WO2013099376A1 - Mature ovum marker for use in in vitro fertilization, and use thereof - Google Patents
Mature ovum marker for use in in vitro fertilization, and use thereof Download PDFInfo
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- WO2013099376A1 WO2013099376A1 PCT/JP2012/074486 JP2012074486W WO2013099376A1 WO 2013099376 A1 WO2013099376 A1 WO 2013099376A1 JP 2012074486 W JP2012074486 W JP 2012074486W WO 2013099376 A1 WO2013099376 A1 WO 2013099376A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
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- 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
- G01N33/689—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 pregnancy or the gonads
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- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Brain natriuretic peptide [BNP, proBNP]; Cardionatrin; Cardiodilatin
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/56—Staging of a disease; Further complications associated with the disease
Definitions
- the present invention relates to a mature egg marker for selecting an embryo suitable for in vitro fertilization, a method for selecting a mature egg using the marker as an index, a kit for the same, and the like.
- Natriuretic peptides are a group of peptide hormones discovered as natural bioactive peptides with antihypertensive diuretic action. To date, it is known that there are three types of peptides called ANP, BNP and CNP having homology with each other. ANP and BNP have already been widely clinically applied as therapeutic agents for acute heart failure and diagnostic agents for heart failure, respectively.
- Non-patent Documents 1 and 2 Recently, it has been revealed that CNP is expressed in the central nervous system, reproductive organs, bones, and vascular endothelium (Non-patent Document 3). However, there are many unclear points about its physiological function, and its application to medical treatment is not progressing.
- Non-Patent Document 4 Non-Patent Document 4
- CNP peptide is present in the follicular fluid at a detectable level, and further, the relationship between the CNP concentration in the follicular fluid and the possibility of establishment of pregnancy has never been known.
- An object of the present invention is to provide means for determining whether or not an ovum collected from a subject is a mature ovum that has a high probability of becoming pregnant when used for in vitro fertilization.
- the present inventors conducted an analysis focusing on CNP. Since CNP is a peptide hormone and thought to act in a very small amount, an extremely sensitive radioimmunoassay (RIA) system was constructed to detect CNP in the follicular fluid and used in human follicular fluid. We succeeded in detecting extremely small amount of CNP and measuring its concentration for the first time. As a result, surprisingly, the CNP concentration in each follicular fluid obtained at the time of one egg collection varied, and the egg could not be collected (meaning that the egg was immature).
- RIA radioimmunoassay
- the CNP concentration in the follicular fluid in which the ovum could be collected (meaning that the egg was mature) was significantly lower. Furthermore, the present inventors have found that an egg collected from a follicle having a low CNP concentration in the follicular fluid is more likely to become pregnant when an embryo after in vitro fertilization is transplanted into the uterus. As a result of further studies based on these findings, the present inventors have completed the present invention.
- the present invention is as follows.
- a test method for predicting whether or not an ovum collected from a subject will become pregnant when the ovum is used in in vitro fertilization, wherein CNP in follicular fluid collected together with the ovum at the time of egg collection A method characterized by measuring a concentration.
- the present invention by measuring the CNP concentration in the follicular fluid, it is possible to objectively discriminate excellent mature eggs having a high possibility of reaching pregnancy when used in in vitro fertilization.
- FIG. 1 shows a model that explains the role of CNP in meiosis control of an egg.
- FIG. 2 shows a standard curve in CNP concentration measurement by RIA.
- FIG. 3 shows the result of measuring the CNP concentration in the follicular fluid collected from one subject during one in vitro fertilization treatment by RIA.
- Fig. 4 shows the flow from egg collection to embryo transfer (upper panel), and the results of measuring the CNP concentration in the follicular fluid collected with the ovum in cases where pregnancy was established (black) and cases where pregnancy was not established (white) (Lower panel).
- the present invention provides an inspection method for selecting eggs suitable for in vitro fertilization.
- the method is characterized in that the CNP concentration in a follicular fluid derived from a subject collected together with an egg at the time of egg collection is measured.
- the CNP concentration in the follicular fluid As shown in the examples below, the lower the CNP concentration in the follicular fluid, the more mature the ovum in the follicle, and the oocytes collected from the follicle with a lower CNP concentration in the follicular fluid are used for in vitro fertilization. When used, there was a tendency for pregnancy to be easily established.
- the present invention also includes (1) a step of measuring CNP concentration in a follicular fluid derived from a subject collected together with an egg at the time of egg collection, and (2) when the egg is used in in vitro fertilization based on the CNP concentration.
- the follicle is composed of an ovum and cumulus cells surrounding it, granulosa cells stratified along the follicle wall, and follicular fluid filled with substances secreted from the granulosa cells.
- CNP is secreted from granulosa cells into the follicular fluid and binds to the CNP receptor expressed on cumulus cells to promote cGMP production in cumulus cells.
- the produced cGMP is mediated by tight junctions.
- the theory has been proposed that the transition to an oocyte (egg) and suppression of the meiosis resumption of the ovum is suppressed (see FIG. 1, Non-Patent Document 4 above).
- follicular fluid (ovum) is collected from multiple follicles at the time of egg collection, but even if CNP in follicular fluid can be detected, the CNP concentration in each follicular fluid obtained by one egg collection in the same individual. It was totally unpredictable how much variation there was.
