Compositions for diagnosis of endometriosis

Abstract

The present invention can diagnose endometriosis early by measuring the relative abundance of various microbiomes within the female reproductive system corresponding to one or more classification manipulation units (OTUs).

Description

Technology Field

[0001] The present invention relates to a composition for diagnosing endometriosis using the analysis of microbial communities within the female reproductive system, and a method for diagnosing using the same. Background Technology

[0002] Endometriosis is a common gynecological disease that affects adolescent and reproductive women. It is characterized by the deposition and proliferation of endometrial cells outside the uterine cavity, in which endometrial tissue, which should be lining the inside of the uterus, is located outside the cavity. While endometriosis is often asymptomatic, some patients may experience symptoms such as menstrual pain, dyspareunia, and irregular bleeding. Furthermore, reports indicate that endometriosis was diagnosed in 9 to 50% of patients who underwent surgery for infertility, leading to its identification as a cause of infertility. Although endometriosis itself does not necessarily cause infertility, severe endometriosis can be considered a disease that hinders pregnancy in various ways.

[0003] Currently, standard tests available for diagnosing endometriosis include laparoscopic examination and histological examination of lesions through it, as well as various other tests such as physical examination, blood marker tests, ultrasound, CT, or MRI. However, imaging techniques such as ultrasound are unreliable in the diagnosis and staging of endometriosis, and a definitive diagnosis of endometriosis by laparoscopic examination is only confirmed when the disease has progressed to the point where lesions can be directly visualized.

[0004] Accurate diagnosis in the early stages is essential for the successful treatment of endometriosis. While serum CA-125 testing, an existing diagnostic method, shows elevated levels in some women with endometriosis, it is not specific and has limitations due to its low sensitivity and specificity. Thus, there is currently a lack of technology capable of accurately diagnosing endometriosis in its early stages.

[0005] With the recent development of Next-Generation Sequencing (NGS), it has become possible to analyze all microorganisms present in a specific environment by determining the nucleotide sequence of microbial communities based on the 16S rRNA gene, which preserves the most microbial information for the diagnosis of various diseases. Consequently, therapeutic methods such as probiotics, fecal transplants, and antibiotics are being developed to improve diseases by maintaining the balance of microbial community composition, including regulating the composition of microbial communities contained in various organs within the human body.

[0006] Accordingly, the inventor analyzed the bacterial environment in the endometrium or vagina of the female reproductive system and, through this, determined that endometriosis can be diagnosed by quantifying microorganisms at each location of the female reproductive system, thereby completing the present invention. The problem to be solved

[0007] One objective of the present invention is to provide a composition for diagnosing endometriosis.

[0008] Another objective of the present invention is to provide a kit for diagnosing endometriosis.

[0009] Another objective of the present invention is to provide a method for providing information regarding the diagnosis of endometriosis.

[0010] Another objective of the present invention is to provide a device for diagnosing endometriosis.

[0011] delete

[0012] delete

[0013] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below. means of solving the problem

[0014] Various embodiments described herein are described with reference to the drawings. In the following description, for a complete understanding of the invention, various specific details, such as specific forms, compositions, and processes, are described. However, specific embodiments may be practiced without one or more of these specific details, or in combination with other known methods and forms. In other examples, known processes and manufacturing techniques are not described as specific details so as not to unnecessarily obscure the invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that the particular features, forms, compositions, or characteristics described in association with the embodiment are included in one or more embodiments of the invention. Accordingly, the context of "in one embodiment" or "an embodiment" expressed at various places throughout this specification does not necessarily represent the same embodiment of the invention. Additionally, particular features, forms, compositions, or characteristics may be combined in any suitable way in one or more embodiments.

[0015] Unless otherwise specifically defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.

[0017] According to one embodiment of the present invention, the invention relates to a composition for diagnosing endometriosis.

[0018] In the present invention, the composition may include a preparation for quantifying strains of the genus Sutterella.

[0019] In the present invention, the Sutterella genus strain may be at least one strain selected from the group consisting of Sutterella massiliensis, Sutterella stercoricanis, and Sutterella wadsworthensis, but is not limited thereto.

[0020] In the present invention, the composition comprises Rhodanobacter glycinis, Prevotella timonensis, Corynebacterium tuberculostearicum, Howardella ureilytica, Acidibrevibacterium fodinaquatile, Actinomadura rifamycini, Aeromicrobium panaciterrae, Afifella pfennigii, Altererythrobacter rigui, Arenimonas subflava, and Campylobacter ureoliticus ureolyticus), Chryseobacterium greenlandense, Desulfonatronum alkalitolerans, Desulfovibrio piger, Dongia mobilis, Edaphobacter dinghuensis, Insolitispirillum peregrinum, Lysobacter dokdonensis, Lysobacter niabensis, Mycobacterium marinum, Natranaerovirga pectinivora, Nocardioides alpinus, Pelobacter carbinolicus, Povalibacter uvarum,It may further include, but is not limited to, a preparation for quantifying at least one strain selected from the group consisting of Pseudoduganella violaceinigra, Pseudoflavonifractor capillosus, Pseudoxanthomonas wuyuanensis, Smithella propionica, Sphingobium mellinum, Streptomyces aomiensis, and Streptomyces chlorus.

[0021] In the present invention, the "sutterella genus strain" is a genus of Gram-negative, anaerobic, non-spore-forming bacteria of the Sutterellaaceae lineage belonging to the Betaproteobacteria class. The sutterella genus strain includes species such as Sutterella parvirubra, Sutterella massiliensis, Sutterella stercoricanis, and Sutterella wadsworthensis.

[0022] In the present invention, the "Rhodanobacter glycinis strain" is a species of the genus Rhodanobacter and is a Gram-negative, non-spore-forming, and motile bacterium.

[0023] In the present invention, the "Prevotella timonensis strain" corresponds to a bacterium belonging to the genus Prevotella, which is a Gram-negative bacterium.

