WO2013068486A1 - Méthodes pour le diagnostic et le traitement de l'infertilité masculine - Google Patents

Méthodes pour le diagnostic et le traitement de l'infertilité masculine Download PDF

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WO2013068486A1
WO2013068486A1 PCT/EP2012/072171 EP2012072171W WO2013068486A1 WO 2013068486 A1 WO2013068486 A1 WO 2013068486A1 EP 2012072171 W EP2012072171 W EP 2012072171W WO 2013068486 A1 WO2013068486 A1 WO 2013068486A1
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mir
mirna
family
expression level
patient
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PCT/EP2012/072171
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Mohamed BENAHMED
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INSERM (Institut National de la Santé et de la Recherche Médicale)
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the invention relates to the diagnosis and treatment of male infertility.
  • male infertility represents an ever-increasingly current and complicated problem in view of the fact that many causes may concur to present this disorder. Only recently have the biological processes at the base of spermatogenesis been studied, contributing significantly to a wider comprehension of male fertility, emphasizing how genetic and molecular causes can be directly responsible for the different clinical aspects of male infertility. Thus, the male infertility can be caused by disorders relating to spermatozoa production, emission or functionality and represents approximately 35% of couple infertility causes.
  • the diagnosis of male infertility mostly relies on microscopic assays of semen quality including sperm concentration, motility and morphology. In the case that infertility is suspected to be related to the male, a few types of tests are used to assess the quality of male sperm. Five parameters that are frequently used to analyze sperm quality, and which are recommended by the World Health Organization (WHO) are: (1) sperm count; (2) sperm motility; (3) sperm viability; (4) white blood cell (WBC) count; (5) sperm head morphology.
  • WHO World Health Organization
  • Such assays are time-consuming and expensive since they require spermogram with microscopy, echography and semen biochemical analysis and even in certain cases chirurgy. Therefore, there is still a need for identifying marker genes with expression profiles that can be used in a reliable, non- invasive and quick manner to identify male infertility.
  • microR As play essential roles in spermatogenesis
  • the measurement of miRNAs in seminal plasma provides an interesting approach for diagnosing male infertility since 7 miRNAs (miR-34c-5p, miR-122, miR-146b-5p, miR-181a, miR-374b, miR-509-5p, and miR-513a-5p) were identified as markedly decreased in azoospermia but increased in asthenozoospermia (Wang et al., 2011).
  • the present invention is based on the discovery that the miR-29 family, which is up- regulated in the seminal and blood plasma, regulates level expression of DNA methyltransferases (DNMT1, -3 a and -3b) and anti-apoptotic Mcl-1 protein and leads to the apoptosis of testicular germ cells.
  • Up-regulation of miR-29a-c expression or function thus results for notably in no sperm in the ejaculate (azoospermia) or in poor sperm quantity (oligozoospermia) leading to male infertility.
  • level expression of the members of the miR-29 family are directly correlated with the number of spermatozoa in semen (or sperm) evaluated by spermogram useful for stratifying patients.
  • miR-29 family is a new blood biomarker for determining the apoptosis in testes of germ cells but also represents a relevant target for preventing or treating male infertility.
  • This new biomarker offers the promise of earlier and more accurate than those currently used (such as blood follicle stimulating hormone (FSH) since the increase of expression level of the miR-29 family would lead to the decrease of germ cells, whereas increase of expression of FSH would be only a consequence of said decrease of germ cells.
  • FSH blood follicle stimulating hormone
  • the invention relates to a method of identifying whether a patient has, or is at risk of having or developing male infertility, comprising a step of measuring in a sample obtained from said patient the expression level of at least one member of the miR-29 family.
  • the invention also relates to a method of stratifying a patient having, or being at risk of having or developing male infertility, comprising a step of measuring in a sample obtained from said patient the expression level of at least one member of the miR-29 family.
  • kits for performing the methods of the invention comprise means for measuring the expression level of at least one member of the miR-29 family in the sample obtained from the patient.