- the present invention is used for quantification of PACAP, etc., with extremely high sensitivity (50% inhibitory dose (ID50) of about several tens of fmol / ml (about 1 fmol / mg protein in human follicular fluid), detection limit of several fmol.
- ID50 inhibitory dose
- CNP C-type natriuretic peptide
- CNP-22 a known in vivo bioactive peptide consisting of 22 amino acid residues and having a hypotensive diuretic action, and a precursor thereof.
- a CNP precursor called NPPC Natriuretic Peptide Precursor C
- NPPC Natriuretic Peptide Precursor C
- the CNP concentration in step (1) is the concentration of the mature peptide (CNP-22) and its precursors that have physiological activity (activity that can interact with the receptor) and can exist in the follicular fluid. Is defined as
- the amino acid sequence of CNP has been published in several animals.
- the CNP prepropolypeptide is a polypeptide containing the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2 (GenBank Accession No. NP — 077720.1).
- pro-CNP is amino acid numbers 24-126
- CNP-53 is amino acid numbers 74-126.
- CNP-22 is a polypeptide containing an amino acid sequence identical or substantially identical to the amino acid sequence shown in SEQ ID NO: 1, and a cyclic domain is formed by a disulfide bond between Cys at position 6 and Cys at position 22. Forming.
- the amino acid sequence substantially the same as the amino acid sequence shown in SEQ ID NO: 1 or 2 is the amino acid sequence of each of the orthologs of mature CNP or a prepropolypeptide thereof in mammals other than human, or found in humans or other mammals.
- public databases well known in the art eg, HomoloGene (http://www.ncbi.nlm.nih.gov/ HomoloGene /) and UniProtKB / Swiss-Prot database
- HomoloGene http://www.ncbi.nlm.nih.gov/ HomoloGene /
- UniProtKB / Swiss-Prot database can be used to obtain sequence information.
- the subject in the present invention is not particularly limited as long as it is a mammal capable of pregnancy.
- Mammals include, for example, rodents such as mice, rats, hamsters, guinea pigs, laboratory animals such as rabbits, pets such as dogs and cats, domestic animals such as cows, pigs, goats, horses and sheep, monkeys, orangutans and Examples include primates such as chimpanzees and humans, with humans being particularly preferred.
- the follicular fluid used for CNP concentration measurement can be collected from the subject together with the ovum at the time of egg collection by a conventional method.
- follicular fluid can be collected by the following procedure.
- the follicles are developed by injecting the subject with the gonadotropin hMG. The growth state of the ovary is observed with an ultrasonic monitor or the like, and when the follicle diameter grows to 17 mm or more, the hMG injection is switched to the hCG ovulation stimulant injection, and the egg is collected 36 hours later.
- Egg collection may be performed by laparotomy, laparoscope, ultrasonic egg collection, or the like, but ultrasonic egg collection that is usually less invasive is performed.
- Ultrasonic egg collection is classified into transvesical egg collection using an abdominal wall probe, transvaginal egg collection using an abdominal wall probe or vaginal probe, and transurethral egg collection using an abdominal wall probe, depending on the needle insertion method. Any of them may be used.
- the collected follicular fluid can be used for CNP concentration measurement as it is after the ovum has been collected. Further, the supernatant after removing blood cells and the like by centrifugation (eg, 3,500 rpm, 30 minutes) may be used.
- the CNP concentration in the follicular fluid may be measured by any method as long as it has a detection sensitivity capable of quantitatively measuring the concentration of CNP that can be present in the follicular fluid. Since follicular fluid usually contains CNP in the order of 100 fmol / ml, it is preferable to use a measurement system having at least a detection limit of 100 fmol / ml or less, preferably several 10 (10- 50) fmol / ml or less, measurement system of several hundred (100-500) fmol / ml or less at 50% inhibitory dose (ID50).
- the follicular fluid usually contains CNP in the order of 1 fmol / mg protein
- a measurement system with at least a detection limit of 1 fmol / mg protein or less, and desirably a detection limit of 0.2
- a measurement system with fmol / mg protein or less and 50% inhibitory dose (ID50) of 2 fmol / mg protein or less can be mentioned.
- ID50 inhibitory dose
- it can be performed by various immunological assays using an antibody that recognizes CNP (anti-CNP antibody).
- the amount of antibody or antibody-antigen complex corresponding to the amount of CNP in the follicular fluid is detected by chemical or physical means, and the result is used to prepare a standard solution containing a known amount of antigen.
- Any measurement method may be used as long as it is a measurement method for calculating the CNP concentration using the prepared standard curve.
- immunological assays include radioimmunoassay (RIA), enzyme immunoassay (EIA method), fluorescent immunoassay (FIA), immunochromatography, Western blotting, etc. It is well known.
- RIA radioimmunoassay
- EIA method enzyme immunoassay
- FIA fluorescent immunoassay
- immunochromatography Western blotting
- the anti-CNP antibody can be prepared by a method known per se. For example, it can be prepared by immunizing an animal using CNP or a partial peptide thereof as an antigen after isolation and purification from a CNP-expressing sample (eg, cell, tissue, etc.).
- CNP or its partial peptide used as an antigen may be synthesized chemically or biochemically using a cell-free protein synthesis system, or a nucleotide sequence encoding the amino acid sequence of CNP or its partial peptide. You may produce from the transformed cell into which the nucleic acid to contain was introduce
- the anti-CNP antibody may be an antibody against CNP derived from any species as long as it can specifically recognize CNP in the follicle derived from the subject, but from the viewpoint of specificity, it may be directed against CNP derived from the same species as the subject. It is preferably an antibody (for example, an anti-human CNP antibody when the subject is a human).