[0024] In the present invention, the "Corynebacterium tuberculostearicum strain" corresponds to a bacterium belonging to the genus Corynebacterium that is Gram-positive and mostly aerobic.

[0025] In the present invention, the "Howardella ureilytica strain" is a species of the genus Howardella and is a Gram-positive bacterium.

[0026] In the present invention, the "Acidibrevibacterium fodinaquatile strain" corresponds to a bacterium belonging to the genus Acidibrevibacterium that is acidophilic within the lineage of the family Acetobacteriaceae.

[0027] In the present invention, the "Actinomadura rifamycini strain" corresponds to an aerobic, Gram-positive bacterium belonging to the genus Actinomadura, which is one of the four genera of actinomycetes belonging to the Thermomonosporaceae lineage.

[0028] In the present invention, the "Aeromicrobium panaciterrae strain" is a bacterium belonging to the genus Aeromicropium, which is a Gram-positive, aerobic, non-spore-forming, and non-motile bacterium of the Nocardioidaceae lineage.

[0029] In the present invention, the "Afifella pfennigii strain" corresponds to a bacterium belonging to the genus Afifella, which is an anaerobic microorganism.

[0030] In the present invention, the "Altererythrobacter rigui strain" corresponds to a Gram-negative, aerobic, non-motile bacterium belonging to the genus Altererythrobacter.

[0031] In the present invention, the "Arenimonas subflava strain" is a Gram-negative, aerobic, and non-motile bacterium of the genus Arenimonas.

[0032] In the present invention, the "Campylobacter ureolyticus strain" is also referred to as Bacterioides ureolyticus and corresponds to a Gram-negative, obligate anaerobic bacterium.

[0033] In the present invention, the "Chryseobacterium greenlandense strain" is Gram-negative, lacks flagella, and has an average cell volume of 0.08 μm 3 It corresponds to rod-shaped bacteria.

[0034] In the present invention, the "Desulfonatronum alkalitolerans strain" is a bacterium belonging to the genus Desulfonatronum, which is a Gram-negative and extremely alkaline bacterium of the Desulfovibrionaceae lineage.

[0035] In the present invention, the "Desulfovibrio piger strain" corresponds to a bacterium belonging to the genus Desulfovibrio, which is a Gram-negative sulfate-reducing bacterium.

[0036] In the present invention, the "Dongia mobilis strain" corresponds to a Gram-negative, aerobic, and heterotrophic bacterium belonging to the Rhodospirillaceae lineage.

[0037] In the present invention, the "Edaphobacter dinghuensis strain" is an acidophilic, mesophilic bacterium belonging to the genus Edaphobacter.

[0038] In the present invention, the "Insolitispirillum peregrinum strain" corresponds to a Gram-negative, spiral-shaped bacterium belonging to the genus Insolitispirillum.

[0039] In the present invention, the "Lysobacter dokdonensis strain" corresponds to a bacterium included in the genus Lysobacter, which belongs to the family Xanthomonadaceae within Gammaproteobacteria.

[0040] In the present invention, the "Lysobacter niabensis strain" corresponds to bacteria included in the genus Lysobacter, which belongs to the family Xanthomonadaceae within Gammaproteobacteria.

[0041] In the present invention, the "Mycobacterium marinum strain" corresponds to a bacterium belonging to the genus Mycobacterium, previously known as Mycobacterium balnei.

[0042] In the present invention, the "Natranaerovirga pectinivora strain" is a bacterium belonging to the genus Natranaerovirga.

[0043] In the present invention, the "Nocardioides alpinus strain" corresponds to a Gram-positive, rod-shaped, psychrophilic, and non-motile bacterium belonging to the genus Nocardioides.

[0044] In the present invention, the "Pelobacter carbinolicus strain" is a type of bacterium that ferments 2,3-butanediol and acetoin and corresponds to a Gram-negative, anaerobic bacterium.

[0045] In the present invention, the "Povalibacter uvarum strain" corresponds to a Gram-negative, rod-shaped, motile bacterium belonging to the genus Povalibacter.

[0046] In the present invention, the "Pseudoduganella violaceinigra strain" is a mesophilic bacterium of the genus Duganella of the family Oxalobacteriaceae isolated from forest soil in Yunnan Province, China.

[0047] In the present invention, the "Pseudoflavonifractor capillosus strain" corresponds to a Gram-negative anaerobic bacterium belonging to the genus Pseudoflavonifractor.

[0048] In the present invention, the "Pseudoxanthomonas wuyuanensis strain" corresponds to a Gram-negative, aerobic, motile bacterium having a single polar flagellum.

[0049] In the present invention, the "Smithella propionica strain" corresponds to an anaerobic bacterium belonging to the genus Smithella.

[0050] In the present invention, the "Sphingobium mellinum strain" is a Gram-negative bacterium belonging to the genus Sphingobium.

[0051] In the present invention, the "Streptomyces aomiensis strain" corresponds to a Gram-positive, aerobic bacterium belonging to the genus Yinghuangia.

[0052] In the present invention, the "Streptomyces chlorus strain" corresponds to a Gram-positive bacterium belonging to the genus Streptomyces isolated from soil.

[0053] The present invention can predict the risk of developing endometriosis in advance by metagenome analysis of the strain within the female reproductive system.

[0054] In the present invention, the metagenome is defined by Handelsman et al. as a set of genomes of all microorganisms existing in a specific natural environment, and refers to the sum of genomes including viruses, bacteria, fungi, etc., within a specific isolated range. In particular, the metagenome does not refer to a single species' genome or genome, but rather to a type of mixed genome consisting of the genomes of all species within a single environmental system. It is the subject of a technique to identify all species within a single environment and elucidate their interactions and metabolic processes. In the present invention, metagenome analysis was performed using a biological sample isolated from a female reproductive organ.

