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WO2013099376A1 - Marqueur d'ovule mature pour l'utilisation dans la fécondation in vitro et utilisation de ce marqueur - Google Patents

Marqueur d'ovule mature pour l'utilisation dans la fécondation in vitro et utilisation de ce marqueur Download PDF

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Publication number
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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cnp
follicular fluid
concentration
egg
collected
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Japanese (ja)
Inventor
薫 宮本
哲也 水谷
誠 折坂
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University of Fukui NUC
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University of Fukui NUC
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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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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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PCT/JP2012/074486 2011-12-26 2012-09-25 Marqueur d'ovule mature pour l'utilisation dans la fécondation in vitro et utilisation de ce marqueur Ceased WO2013099376A1 (fr)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
WO2015016036A1 (fr) * 2013-07-29 2015-02-05 国立大学法人福井大学 Biomarqueur pour analyser la possibilité de fertilisation d'ovule et détermination l'utilisant

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000121639A (ja) * 1994-09-23 2000-04-28 Unipath Ltd 監視方法およびその方法で使用される装置
WO2009142307A1 (fr) * 2008-05-23 2009-11-26 アスビオファーマ株式会社 Peptide capable de prolonger la demi-vie de peptide d’intérêt dans le plasma
WO2010061613A1 (fr) * 2008-11-28 2010-06-03 株式会社日立製作所 Procédé, système et kit d'assistance à la grossesse

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000121639A (ja) * 1994-09-23 2000-04-28 Unipath Ltd 監視方法およびその方法で使用される装置
WO2009142307A1 (fr) * 2008-05-23 2009-11-26 アスビオファーマ株式会社 Peptide capable de prolonger la demi-vie de peptide d’intérêt dans le plasma
WO2010061613A1 (fr) * 2008-11-28 2010-06-03 株式会社日立製作所 Procédé, système et kit d'assistance à la grossesse

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KAZUHIRO KAWAMURA: "Pre-ovulatory LH/hCG surge decreases C-type natriuretic peptide secretion by ovarian granulosa cells to promote meiotic resumption of pre-ovulatory oocytes", HUMAN REPRODUCTION, vol. 26, no. 11, November 2011 (2011-11-01), pages 3094 - 3101, XP055073895 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015016036A1 (fr) * 2013-07-29 2015-02-05 国立大学法人福井大学 Biomarqueur pour analyser la possibilité de fertilisation d'ovule et détermination l'utilisant
JPWO2015016036A1 (ja) * 2013-07-29 2017-03-02 国立大学法人福井大学 卵子の受精可能性の検査のためのバイオマーカーおよびそれを用いた判定
JP2019007971A (ja) * 2013-07-29 2019-01-17 株式会社プロトセラ 卵子の受精可能性の検査のためのバイオマーカーおよびそれを用いた判定

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