- the anti-CNP antibody may be either a polyclonal antibody or a monoclonal antibody, and can be prepared by a well-known immunological technique.
- the antibody includes not only a complete antibody molecule but also a fragment thereof, and examples thereof include Fab, F (ab ′) 2 , ScFv, and minibody.
- Polyclonal antibodies include, for example, CNP or a partial peptide thereof (which may be a complex cross-linked to a carrier protein such as bovine serum albumin or KLH (Keyhole Limpet Hemocyanin), if necessary, together with a commercially available adjuvant.
- a carrier protein such as bovine serum albumin or KLH (Keyhole Limpet Hemocyanin)
- animals to which the antigen is administered include mammals such as rats, mice, rabbits, goats, guinea pigs, and hamsters.
- Monoclonal antibodies can be obtained by cell fusion methods (for example, Takeshi Watanabe, Principles of Cell Fusion Methods and Production of Monoclonal Antibodies, Akira Taniuchi, Toshitada Takahashi, “Monoclonal Antibodies and Cancer: Basic and Clinical”, 2-14). Page, Science Forum Publishing, 1985).
- CNP or a partial peptide thereof is administered to a mouse 2-4 times subcutaneously or intraperitoneally with a commercially available adjuvant, and the spleen or lymph node is collected about 3 days after the final administration, and lymphocytes are collected.
- the lymphocytes and myeloma cells are cell-fused to obtain a hybridoma that produces a monoclonal antibody against CNP.
- the cell fusion may be PEG method [J. Immunol. Methods, 81 (2): 223-228 (1985)] or voltage pulse method [Hybridoma, 7 (6): 627-633 (1988)].
- a hybridoma producing a desired monoclonal antibody can be selected by detecting an antibody that specifically binds to an antigen from the culture supernatant using a known EIA or RIA method or the like.
- the culture of the hybridoma producing the monoclonal antibody can be performed in vitro or in vivo such as mouse or rat, preferably mouse ascites, and the antibody can be obtained from the culture supernatant of the hybridoma and the ascites of the animal, respectively.
- the antibody may be directly or indirectly labeled with a labeling substance.
- Labeling substances include fluorescent substances (eg, FITC, rhodamine), radioactive substances (eg, 125 I, 131 I, 3 H), enzymes (eg, alkaline phosphatase, peroxidase), colored particles (eg, metal colloid particles, colored) Latex), biotin and the like.
- Solid phase includes plates (eg, microwell plates), tubes, beads (eg, plastic beads, magnetic beads), chromatographic carriers (eg, water-absorbing substrates such as nitrocellulose membranes, Sepharose), membranes (Eg, nitrocellulose membrane, PVDF membrane), gel (eg, polyacrylamide gel), metal membrane (eg, gold membrane) and the like.
- the bond include covalent bond, ionic bond, physical adsorption, and the like, and are not particularly limited. However, covalent bond and / or physical adsorption are preferable because sufficient bond strength can be obtained.
- the solid phase may be directly bonded to the solid phase or indirectly bonded to the solid phase using a substance known per se.
- a CNP concentration measurement system may be constructed by adding ordinary technical considerations to those skilled in the art to the usual conditions and operation methods in each method.
- a CNP concentration measurement system may be constructed by adding ordinary technical considerations to those skilled in the art to the usual conditions and operation methods in each method.
- Hiroshi Irie “Radioimmunoassay” Kelsha, published in 1974
- Hiroshi Irie “Continue Radioimmunoassay” published in Kodansha, 1979
- Enzyme Immunoassay edited by Eiji Ishikawa et al. 53
- the CNP concentration in the follicular fluid can be measured using various molecular weight measuring methods such as gel electrophoresis (eg, SDS-PAGE), various separation and purification methods (eg, size exclusion chromatography, ion exchange).
- gel electrophoresis eg, SDS-PAGE
- separation and purification methods eg, size exclusion chromatography, ion exchange.
- step (2) based on the CNP concentration in the follicular fluid measured in step (1), it is predicted whether pregnancy will be established when the ovum collected with the follicular fluid is used in in vitro fertilization. This determination is made based on a negative correlation between the CNP concentration and the possibility of reaching pregnancy.
- the determination can be made by setting a reference value for the CNP concentration in advance and comparing the measured CNP concentration with this reference value.
- the measured CNP concentration is below the reference value, it can be determined that there is a high possibility of reaching pregnancy.
- the relationship between the CNP concentration and whether or not the pregnancy has been established is examined, and if the concentration is equal to or less than that concentration, the concentration at which the possibility of pregnancy establishment is statistically significantly high can be set as a reference value.
- fmol / mg protein or less can be set as a reference value.
- the follicular fluid may be diluted due to the restriction of the operation when collecting the follicular fluid.
- the measurement value varies, and depending on the sample, there is a risk that the CNP concentration in the follicular fluid is not accurately evaluated, so expressing it in moles per unit protein will result in errors due to egg collection operations in the measurement results. It can be prevented from being reflected.
- the screening method of the present invention may be used in combination with a method based on another standard for discriminating high-quality mature eggs.
- Other criteria include those that have been used in the past, such as the appearance (eg, shape, size, etc.) of the egg.