[0055] In the present invention, the preparation for quantifying the strain may be included in a biological sample of a target individual for the diagnosis of endometriosis. In the present invention, the quantification may be performed by measuring the relative abundance of the strain.

[0056] In the present invention, the relative abundance is obtained from the composition of microbial communities within a specific organ of the target individual, and refers to the value obtained by dividing the count of each OTU of the diagnostic target strain identified in the specific organ of the target individual by the total number of strains included in the total strain population within the specific organ.

[0057] In the present invention, the term "desired individual" refers to an individual that has developed or is highly likely to develop endometriosis, and may be a mammal including humans, and may be selected from the group consisting of, for example, humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cattle, horses, pigs, sheep, and goats, and preferably may be a human, but is not limited thereto.

[0058] In the present invention, the organ refers to a unit constituting the body of a multicellular organism, which forms an integrated structure composed of various tissues and performs a specific function. Examples include reproductive organs, circulatory organs, digestive organs, endocrine organs, immune organs, integumentary organs, lymphatic organs, musculoskeletal organs, nervous organs, respiratory organs, skeletal organs, or urinary organs. The specific organ in the present invention may be a reproductive organ, preferably a female reproductive organ, but is not limited thereto.

[0059] In the present invention, the female reproductive organ may include at least one selected from the group consisting of the endometrium, cervix, ovary, fallopian tube, vagina, and abdominal cavity, but is not limited thereto.

[0060] In the present invention, the strains of the genus Sutterella, Rhodanobacter glycinis, Prevotella timonensis, Corynebacterium tuberculostearicum, Howardella ureilytica, Acidibrevibacterium fodinaquatile, Actinomadura rifamycini, Aeromicrobium panaciterrae, Afifella pfennigii, Altererythrobacter rigui, Arenimonas subflava, and Campylobacter Campylobacter ureolyticus, Chryseobacterium greenlandense, Desulfonatronum alkalitolerans, Desulfovibrio piger, Dongia mobilis, Edaphobacter dinghuensis, Insolitispirillum peregrinum, Lysobacter dokdonensis, Lysobacter niabensis, Mycobacterium marinum, Natranaerovirga pectinivora, Nocardioides alpinus, Pelobacter Pelobacter carbinolicus, Povalibacter uvarum,At least one strain selected from the group consisting of Pseudoduganella violaceinigra, Pseudoflavonifractor capillosus, Pseudoxanthomonas wuyuanensis, Smithella propionica, Sphingobium mellinum, Streptomyces aomiensis, and Streptomyces chlorus may be a strain present in a specific organ of the target individual, preferably a strain present in the female reproductive organs, and most preferably a strain present in the vagina or endometrium, but is not limited thereto.

[0061] In one example of the present invention, the genus Suterella strain may be a strain present in the female reproductive organs, preferably a strain present in the vagina, but is not limited thereto. In the present invention, the genus Suterella strain may be at least one strain selected from the group consisting of Suterella masiliensis, Suterella stercoricanis, and Suterella wadswottensis, but is not limited thereto.

[0062] In another example of the present invention, Rhodanobacter glycinis, Prevotella timonensis, Corynebacterium tuberculostearicum, Hoardella ureilitica, Acidibrevibacterium podinaquatile, Actinomadura rifamaicini, Aeromicropium panasitherae, Apipella penigii, Alterellitrobacter ligui, Arenemonas subflava, Campylobacter ureoliticus, Cryseobacterium chenlandens, Desulfonatronum alkalitorans, Desulfovibrio fiser, Dongia mobilis, Edapobacter dinghuensis, Insoritis spirillum peregrinum, Lysobacter dokdonensis, Lysobacter niabensis, Mycobacterium marinum, Natran-aerobigra pectiniphora, Nacardioides At least one strain selected from the group consisting of Alpinus, Pelobacter carbinoricus, Povaribacter uvarum, Pseudoduganella violasenigra, Pseudoflavonifractor capillosus, Pseudoxanthomonas milganesis, Smitella propionica, Sphingopium melinum, Streptomyces aomiensis, and Streptomyces chlorus may be a strain present in the female reproductive system, preferably a strain present in the endometrium, but is not limited thereto.

[0063] In the present invention, the operational taxonomic units (OTUs) may be obtained by performing cluster analysis in which 94 to 97% or more similarity is confirmed in the 16s rRNA gene of the strain to be diagnosed, taking the most representative sequence as a seed sequence, and analyzing the seed sequence from a biological sample of a target individual associated with endometriosis among them; preferably, they may be obtained from the operational taxonomic units (OTUs) by performing cluster analysis in which 94 to 97% or more similarity is confirmed in the V3-V4 region of the 16s rRNA gene of the strain.

[0064] In the present invention, the OTU analysis is one of the methods for classifying nucleotide sequences based on similarity between nucleotide sequences obtained from samples of an individual or group of organisms to be classified, and evaluating microbial diversity within a sample using this as a unit. Recently, it has become possible to serialize the bacterial composition of the human microbiome using a method based on 16S ribosomal RNA (16S rRNA) nucleotide sequences, and it has become possible to analyze the nucleotide sequences of strains through next-generation sequencing (NGS) platforms, thereby enabling the evaluation of microbial diversity within a sample and the comparison of microbial community compositions of different samples.

[0065] In the present invention, the relative abundance can be obtained through analysis of OTUs, and each OTU is considered as one strain species. In the present invention, the microbial diversity and the relative abundance of a specific microorganism in a sample can be obtained through analysis of OTUs, but are not limited thereto.

[0066] In the present invention, the agent for quantifying the strain may include a agent for measuring relative abundance, which may include one or more selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to the 16S rRNA gene of the strain, and preferably may include one or more selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to the V3-V4 region of the 16S rRNA gene of the strain, but is not limited thereto.

