatment for elderly women, comprising the step of selecting a test substance that increases the expression or activity level of the LDLR compared to an untreated control group as a treatment for infertility.
PCT/KR2023/013493 2022-09-08 2023-09-08 Biomarker for diagnosing infertility in elderly women and use thereof WO2024054087A1 (en)

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