- eggs that are closer to a true sphere are suitable for in vitro fertilization, and a plurality of follicular fluids collected with such eggs are further selected by applying the selection method of the present invention. It can also be done.
- the oocytes selected as described above are separated from the follicular fluid after egg collection, and immediately cultured in an incubator at 37 ° C., and then subjected to in vitro fertilization with separately prepared sperm.
- the method of in vitro fertilization can be performed by any conventionally used method. If there is a problem on the sperm side, such as a small number of motor spermatozoa, it is preferable to fertilize by microinjection. Fertilization is determined by observing pronuclear formation under a stereomicroscope or an inverted phase contrast microscope.
- Culture is carried out for 3-5 days after fertilization, development proceeds from an 8-cell stage embryo to an expanded blastocyst, embryos showing good morphology are selected by microscopic observation, and transplanted to the uterus by a conventional method. If the CNP concentration in the follicular fluid is low and multiple good embryos are obtained from the ova selected by the above selection method, the embryo that is judged to be the most favorable from other criteria is transplanted, and the surplus embryo is frozen It is desirable to keep it.
- the screening method of the present invention it is possible to objectively discriminate in advance mature eggs that are likely to become pregnant when used in in vitro fertilization, thus improving the success rate of in vitro fertilization and reducing the burden on patients. It is useful in that it leads to
- the present invention also provides an egg test kit suitable for in vitro fertilization, comprising an anti-CNP antibody.
- the anti-CNP antibody is an antibody for measuring the CNP concentration in the follicular fluid derived from a subject, and is the antibody described in relation to the test method of the present invention.
- the subject is not particularly limited as long as it is a mammal capable of pregnancy, and includes the biological species described in relation to the test method of the present invention, but preferably a human.
- the kit may further contain other components preferable for carrying out the above-described test method of the present invention, such as a reaction buffer solution, a washing solution, a solid phase carrier, a labeling agent, and a CNP preparation.
- a reaction buffer solution a washing solution
- a solid phase carrier a labeling agent
- a CNP preparation a CNP preparation for measurement of CNP concentration
- a radioisotope for example, radioactive iodine
- test kit of the present invention is useful because it enables provision of a simple means for objectively distinguishing mature eggs that are likely to become pregnant when used in in vitro fertilization.
- the subjects were 5 infertile patients (33 to 40 years old) who obtained informed consent. Subjects were injected with hMG (HMG Fujiseiyaku, Fuji Pharmaceutical, 150-300 units, 7-10 days) to artificially develop follicles, and the ovarian growth state was observed with an ultrasonic monitor. After growing until the principal follicle diameter reached 17 mm or more, the CG was switched to an hCG ovulation stimulant (gonatropin, Asuka Pharmaceutical, 5000-10000 units). Eggs were collected by a conventional method 36 hours after switching. Eggs obtained by in vitro fertilization and embryo transfer were fertilized in vitro.
- hMG HMG Fujiseiyaku, Fuji Pharmaceutical, 150-300 units, 7-10 days
- Oocytes were collected from the collected follicular fluid and transferred to the culture solution (Complete HTF, Irvine Scientific, 0.5 mL) in the culture dish. After 3 to 4 hours of pre-culture, the moving sperm was fertilized and fertilized toward the egg. Fertilized eggs were cultured in a petri dish for 2 to 3 days (37.0 ° C., O 2 5%, CO 2 5%). Fertilized excellent eggs divide into 8 cells after repeated cell division in 3 days of culture. Cells at this stage were returned to the maternal uterus. Radioimmunoassay (RIA) Synthetic CNP-22 was used as a standard product and diluted with RIA buffer (described later) to prepare a dilution series.
- RIA Radioimmunoassay
- Example 2 Discrimination of mature eggs by CNP concentration in human follicular fluid Since it was possible to measure CNP concentration in human follicular fluid, the relationship between CNP concentration in human follicular fluid and the degree of maturation of eggs was investigated. At the time of egg collection, mature eggs can be collected, but immature eggs cannot be collected because they are strongly adhered to the follicular wall. Therefore, the follicular fluid from which the ovum could be collected is related to the mature ovum, and the follicular fluid from which the ovum could not be collected is related to the immature ovum.
- concentration was represented as the number of moles per protein amount (mg).
- the CNP concentration in the follicular fluid from which the ovum could be collected showed a significantly lower value. This result shows that the maturity of the ovum can be determined by measuring the CNP concentration in the follicular fluid.
- Example 3 Correlation between CNP concentration in human follicular fluid and pregnancy establishment
- the correlation between CNP concentration in human follicular fluid and pregnancy establishment was examined.
- the collected egg was fertilized in a test tube, the fertilized egg was cultured for 3 days and divided to 8 cells. The cells at this stage were returned to the mother's uterus, and it was observed whether pregnancy was established (upper panel in FIG. 4).
- the lower panel of FIG. 4 shows the CNP concentration in the follicular fluid (7 samples) collected together with the ovum used for fertilized egg preparation.
- the CNP concentration in the follicular fluid collected with the ovum of the case where pregnancy was established is shown in black (pregnancy example).
- the CNP concentration in this follicular fluid was 0.5 fmol / mg protein, which was lower than the values of the other 6 samples. This suggests that measurement of the CNP concentration in the follicular fluid is useful for determining whether or not pregnancy is achieved.