[0067] In the present invention, the 16S rRNA gene is an rRNA that constitutes the 30S subunit of a prokaryotic ribosome, includes hypervariable regions V1 to V9, and has a length of approximately 1,500 nucleotides. Prokaryotes can be identified by comparing the sequences of the 16S rRNA, and while the sequences of the RNA are mostly significantly conserved, high nucleotide sequence diversity is observed in some regions. In particular, since there is almost no diversity among the same species but diversity is observed among different species, it is used for biological identification. It is particularly useful for the identification and classification of organisms that are impossible or difficult to culture, or organisms that have never been reported. In one embodiment of the present invention, the V3 and V4 regions of the 16S rRNA gene may be used to characterize OTUs, but are not limited thereto.

[0068] In the present invention, the "primer" is a fragment that recognizes a target gene sequence and includes a forward and reverse primer pair, but preferably is a primer pair that provides analysis results having specificity and sensitivity. High specificity can be conferred 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 it amplifies only the target gene sequence containing the complementary primer binding site and does not induce non-specific amplification.

[0069] In the present invention, the term "probe" refers to a substance capable of specifically binding to a target substance to be detected within a sample, and means a substance capable of specifically confirming the presence of the target substance within the sample through said binding. The type of probe is not limited to substances commonly used in the industry, but preferably may be PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA, or DNA, and most preferably PNA. More specifically, the probe may be a biomaterial derived from an organism or similar, or manufactured in vitro, and may be, for example, 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.

[0070] In the present invention, "LNA (Locked nucleic acids)" refers to nucleic acid analogs containing a 2'-O, 4'-C methylene bridge [J Weiler, J Hunziker and J Hall Gene Therapy (2006) 13, 496.502]. LNA nucleosides contain common nucleic acid bases of DNA and RNA and can form base pairs according to the Watson-Crick base pairing rule. However, due to the 'locking' of the molecule caused by the methylene bridge, LNAs are unable to form an ideal shape in Watson-Crick bonding. When LNAs are included in DNA or RNA oligonucleotides, LNAs can pair more quickly with complementary nucleotide chains, thereby increasing the stability of the double helix.

[0071] In the present invention, "antisense" refers to an oligomer having a backbone between nucleotide base sequences and subunits, wherein the antisense oligomer hybridizes with a target sequence within RNA by Watson-Crick base pairing, thereby allowing the formation of a mRNA and RNA:oligomer heterodimer within the target sequence. The oligomer may have exact sequence complementarity or approximate complementarity with respect to the target sequence.

[0072] Since the 16s rRNA gene information of the strain according to the present invention is known, a person skilled in the art can easily design a primer, probe, or antisense nucleotide that specifically binds to the gene based on this.

[0073] In the present invention, "endometriosis" refers to a disease in which glandular and stromal tissue structures observed in the endometrium appear in organs or other parts of the body outside the uterus; it is a chronic disease that causes dysmenorrhea, chronic pelvic pain, and reduced reproductive capacity. To date, the pathophysiology and causes have not been clearly elucidated, and there is no complete cure; consequently, if the diagnosis and treatment of endometriosis are delayed, complications such as chronic pelvic pain or infertility may occur. Despite being a relatively common gynecological disease, there is a lack of diagnostic tools with sufficient sensitivity and specificity, leading to ongoing research. Currently, the most standard method for diagnosing endometriosis is considered to be visualizing lesions within the abdominal cavity via laparoscopy and, if necessary, performing a histological examination; however, this method has limitations in that it is difficult to diagnose early.

[0074] The present invention predicts the risk of developing endometriosis in advance through metagenomic analysis of specific strains present in the female reproductive system, thereby enabling early diagnosis and prediction of risk groups for endometriosis. Through appropriate management, the onset of the disease can be delayed or prevented, and furthermore, even after the disease develops, treatment efficacy can be improved through early diagnosis.

[0076] According to another embodiment of the present invention, the invention relates to a kit for diagnosing endometriosis comprising the diagnostic composition of the present invention.

[0077] In the present invention, the "kit" refers to a tool capable of evaluating the expression level of a biomarker by labeling a probe or antibody that specifically binds to a biomarker component with a detectable label. It includes not only direct labeling of a detectable substance related to a probe or antibody through reaction with a substrate, but also indirect labeling in which a label that develops color through reactivity with another directly labeled reagent is conjugated. It may include a color-developing substrate solution, washing solution, and other solutions that react with the label for color development, and may be manufactured to include the reagent components used. In the present invention, the kit may be a kit containing essential elements necessary for performing RT-PCR, and in addition to specific primer pairs for the marker gene, it may include test tubes, reaction buffer, deoxyribonucleotides (dNTPs), Taq polymerase, reverse transcriptase, DNase, RNase inhibitor, sterile water, etc. Furthermore, the kit may be a kit for detecting genes of various strains for the diagnosis of endometriosis, containing essential elements necessary for performing DNA chip analysis. A DNA chip kit comprises a substrate to which cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may comprise cDNA corresponding to a quantitative control gene or a fragment thereof. The kit of the present invention is not limited thereto, provided that it is known in the art.

[0078] In the present invention, the kit may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.

[0079] The kit of the present invention may further include one or more other component compositions, solutions, or devices suitable for the analysis method. For example, the kit of the present invention may further include essential elements necessary to perform a reverse transcription polymerase chain reaction. The reverse transcription polymerase chain reaction kit includes a primer pair specific to a gene encoding a marker protein. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of the gene and may have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also include a primer specific to the nucleic acid sequence of a control gene. Furthermore, the reverse transcription polymerase chain reaction kit may include a test tube or other suitable container, a reaction buffer (with varying pH and magnesium concentration), deoxyribonucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC-water, sterile water, etc.

[0080] In addition, the diagnostic kit of the present invention may include essential elements necessary for performing DNA chip operations. The DNA chip kit may include a substrate to which cDNA or oligonucleotides corresponding to a gene or a fragment thereof are attached, and reagents, preparations, enzymes, etc., for producing fluorescently labeled probes. Additionally, the substrate may include cDNA or oligonucleotides corresponding to a control gene or a fragment thereof.