  • the invention further relates to a compound that inhibits the expression level of at least one member of the miR-29 family for use in the prevention or the treatment of male infertility.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound that inhibits the expression level of at least one member of the miR-29 family or a vector encoding thereof for use in the prevention or the treatment of male infertility.
  • the invention relates further to a method for screening a compound that inhibits the expression level of at least one member of the miR-29 family for use in the prevention or the treatment of male infertility.
  • miRNAs has its general meaning in the art and refers to microRNA molecules that are generally 21 to 22 nucleotides in length, even though lengths of 19 and up to 23 nucleotides have been reported. miRNAs are each processed from a longer precursor RNA molecule ("precursor miRNA"). Precursor miRNAs are transcribed from non-protein- encoding genes. The precursor miRNAs have two regions of complementarity that enables them to form a stem-loop- or fold-back- like structure, which is cleaved in animals by a ribonuclease Ill-like nuclease enzyme called Dicer. The processed miRNA is typically a portion of the stem. The processed miRNA (also referred to as "mature miRNA”) become part of a large complex to down-regulate a particular target gene.
  • Table A list of the miRNAs according to the invention
  • MicroRNA 29 is a family of microRNAs based on their sequence homology that consists of 3 known members, miR-29a, b and c. While miR-29b and 29a stem from the same transcript originating from chromosome 7 in humans and chromosome 6 in mice, the miR A cluster containing miR29c is transcribed from chromosome 1 in both species.
  • measuring encompasses detecting or quantifying.
  • detecting means determining if at least one member of the miR-29 family is present or not in a sample and “quantifying” means determining the amount of at least one member of the miR-29 family in a sample.
  • sample refers to any tissue sample derived from the patient that contains nucleic acid materials. Said tissue sample is obtained for the purpose of the in vitro evaluation.
  • the sample can be fresh, frozen, fixed (e.g., formalin fixed), or embedded (e.g., paraffin embedded).
  • the sample can be is a body fluid such as blood, plasma, serum, semen or seminal plasma (obtained by separating seminal fluid from the sperm).
  • the sample results from biopsy performed in the tissue sample of the patient. For example, a testicular biopsy performed in a patient affected with obstructive azoopermia (also called excretory azoospermia).
  • obstructive azoopermia also called excretory azoospermia
  • a patient denotes a mammal.
  • a patient according to the invention refers to any patient (preferably human) afflicted with or susceptible to be afflicted with male infertility.
  • a patient refers to any patient afflicted with a disorder related to an impaired spermatogenesis, underdevelopment of testes, decrease of testicular weight and excess apoptosis in testes (including germ cells apoptosis, such as adult testicular germ cells).
  • disorders involving to an impairment of spermatogenesis leading to infertility are selected from the group consisting of:
  • a first aspect of the invention relates to a method of identifying whether a patient has, or is at risk of having or developing male infertility, comprising a step of measuring in a sample obtained from said patient the expression level of at least one member of the miR-29 family.
  • a second aspect of the invention relates to a method of stratifying a patient having, or being at risk of having or developing male infertility, comprising a step of measuring in a sample obtained from said patient the expression level of at least one member of the miR-29 family.
  • plasma miR29a levels could be increased by more than 100-fold in oligo or azoospermia patients (who have number of spermatozoa lower than 15 millions/ml of seminal liquid, e.g.. no spermatozoa in azoospermia patients or less than 5 millions/ml of seminal liquid in severe azoospermia patients) compared to fertile ones.
  • said member is selected from the group consisting of miR-29a, miR-29b and miR-29c.
  • one, two or three miRNAs are measured.
  • Combinations of two miRNAs are selected from the group consisting of miR-29a and miR-29b, miR-29a and miR-29c, miR-29a and miR-29c.
  • Combinations of three miRNAs are miR-29a, miR-29b and miR-29c.