- the Japanese Society of Obstetrics and Gynecology strongly recommends that only one fertilized egg is returned to the maternal uterus. This is because if many fertilized eggs are returned to the uterus at the same time, multiple pregnancy is likely to occur, and the risk to the mother increases. From such a background, when returning the fertilized egg in the 8-cell stage to the uterus, a criterion for determining which fertilized egg can be returned with a good result is required. This example shows that the level of CNP concentration in the follicular fluid can be the standard.
- the present invention it becomes possible to discriminate excellent matured ova that have a high probability of reaching pregnancy when used in in vitro fertilization, and can significantly increase the success rate of infertility treatment. Therefore, the burden on the mother can be reduced and the medical economic efficiency can be increased.
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Abstract
Description
しかしながら、卵胞液中にCNPペプチドが検出可能なレベルで存在するか否かについての報告はなく、ましてや卵胞液中のCNP濃度と妊娠成立の可能性との関係については全く知られていなかった。 Recently, in mouse follicles, the CNP gene is expressed in granulosa cells and its receptor gene is expressed in cumulus cells, and CNP is matured in oocytes surrounded by cumulus cells in a concentration-dependent manner in vitro. On the other hand, in Graf's follicles of mutant mice with CNP or its receptor, it is reported that meiosis resumes early, and that CNP produced by granulosa cells is involved in the regulation of oocyte maturation. It has been suggested (Non-Patent Document 4).
However, there has been no report as to whether or not CNP peptide is present in the follicular fluid at a detectable level, and further, the relationship between the CNP concentration in the follicular fluid and the possibility of establishment of pregnancy has never been known.
本発明者らは、これらの知見に基づいて更に研究を重ねた結果、本発明を完成するに至った。 In order to achieve the above-mentioned object, the present inventors conducted an analysis focusing on CNP. Since CNP is a peptide hormone and thought to act in a very small amount, an extremely sensitive radioimmunoassay (RIA) system was constructed to detect CNP in the follicular fluid and used in human follicular fluid. We succeeded in detecting extremely small amount of CNP and measuring its concentration for the first time. As a result, surprisingly, the CNP concentration in each follicular fluid obtained at the time of one egg collection varied, and the egg could not be collected (meaning that the egg was immature). Compared with the CNP concentration in the liquid, the CNP concentration in the follicular fluid in which the ovum could be collected (meaning that the egg was mature) was significantly lower. Furthermore, the present inventors have found that an egg collected from a follicle having a low CNP concentration in the follicular fluid is more likely to become pregnant when an embryo after in vitro fertilization is transplanted into the uterus.
As a result of further studies based on these findings, the present inventors have completed the present invention.
[1]被験者から採取した卵子が、体外受精において当該卵子を使用した場合に妊娠成立に至るか否かを予測するための検査方法であって、採卵時に卵子と共に採取された卵胞液中のCNP濃度を測定することを特徴とする方法。
[2]CNP濃度が免疫学的アッセイによって測定される、[1]に記載の方法。
[3]免疫学的アッセイがRIAである、[2]に記載の方法。
[4]被験者がヒトである、[1]~[3]のいずれかに記載の方法。
[5]抗CNP抗体を含む、体外受精に適した卵子の検査用キット。
[6]被験者がヒトである、[5]に記載のキット。 That is, the present invention is as follows.
[1] A test method for predicting whether or not an ovum collected from a subject will become pregnant when the ovum is used in in vitro fertilization, wherein CNP in follicular fluid collected together with the ovum at the time of egg collection A method characterized by measuring a concentration.
[2] The method according to [1], wherein the CNP concentration is measured by an immunological assay.
[3] The method according to [2], wherein the immunological assay is RIA.
[4] The method according to any one of [1] to [3], wherein the subject is a human.
[5] An egg test kit suitable for in vitro fertilization, comprising an anti-CNP antibody.
[6] The kit according to [5], wherein the subject is a human.
本発明は、例えばPACAPなどの定量に用いられるような、極めて高感度(50%阻害用量(ID50)で数10fmol/ml程度(ヒト卵胞液では1 fmol/mg protein程度)、検出限界で数fmol/ml程度(ヒト卵胞液では0.1 fmol/mg protein程度))のラジオイムノアッセイ(RIA)系を用いることで、卵胞液中の極めて微量のCNPを定量的に測定し得ることを初めて見出し、そして、1回の採卵で得られる各卵胞液中のCNP濃度には、意外にもかなりのばらつきがあり、CNP濃度がより低い方が、採卵が可能な(即ち、卵子の成熟が進んでいる)場合が多く、しかも、採取され、体外受精により受精が成立し、移植可能な段階まで正常に分裂した胚においても、採卵時の卵胞液中のCNP濃度が低いほど、妊娠成立の可能性が高いことを、初めて見出したことに基づくものである。 The follicle is composed of an ovum and cumulus cells surrounding it, granulosa cells stratified along the follicle wall, and follicular fluid filled with substances secreted from the granulosa cells. CNP is secreted from granulosa cells into the follicular fluid and binds to the CNP receptor expressed on cumulus cells to promote cGMP production in cumulus cells. The produced cGMP is mediated by tight junctions. The theory has been proposed that the transition to an oocyte (egg) and suppression of the meiosis resumption of the ovum is suppressed (see FIG. 1, Non-Patent Document 4 above). However, although this document shows the expression of CNP mRNA in granulosa cells and the expression of CNP receptor mRNA in cumulus cells, and the effects of CNP addition and CNP deficiency on ovarian maturation, follicular fluid, In particular, it has not been confirmed that CNP is actually present (ie at a detectable level) in human follicular fluid. CNP is one of the peptide hormones and is considered to exert physiological activity in a very small amount in vivo. Therefore, whether or not CNP in follicular fluid can be detected by a conventional immunoassay system is quantitative. It was completely unknown whether there was. Furthermore, follicular fluid (ovum) is collected from multiple follicles at the time of egg collection, but even if CNP in follicular fluid can be detected, the CNP concentration in each follicular fluid obtained by one egg collection in the same individual. It was totally unpredictable how much variation there was.