[0081] In addition, the diagnostic kit of the present invention may include essential elements necessary for performing ELISA. The ELISA kit includes an antibody specific to the protein. The antibody is an antibody that has high specificity and affinity for the marker protein and has little cross-reactivity with other proteins, and is a monoclonal antibody, a polyclonal antibody, or a recombinant antibody. Additionally, the ELISA kit may include an antibody specific to a control protein. Furthermore, the ELISA kit may include reagents capable of detecting the bound antibody, such as a labeled secondary antibody, chromophores, an enzyme (e.g., conjugated with the antibody) and its substrate, or other substances capable of binding to the antibody.

[0082] In the diagnostic kit of the present invention, a nitrocellulose membrane, a PVDF membrane, a well plate synthesized from polyvinyl resin or polystyrene resin, a glass slide glass, etc., may be used as a fixative for the antigen-antibody binding reaction, but is not limited thereto.

[0083] In addition, the label of the secondary antibody in the diagnostic kit of the present invention is preferably a conventional chromogenic agent that produces a color reaction, and labels such as fluorescein and dyes such as HRP (horseradish peroxidase), alkaline phosphatase, colloid gold, FITC (poly L-lysine-fluorescein isothiocyanate), and RITC (rhodamine-B-isothiocyanate) may be used, but are not limited thereto.

[0084] In addition, for the diagnostic kit of the present invention, it is preferable to use a chromogenic substrate to induce color development depending on the label that performs the color reaction, and TMB (3,3',5,5'-tetramethylbezidine), ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)], OPD (o-phenylenediamine), etc. may be used. At this time, it is more preferable that the chromogenic substrate be provided in a state dissolved in a buffer solution (0.1 M NaAc, pH 5.5). A chromogenic substrate such as TMB is degraded by HRP used as a label for the secondary antibody conjugate to produce a chromogenic precipitate, and the presence or absence of the marker proteins is detected by visually confirming the degree of precipitation of this chromogenic precipitate.

[0085] In the diagnostic kit of the present invention, the washing solution preferably comprises phosphate buffer solution, NaCl, and Tween 20, and a buffer solution (PBST) composed of 0.02 M phosphate buffer solution, 0.13 M NaCl, and 0.05% Tween 20 is more preferably used. After the antigen-antibody binding reaction, a secondary antibody is reacted with the antigen-antibody conjugate, and then an appropriate amount of the washing solution is added to the immobilizer to wash 3 to 6 times. A sulfuric acid solution (H2SO4) may preferably be used as the reaction stopping solution.

[0087] According to another embodiment of the present invention, the invention relates to a method for providing information for the diagnosis of endometriosis.

[0088] The method of the present invention may include the step of quantifying a specific strain in a biological sample isolated from a specific organ of the target individual.

[0089] The quantifying step of the present invention may be a step performed by measuring the relative abundance of the strain.

[0090] The above method of the present invention may be used to screen for the presence or absence of endometriosis in a biological sample separated from the reproductive organ of a target individual.

[0091] In the present invention, the term "desired individual" refers to an individual that has developed or is highly likely to develop endometriosis, and may be a mammal including humans, and may be selected from the group consisting of, for example, humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cattle, horses, pigs, sheep, and goats, and preferably may be a human, but is not limited thereto.

[0092] In the present invention, the reproductive organ may be at least one selected from the group consisting of the endometrium, cervix, ovary, fallopian tube, vagina, and abdominal cavity, but is not limited thereto.

[0093] In the present invention, the biological sample refers to any substance, biological body fluid, tissue, or cell obtained from or derived from an individual, including whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, and bronchial aspirate. It may be one or more selected from the group consisting of synovial fluid, joint aspirate, organ secretions, cell, cell extract and cerebrospinal fluid, and preferably may be obtained from tissue or cell within the reproductive organ, but is not limited thereto.

[0094] In the present invention, the specific strains are strains of the genus Sutterella, Rhodanobacter glycinis, Prevotella timonensis, Corynebacterium tuberculostearicum, Howardella ureilytica, Acidibrevibacterium fodinaquatile, Actinomadura rifamycini, Aeromicrobium panaciterrae, Afifella pfennigii, Altererythrobacter rigui, Arenimonas subflava, and Campylobacter Campylobacter ureolyticus, Chryseobacterium greenlandense, Desulfonatronum alkalitolerans, Desulfovibrio piger, Dongia mobilis, Edaphobacter dinghuensis, Insolitispirillum peregrinum, Lysobacter dokdonensis, Lysobacter niabensis, Mycobacterium marinum, Natranaerovirga pectinivora, Nocardioides alpinus, Pelobacter Pelobacter carbinolicus, Povalibacter uvarum,It may be at least one strain selected from the group consisting of Pseudoduganella violaceinigra, Pseudoflavonifractor capillosus, Pseudoxanthomonas wuyuanensis, Smithella propionica, Sphingobium mellinum, Streptomyces aomiensis, and Streptomyces chlorus.

[0095] In one example of the present invention, the specific strain may include a strain of the genus Suterella present in the female reproductive organs, preferably in the vagina.

[0096] In another example of the present invention, the specific strain is Rhodanobacter glycinis, Prevotella timonensis, Corynebacterium tuberculostearicum, Hoardella ureilitica, Acidibrevibacterium podina quatile, Actinomadura rifamaicini, Aeromicropium panasitherae, Apipella penigii, Alteritrobacter ligui, Arenemonas subflava, Campylobacter ureoliticus, Cryseobacterium greenlandens, Desulfonatronum alkalitorans, Desulfovibrio fischer, Dongia mobilis, Edapobacter dinghuensis, Insoritis spirillum peregrinum, Lysobacter dokdonensis, Lysobacter niabensis, Mycobacterium marinum, present in the female reproductive tract, preferably in the endometrium. It may include at least one strain selected from the group consisting of Natran-aerobigra pectiniphora, Nacardioides alpinus, Pelobacter carbinoricus, Povaribacter uvarum, Pseudoduganella violasenigra, Pseudoflavonifractor capillosus, Pseudocanthomonas yuganesis, Smitella propionica, Sphingopium melinum, Streptomyces aomiensis, and Streptomyces chlorus.

[0097] The preparation used in the step of quantifying the strain in the present invention may include one or more selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to the 16S rRNA gene of the strain, but is not limited thereto.

[0098] In the present invention, the measurement of the expression level of the 16S rRNA gene of the strain may be performed by reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase chain reaction (Competitive RT-PCR), real-time reverse transcription polymerase chain reaction (Real-time RT-PCR), RNase protection assay (RPA), Northern blotting, or DNA chip.

[0099] In the present invention, if the relative abundance of a specific strain measured for a biological sample of the target individual is higher or lower than that of the control group, it can be predicted that endometriosis has developed in the target individual or is highly likely to develop.

[0100] In one example of the present invention, if the relative abundance of strains of the genus Suterella measured in a biological sample within the vagina of the target individual is higher than that of the control group, it can be predicted that endometriosis has developed in the target individual or is highly likely to develop.

[0101] In another example of the present invention, if the relative abundance of Rhodanobacter glycinis strains measured in a biological sample within the endometrium of the target individual is lower than that of the control group, it can be predicted that the target individual has developed or is highly likely to develop endometriosis.

[0102] In another example of the present invention, if the relative abundance of the Prevotella timonensis strain measured in a biological sample within the endometrium of the target individual is higher than that of the control group, it can be predicted that the target individual has developed endometriosis or is highly likely to develop it.

[0103] In another example of the present invention, if the relative abundance of the Corynebacterium tuberculostearicum strain measured in the endometrium of the target individual or in a biological sample of the endometrium is higher than that of the control group, it can be predicted that the target individual has developed endometriosis or is highly likely to develop it.

[0104] In another example of the present invention, if the relative abundance of the Hoardella ureilitica strain measured in a biological sample within the endometrium of the target individual is higher than that of the control group, it can be predicted that the target individual has developed endometriosis or is highly likely to develop it.

[0105] In another example of the present invention, if the relative abundance of the Cryseobacterium greenlandens strain measured in a biological sample within the endometrium of the target individual is lower than that of the control group, it can be predicted that the target individual has developed endometriosis or is highly likely to develop it.

[0106] In another example of the present invention, if the relative abundance of Campylobacter ureoliticus strains measured in biological samples within the endometrium of the target individual is higher than that of the control group, it can be predicted that the target individual has developed or is highly likely to develop endometriosis.

[0107] In the present invention, the term "control group" may refer to a normal control group in which endometriosis has not occurred, or to the average or median value of the relative abundance of the said strain in a sample from an individual that has a teratoma, which is an ovarian tumor that does not respond to hormones, but has not developed endometriosis. The relative abundance of a specific strain in the control group can be compared with the relative abundance of a specific strain in a biological sample derived from an endometriosis patient that is the subject of analysis, and the presence of endometriosis can be diagnosed by determining whether there is a significant change in the said relative abundance.

[0108] In the information provision method of the present invention, descriptions regarding endometriosis, strains, relative abundance, primers, probes, antisense nucleotides, etc. are duplicated from what was previously described, so in order to avoid excessive complexity of the specification, detailed descriptions thereof are omitted below.

[0110] According to another embodiment of the present invention, the invention relates to a device for diagnosing endometriosis.

[0111] The measuring unit of the diagnostic device of the present invention can measure the relative abundance of a specific strain corresponding to one or more operational taxonomic units (OTUs) for a biological sample obtained from a target individual.

[0112] In the present invention, the strains are strains of the genus Suterella, Rhodanobacter glycinis, Prevotella timonensis, Corynebacterium tuberculosteacum, Hoardella ureilitica, Acidibrevibacterium podina quatile, Actinomadura rifamaicini, Aeromicropium panasitherae, Apipella penigii, Alteritrobacter ligui, Arenemonas subflava, Campylobacter ureoliticus, Cryseobacterium greenlandens, Desulfonatronum alkalitorans, Desulfovibrio fischer, Dongia mobilis, Edapobacter dinghuensis, Insoritis spirillum peregrinum, Lysobacter dokdonensis, Lysobacter niabensis, Mycobacterium marinum, and Natran aerovigra At least one strain selected from the group consisting of Pectiniphora, Nacardioides alpinus, Pelobacter carbinoricus, Povaribacter uvarum, Pseudoduganella violasenigra, Pseudoflavonifractor capillosus, Pseudocanthomonas milganesis, Smitella propionica, Sphingopium melinum, Streptomyces aomiensis, and Streptomyces chlorus may be a strain present in the female reproductive system, preferably a strain present in the endometrium or vagina, but is not limited thereto.

[0113] In the present invention, the genus Suterella strain may include species such as Suterella parvirubra, Suterella masuiliensis, Suterella stercoricanis, and Suterella wazwottensis, and preferably may be Suterella masuiliensis, Suterella stercoricanis, or Suterella wazwottensis, but is not limited thereto.

[0114] In the present invention, the object of the above invention may be selected from the group consisting of humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cattle, horses, pigs, sheep, and goats, and preferably may be humans, but is not limited thereto.

[0115] In the present invention, the biological samples are whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, and trachea. It may be one or more selected from the group consisting of organ secretions, cells, cell extracts and cerebrospinal fluid, but is not limited thereto.

[0116] The preparation used in the measuring unit of the diagnostic device of the present invention may include one or more selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to the 16S rRNA gene of the strain, but is not limited thereto.

[0117] In the measurement unit of the present invention, the presence or absence of endometriosis or the possibility of developing endometriosis can be predicted by confirming the relative abundance of the corresponding strain using the preparation.