The present invention, for example, is used for quantification of PACAP, etc., with extremely high sensitivity (50% inhibitory dose (ID50) of about several tens of fmol / ml (about 1 fmol / mg protein in human follicular fluid), detection limit of several fmol. for the first time it was possible to quantitatively measure very small amounts of CNP in the follicular fluid by using a radioimmunoassay (RIA) system of about / ml (about 0.1 fmol / mg protein for human follicular fluid), and Surprisingly, the CNP concentration in each follicular fluid obtained from a single egg collection varies considerably, and if the CNP concentration is lower, egg collection is possible (ie, the ovum is more mature) Even in embryos that have been collected, fertilized by in vitro fertilization, and have normally divided to the stage of transplantation, the lower the CNP concentration in the follicular fluid at the time of egg collection, the higher the chance of pregnancy. Is based on the first finding.
従って、工程(1)におけるCNP濃度とは、生理活性(受容体と相互作用し得る活性)を有し、かつ卵胞液中に存在し得る成熟ペプチド(CNP-22)及びその前駆体全体の濃度として定義される。 In the present invention, CNP (C-type natriuretic peptide) means a known in vivo bioactive peptide (CNP-22) consisting of 22 amino acid residues and having a hypotensive diuretic action, and a precursor thereof. A CNP precursor called NPPC (Natriuretic Peptide Precursor C) is first produced in vivo as a prepropolypeptide consisting of 126 amino acid residues, becomes pro-CNP by removal of the signal peptide, and is further cleaved by furin, an endoprotease. Produces a peptide consisting of 53 active amino acid residues (CNP-53). CNP-53 is secreted and further cleaved by an unknown enzyme to produce a mature peptide (CNP-22) consisting of 22 amino acid residues.
Therefore, the CNP concentration in step (1) is the concentration of the mature peptide (CNP-22) and its precursors that have physiological activity (activity that can interact with the receptor) and can exist in the follicular fluid. Is defined as
例えば、入江 寛編「ラジオイムノアッセイ」(講談社、昭和49年発行)、入江 寛編「続ラジオイムノアッセイ」(講談社、昭和54年発行)、石川栄治ら編「酵素免疫測定法」(医学書院、昭和53年発行)、石川栄治ら編「酵素免疫測定法」(第2版)(医学書院、昭和57年発行)、石川栄治ら編「酵素免疫測定法」(第3版)(医学書院、昭和62年発行)、「Methods in ENZYMOLOGY」 Vol. 70 (Immunochemical Techniques (Part A))、同書 Vol. 73 (Immunochemical Techniques (Part B))、同書 Vol. 74 (Immunochemical Techniques (Part C))、同書 Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays))、同書 Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods))、同書 Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) (以上、アカデミックプレス社発行)などを参照することができる。 In applying the above immunological assay to the present invention, special conditions, operations, and the like are not required to be set. A CNP concentration measurement system may be constructed by adding ordinary technical considerations to those skilled in the art to the usual conditions and operation methods in each method. For details of these general technical means, it is possible to refer to reviews, books and the like.
For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Continue Radioimmunoassay” (published in Kodansha, 1979), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. 53), edited by Eiji Ishikawa et al. "Enzyme Immunoassay" (2nd edition) (Medical Shoin, published in 1982), edited by Eiji Ishikawa et al. "Enzyme Immunoassay" (3rd edition) (Medical School, Showa 62)), "Methods in ENZYMOLOGY" Vol. 70 (Immunochemical Techniques (Part A)), ibid Vol. 73 (Immunochemical Techniques (Part B)), ibid Vol. 74 (Immunochemical Techniques (Part C)), ibid Vol 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid. Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid. )) (Above, published by Academic Press) It is possible to irradiation.
採卵
インフォームドコンセントが得られた5名の不妊患者(33~40歳)を被験者とした。被験者にhMG(HMGフジセイヤク、富士製薬、150~300単位、7~10日間)を注射して、卵胞を人工的に発育させ、超音波モニターで卵巣の発育状態を観察した。首席卵胞径が17mm以上になるまで発育させた後、hCG排卵刺激剤(ゴナトロピン、あすか製薬、5000~10000単位)に切り替えた。切り替えから36時間後に定法により採卵を行なった。
体外受精及び胚移植
採卵した卵子を試験管内で受精させた。採取した卵胞液より卵子を回収し、培養ディッシュ内の培養液(Complete HTF、Irvine Scientific、0.5mL)へ移した。3~4時間の前培養のあと、卵子に向けて運動精子を媒精し、受精させた。
受精卵を2~3日間シャーレで培養した(37.0℃、O2 5%, CO2 5%)。受精した優良な卵子は3日間の培養で細胞分裂を重ねて8細胞にまで分裂する。この段階の細胞を母体の子宮に戻した。
ラジオイムノアッセイ(RIA)
合成CNP-22を標準品として、RIAバッファー(後述)により希釈して、希釈系列を作製して用いた。抗体は、BSA-conjugated CNP-22を家兎に免疫して作製したものを、希釈して用いた。検体および標準品をそれぞれRIAバッファー(50mMリン酸緩衝液pH7.4、0.25% BSA、0.1% Triton X-100、80mM NaCl、25mM EDTA、2% Dextran T-40)100μlに溶解し、希釈した抗体溶液100μl、ヨウ素125標識CNP 100μl(17,000cpm)を加えて24時間インキュベートしたのちB/F分離した。結合した放射性ヨウ素をガンマーカウンターにより計測し、RIA測定用ソフトウェア(アロカRIA Pro ver3)により検体中のCNP濃度を求めた。 Materials and methods
The subjects were 5 infertile patients (33 to 40 years old) who obtained informed consent. Subjects were injected with hMG (HMG Fujiseiyaku, Fuji Pharmaceutical, 150-300 units, 7-10 days) to artificially develop follicles, and the ovarian growth state was observed with an ultrasonic monitor. After growing until the principal follicle diameter reached 17 mm or more, the CG was switched to an hCG ovulation stimulant (gonatropin, Asuka Pharmaceutical, 5000-10000 units). Eggs were collected by a conventional method 36 hours after switching.
Eggs obtained by in vitro fertilization and embryo transfer were fertilized in vitro. Oocytes were collected from the collected follicular fluid and transferred to the culture solution (Complete HTF, Irvine Scientific, 0.5 mL) in the culture dish. After 3 to 4 hours of pre-culture, the moving sperm was fertilized and fertilized toward the egg.
Fertilized eggs were cultured in a petri dish for 2 to 3 days (37.0 ° C.,
Radioimmunoassay (RIA)
Synthetic CNP-22 was used as a standard product and diluted with RIA buffer (described later) to prepare a dilution series. An antibody prepared by immunizing rabbits with BSA-conjugated CNP-22 was used after dilution. Specimens and standards were each diluted and diluted in 100 μl of RIA buffer (50 mM phosphate buffer pH 7.4, 0.25% BSA, 0.1% Triton X-100, 80 mM NaCl, 25 mM EDTA, 2% Dextran T-40) 100 μl of the solution and 100 μl (17,000 cpm) of iodine 125-labeled CNP were added and incubated for 24 hours, followed by B / F separation. The bound radioiodine was measured with a gamma counter, and the CNP concentration in the specimen was determined by RIA measurement software (Aroka RIA Pro ver3).
RIAによるヒト卵胞液中のCNP濃度の定量
従来、CNPは卵巣顆粒膜細胞で産生され、卵胞液中に分泌されると考えられていたが(図1)、実際に卵胞液中にCNPが存在するか否かは不明であった。そこで、採卵時に採取したヒト卵胞液について、高感度のRIAによりCNPの検出及びその濃度の測定を行なった。結果を図2に示す。このRIA系は、ID50=2 fmol/mg proteinと極めて高感度の測定系である。各卵胞から得られた3検体の卵胞液の2倍希釈液、4倍希釈液、及び8倍希釈液を用いて測定すると、それぞれ標準曲線と並行した希釈曲線が得られた。この結果から、これらのヒト卵胞液中のCNP濃度は、0.5 fmol/mg protein~4 fmol/mg protein程度と計算された。これは、ヒト卵胞液中にCNPが存在することを示した初めての結果である。このように、ヒト卵胞液中には極めて微量ではあるが、CNPが分泌されていることが明らかとなった。 Example 1
Quantification of CNP concentration in human follicular fluid by RIA Previously, CNP was thought to be produced in ovarian granulosa cells and secreted into follicular fluid (Fig. 1), but CNP is actually present in follicular fluid Whether or not to do so was unclear. Therefore, CNP was detected and the concentration of human follicular fluid collected at the time of egg collection was detected by highly sensitive RIA. The results are shown in FIG. This RIA system is an extremely sensitive measurement system with ID50 = 2 fmol / mg protein. When two-fold diluted solution, four-fold diluted solution, and 8-fold diluted solution of three specimens of follicular fluid obtained from each follicle were measured, a dilution curve parallel to the standard curve was obtained. From these results, the CNP concentration in these human follicular fluids was calculated to be about 0.5 fmol / mg protein to 4 fmol / mg protein. This is the first result showing that CNP is present in human follicular fluid. As described above, it was revealed that CNP is secreted in human follicular fluid, though in a very small amount.