[0118] The diagnostic device of the present invention may further include a detection unit that predicts and outputs the presence or absence of endometriosis or the likelihood of developing endometriosis in the target individual based on the relative abundance ratio obtained by quantifying the corresponding strain obtained from the measurement unit.

[0119] In the present invention, the detection unit can accurately diagnose endometriosis at an early stage by generating and classifying information regarding the presence or absence of endometriosis or the possibility of developing endometriosis according to the category of the relative abundance of the corresponding strain obtained from the measurement unit.

[0120] The detection unit of the present invention can predict and output that there is a high probability of endometriosis developing in the target individual when the relative abundance level of a specific strain measured by the measurement unit is higher or lower than that of the control group.

[0121] In one example of the present invention, the detection unit may predict and output that there is a high probability of endometriosis developing in the target individual when the relative abundance level of the genus Suterella measured by the measurement unit is higher than that of the control group.

[0122] In another example of the present invention, the detection unit may predict and output that there is a high probability of endometriosis developing in the target individual when the relative abundance level of the Rhodanobacter glycinis strain measured by the measurement unit is lower than that of the control group.

[0123] In another example of the present invention, the detection unit may predict and output that there is a high probability of endometriosis developing in the target individual when the relative abundance level of the Prevotella timonensis strain measured by the measurement unit is higher than that of the control group.

[0124] In another example of the present invention, the detection unit may predict and output that there is a high probability of endometriosis developing in the target individual when the relative abundance level of the Corynebacterium tuberculostearicum strain measured by the measurement unit is higher than that of the control group.

[0125] In another example of the present invention, the detection unit may predict and output that there is a high probability of endometriosis developing in the target individual when the relative abundance level of the Hoardella ureilitica strain measured by the measurement unit is higher than that of the control group.

[0126] In another example of the present invention, the detection unit may predict and output that there is a high probability of endometriosis developing in the target individual when the relative abundance level of the Cryseobacterium greenlandens strain measured by the measurement unit is lower than that of the control group.

[0127] In another example of the present invention, the detection unit may predict and output that there is a high probability of endometriosis developing in the target individual when the relative abundance level of the Campylobacter ureoliticus strain measured by the measurement unit is higher than that of the control group.

[0128] In the diagnostic device of the present invention, descriptions regarding endometriosis, relative abundance, strains, control groups, etc. are the same as those described above, and are therefore omitted to avoid excessive complexity in this specification.

[0129] delete

[0130] delete

[0131] delete

[0132] delete

[0133] delete

[0134] delete

[0135] delete

[0136] delete

[0137] delete

[0138] delete

[0139] delete

[0140] delete

[0141] delete

[0142] delete

[0143] delete

[0144] delete

[0145] delete

[0146] delete

[0147] delete

[0148] delete

[0149] delete

[0150] delete

[0151] delete

[0152] delete

[0153] delete

[0154] delete

[0155] delete

[0156] delete

[0157] delete Effects of the invention

[0158] The present invention predicts the risk of developing endometriosis in advance through metagenomic analysis of the female reproductive system, particularly the endometrium or vaginal microbiome, thereby enabling early diagnosis and prediction of the endometriosis risk group and delaying the onset of the disease or preventing it through appropriate management. Brief explanation of the drawing

[0159] Figure 1 is a figure showing the results of comparing and confirming the relative abundance of the genus Suterella in the vagina of the female reproductive organs of the EMS group and the DER group through OTU analysis according to one embodiment of the present invention. FIGS. 2 to 7 are figures showing the results of comparing and confirming the relative abundance of Rhodanobacter glycinis, Prevotella timonensis, Corynebacterium tuberculostearicum, Hoardella ureilitisca, Criseobacterium greenlandens, or Campylobacter ureoliticus strains in the endometrium of the female reproductive organs of the EMS group and the DER group through OTU analysis according to one embodiment of the present invention. Specific details for implementing the invention

[0160] The present invention will be described in more detail below through examples. These examples are intended solely to explain the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the invention.

[0162] Preparation Example: Preparation of Samples

[0163] All experiments in this study were conducted with the approval of the Institutional Review Board (IRB) of Yonsei University College of Medicine, and all samples were collected after obtaining written consent from patients with endometriosis. Vaginal or endometrial samples were obtained using the OMNIGENE-vaginal swab kit (#OMR-130), a vaginal bacterial collection kit, and peritoneal fluid was collected directly and stored in sterile tubes for preparation.

[0165] Example 1: OTU (operational taxonomic unit) analysis

[0166] After sequencing of the MiSeq raw data was completed, the data was classified by sample using index sequences, and FASTQ files were generated for each sample. Adapter sequences were removed using the Fastp program, and error correction was performed on regions where two reads overlapped. The paired-end data separated by sample were assembled into a single sequence using FLASH (1.2.11). Sequences with a length of less than 400 bp or more than 500 bp were removed. The obtained sequences were then removed using CD-HIT-OTU, a CD-HIT-EST-based OTU analysis program, to remove low-quality sequences, ambiguous sequences, and chimeric sequences considered to be sequencing errors. Subsequently, sequences with a sequence similarity of 97% or more were clustered to form species-level OTUs.

[0167] Representative sequences of each OTU were subjected to BLASTN (v2.9.0+) on the reference DB (NCBI 16S Microbial) to perform taxonomic assignment with the subject organism information that had the highest similarity. At this time, if the query coverage of the best hit matching the DB was less than 85% or the identity of the matched region was less than 85%, the taxonomy was not defined. By targeting the V3-V4 sequences of the 16S rRNA gene, the strains shown in Figures 1 to 5 could be identified.

[0169] Example 2: Comparative analysis of strains based on site within the female reproductive system

[0170] Comparative analysis of various microbial communities was performed using QIIME (v1.9) with the OTU abundance and taxonomy information from Example 1 described above. To confirm the species diversity and evenness of microbial communities within the female reproductive system, the Shannon Index and Inversed Simpson Index were calculated, and alpha diversity information was verified through the Rarefaction curve and Chao1 value. Beta diversity (diversity information between samples within a comparison group) between samples was calculated based on the Weighted / Unweighted UniFrac distance, and the phylogenetic relationships between samples were visualized through PCoA and UPGMA trees. Venn diagrams were constructed to compare the presence or absence of microorganisms between groups. For statistical analysis to compare species diversity (Diversity Index) and microbial community composition (Relative Abundance) among the locations (Vagina / Endometrium / Peritoneum) within each EMS and DER group, the Kruskal-Wallis Rank Sum Test was performed for three-group comparisons and the Wilcoxon Rank Sum Test for two-group comparisons. Additionally, the Wilcoxon Rank Sum Test was performed to compare species diversity (Diversity Index) and microbial community composition (Relative Abundance) between the EMS and DER groups within each site. Here, the EMS group corresponds to the group with endometriosis, and the DER group corresponds to the group with benign teratomas, which are hormone-inresponsive ovarian tumors. LEfSe (Linear discriminant Effect Size) analysis was also performed on the relative abundance of microbial communities between groups to select microorganisms showing significant differences between comparison groups and expressed the degree of difference as an LDA Score.For the DER group, a two-way analysis of variance was performed to examine the effects of Site and Phase factors and their interactions on the microbial community composition.

[0171] As a result, referring to Figure 1, it was confirmed that the relative abundance of *Sterella* strains in the vagina of the female reproductive system was higher in the EMS group, which has endometriosis, compared to the control group, the DER group. Additionally, referring to Figure 2, it was confirmed that the relative abundance of *Rodanobacter glycinis* strains in the endometrium of the female reproductive system was lower in the EMS group compared to the DER group; referring to Figure 3, it was confirmed that the relative abundance of *Prevotella timonensis* strains in the endometrium of the female reproductive system was higher in the EMS group compared to the DER group; referring to Figure 4, it was confirmed that the relative abundance of *Corynebacterium tuberculostearicum* strains was higher in the EMS group compared to the DER group; and referring to Figure 5, it was confirmed that the relative abundance of *Hoardella ureilitica* strains in the endometrium of the female reproductive system was higher in the EMS group compared to the DER group. Referring to Figure 6, it was confirmed that the relative abundance of *Chryseobacterium greenlandens* strains in the female reproductive endometrium was lower in the EMS group compared to the DER group, and referring to Figure 7, it was confirmed that the relative abundance of *Campylobacter ureolyticus* strains in the female reproductive endometrium was higher in the EMS group compared to the DER group.

[0172] Through this, the relative abundance of strains of the genus Suterella in the female reproductive vagina and the relative abundance of strains of Rhodanobacter glycinis, Prevotella timonensis, Corynebacterium tuberculostearicum, Hoardella ureilitisca, Criseobacterium greenlandens, or Campylobacter ureoliticus in the endometrium can be easily utilized for the diagnosis of endometriosis by comparing each with the level of the control group.

[0173] delete

[0175] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 A composition for diagnosing endometriosis comprising a preparation for quantifying strains of the genus Sutterella and strains of Howardella ureilytica in biological samples. Claim 2 A composition according to claim 1, wherein the quantification is performed by measuring the relative abundance of the strain. Claim 3 A composition according to claim 1, wherein the biological sample is isolated from within the female reproductive vagina. Claim 4 A composition according to claim 1, wherein the Sutterella genus strain is at least one strain selected from the group consisting of Sutterella massiliensis, Sutterella stercoricanis, and Sutterella wadsworthensis. Claim 5 The composition according to claim 1, further comprising a preparation for quantifying at least one strain selected from the group consisting of Rhodanobacter glycinis, Prevotella timonensis, Corynebacterium tuberculostearicum, Campylobacter ureolyticus, and Chryseobacterium greenlandense in a biological sample. Claim 6 A composition according to claim 5, wherein the biological sample is separated from the endometrium of the female reproductive system. Claim 7 A composition according to claim 1, wherein the preparation for quantifying the strain comprises one or more selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to the 16S rRNA gene of the strain. Claim 8 A diagnostic kit for endometriosis comprising a composition of any one of claims 1 to 7. Claim 9 A method for providing information for the diagnosis of endometriosis, comprising the step of quantifying strains of the genus Sutterella and strains of Howardella ureilytica from a biological sample isolated from the reproductive organ of a target individual. Claim 10 In claim 9, the reproductive organ is at least one selected from the group consisting of the endometrium, cervix, ovary, fallopian tube, vagina, and abdominal cavity. Claim 11 In Clause 10, the above-mentioned reproductive organ is the vagina, method. Claim 12 The method according to claim 9, wherein the Sutterella genus strain is at least one strain selected from the group consisting of Sutterella massiliensis, Sutterella stercoricanis, and Sutterella wadsworthensis. Claim 13 The method of claim 9 further comprises the step of quantifying at least one strain selected from the group consisting of Rhodanobacter glycinis, Prevotella timonensis, Corynebacterium tuberculostearicum, Campylobacter ureolyticus, and Chryseobacterium greenlandense from a biological sample isolated from the reproductive organ of a target individual. Claim 14 In paragraph 13, the above-mentioned reproductive organ is the endometrium, method. Claim 15 (a) a measuring unit that quantifies strains of the genus Sutterella and strains of Howardella ureilytica from a biological sample obtained from the reproductive organ of a target individual and measures the relative abundance of strains corresponding to one or more operational taxonomic units (OTUs); and (b) a detector unit that outputs information regarding endometriosis of the target individual from the relative abundance of strains measured by the measuring unit; an endometriosis diagnostic device comprising Claim 16 A diagnostic device according to claim 15, wherein the reproductive organ is at least one selected from the group consisting of the endometrium, cervix, ovary, fallopian tube, vagina, and abdominal cavity. Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete

Images