ヒト卵胞液中のCNP濃度による成熟卵子の判別
ヒト卵胞液中のCNP濃度の測定が可能となったことから、ヒト卵胞液中のCNP濃度と卵子の成熟度との関連を調べた。採卵の際に、成熟した卵子は採卵できるが、未成熟な卵子は卵胞壁に強く接着しているために採卵できない。従って、卵子を採取できた卵胞液は成熟した卵子に関連しており、卵子を採取できなかった卵胞液は未成熟な卵子に関連している。
1人の被験者から1回の体外受精治療の際に採取した4~22検体の卵胞液中のCNP濃度を高感度RIAにより測定した結果を図3に示す。尚、採卵操作の際に卵胞液が希釈されるおそれがあるため、CNP濃度はタンパク質量(mg)あたりのモル数として表した。卵子を採取できなかった卵胞液中のCNP濃度と比較して、卵子を採取できた卵胞液中のCNP濃度の方が有意に低い値を示した。この結果は、卵胞液中のCNP濃度を測定することで、卵子の成熟度を判別できることを示している。 Example 2
Discrimination of mature eggs by CNP concentration in human follicular fluid Since it was possible to measure CNP concentration in human follicular fluid, the relationship between CNP concentration in human follicular fluid and the degree of maturation of eggs was investigated. At the time of egg collection, mature eggs can be collected, but immature eggs cannot be collected because they are strongly adhered to the follicular wall. Therefore, the follicular fluid from which the ovum could be collected is related to the mature ovum, and the follicular fluid from which the ovum could not be collected is related to the immature ovum.
FIG. 3 shows the results of measuring the CNP concentration in the follicular fluid of 4 to 22 samples collected from one subject during one in vitro fertilization treatment by high sensitivity RIA. In addition, since there exists a possibility that a follicular fluid may be diluted in the case of egg-collecting operation, CNP density | concentration was represented as the number of moles per protein amount (mg). Compared with the CNP concentration in the follicular fluid from which the ovum could not be collected, the CNP concentration in the follicular fluid from which the ovum could be collected showed a significantly lower value. This result shows that the maturity of the ovum can be determined by measuring the CNP concentration in the follicular fluid.
ヒト卵胞液中のCNP濃度と妊娠成立との相関
本実施例では、ヒト卵胞液中のCNP濃度と妊娠成立との相関について検討した。採取した卵子を試験管内で受精させた後、受精卵を3日間培養し、8細胞にまで分裂させた。この段階の細胞を母体の子宮に戻し、妊娠が成立するか否かを観察した(図4上パネル)。
図4下パネルには、受精卵調製に使用した卵子と共に採取した卵胞液(7検体)中のCNP濃度を示す。妊娠が成立したケースの卵子と共に採取した卵胞液中のCNP濃度を黒(妊娠例)で示す。この卵胞液中のCNP濃度は0.5 fmol/mg proteinであり、他の6検体の値よりも低い値を示した。このことは、妊娠成立に至るか否かの判定に、卵胞液中のCNP濃度の測定が有用であることを示唆している。
現在、日本産婦人科学会では、母体子宮に戻す受精卵の数を1つだけとすることを強く推奨している。これは、数多くの受精卵を同時に子宮に戻すと多胎妊娠を招きやすく、母体へのリスクが高くなると考えられるためである。このような背景から、8細胞期の受精卵を子宮に戻す際に、どの受精卵を戻したら良い結果が得られるかの判断基準が求められている。本実施例は、卵胞液中のCNP濃度の高低がその基準となり得ることを示している。 Example 3
Correlation between CNP concentration in human follicular fluid and pregnancy establishment In this example, the correlation between CNP concentration in human follicular fluid and pregnancy establishment was examined. After the collected egg was fertilized in a test tube, the fertilized egg was cultured for 3 days and divided to 8 cells. The cells at this stage were returned to the mother's uterus, and it was observed whether pregnancy was established (upper panel in FIG. 4).
The lower panel of FIG. 4 shows the CNP concentration in the follicular fluid (7 samples) collected together with the ovum used for fertilized egg preparation. The CNP concentration in the follicular fluid collected with the ovum of the case where pregnancy was established is shown in black (pregnancy example). The CNP concentration in this follicular fluid was 0.5 fmol / mg protein, which was lower than the values of the other 6 samples. This suggests that measurement of the CNP concentration in the follicular fluid is useful for determining whether or not pregnancy is achieved.
At present, the Japanese Society of Obstetrics and Gynecology strongly recommends that only one fertilized egg is returned to the maternal uterus. This is because if many fertilized eggs are returned to the uterus at the same time, multiple pregnancy is likely to occur, and the risk to the mother increases. From such a background, when returning the fertilized egg in the 8-cell stage to the uterus, a criterion for determining which fertilized egg can be returned with a good result is required. This example shows that the level of CNP concentration in the follicular fluid can be the standard.
Claims (6)
- 被験者から採取した卵子が、体外受精において当該卵子を使用した場合に妊娠成立に至るか否かを予測するための検査方法であって、採卵時に卵子と共に採取された卵胞液中のCNP濃度を測定することを特徴とする方法。 A test method for predicting whether or not an egg collected from a subject will become pregnant when the egg is used in in vitro fertilization, and measures the CNP concentration in the follicular fluid collected with the egg at the time of egg collection A method characterized by:
- CNP濃度が免疫学的アッセイによって測定される、請求項1に記載の方法。 The method according to claim 1, wherein the CNP concentration is measured by an immunological assay.
- 免疫学的アッセイがRIAである、請求項2に記載の方法。 The method according to claim 2, wherein the immunological assay is RIA.
- 被験者がヒトである、請求項1~3のいずれか1項に記載の方法。 The method according to any one of claims 1 to 3, wherein the subject is a human.
- 抗CNP抗体を含む、体外受精に適した卵子の検査用キット。 O Egg testing kit suitable for in vitro fertilization, including anti-CNP antibody.
- 被験者がヒトである、請求項5に記載のキット。 The kit according to claim 5, wherein the subject is a human.
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