  • the method of the present invention may further comprise a step consisting of comparing the expression level of at least one miRNA in the sample with a control, wherein detecting differential in the expression level of the miRNA between the sample and the control is indicative of having or a risk of having or developing a male infertility.
  • the control may consist in sample associated with a healthy patient not afflicted with male infertility or in a sample associated with a patient afflicted with male infertility.
  • control or “control value” can be a single value such as a level or a mean expression level of a miRNA of the invention as determined in a healthy patient or a group of patients who are not afflicted with male infertility or who did not develop male infertility.
  • higher expression level of at least one miRNA selected from the group consisting of miR-29a, miR-29b and miR-29c is indicative of patient having or at risk of having or developing an infertility.
  • a level of expression of a miRNA of the invention indicates that the patient has or is at risk of having or developing infertility.
  • a control value e.g. value obtained from a healthy patient or a group of patients who are not afflicted with male infertility or healthy control value
  • the miR-29s expression measured in the sample of the patient may be at least 10%, 20%, 30%>, 40%>, 50%>, 60%, 70%, 80%, 90%, 100%, 150% or 200% higher than the control value.
  • the invention thus provides a method of identifying whether a patient has, or is at risk of having or developing male infertility, comprising the following steps of:
  • the invention further provides a method of stratifying a patient having, or being at risk of having or developing male infertility, comprising the following steps of:
  • the sample obtained from the patient is a blood sample, a semen sample or a seminal plasma sample.
  • the invention provides a method of identifying whether a patient has, or is at risk of having or developing male infertility, comprising a step of measuring in a blood sample, a semen sample or a seminal plasma sample obtained from said patient the expression level of at least one member of the miR-29 family.
  • the invention relates to a method of stratifying a patient having, or being at risk of having or developing male infertility, comprising a step of measuring in a blood sample, a semen sample or a seminal plasma sample obtained from said patient the expression level of at least one member of the miR-29 family.
  • the invention further provides a method of identifying whether a patient has, or is at risk of having or developing male infertility, comprising the following steps of:
  • the invention further provides a method of stratifying a patient having, or being at risk of having or developing male infertility, comprising the following steps of: (a) measuring in a blood sample, a semen sample or a seminal plasma sample obtained from said patient the expression level of at least one member of the miR-29 family;
  • measuring the expression level of the miRNA of the invention in the sample obtained from the patient can be performed by a variety of techniques.
  • the nucleic acid contained in the samples is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions.
  • the extracted miRNAs is then detected by hybridization (e. g., Northern blot analysis) and/or amplification (e.g., RT-PCR).
  • hybridization e. g., Northern blot analysis
  • amplification e.g., RT-PCR
  • RT-PCR e.g., Northern blot analysis
  • RT-PCR e.g., RT-PCR
  • RT-PCR e.g., RT-PCR
  • RT-PCR e.g., RT-PCR
  • RT-PCR e.g., RT-PCR
  • RT-PCR e.g., Northern blot analysis
  • RT-PCR e.g., RT-PCR
  • RT-PCR e.g., RT-PCR
  • the determination comprises contacting the sample with selective reagents such as probes or primers and thereby detecting the presence, or measuring the amount of miRNAs originally in the sample.
  • Contacting may be performed in any suitable device, such as a plate, microtiter dish, test tube, well, glass, column, and so forth.
  • the contacting is performed on a substrate coated with the reagent, such as a miRNA array.
  • the substrate may be a solid or semi- so lid substrate such as any suitable support comprising glass, plastic, nylon, paper, metal, polymers and the like.
  • the substrate may be of various forms and sizes, such as a slide, a membrane, a bead, a column, a gel, etc.
  • the contacting may be made under any condition suitable for a detectable complex, such as a miRNAs hybrid, to be formed between the reagent and the miRNAs of the sample.
  • a detectable complex such as a miRNAs hybrid
  • Nucleic acids exhibiting sequence complementarity or homology to the miRNAs of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95%) identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization.
  • appropriate means such as a detectable label
  • a wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin).
  • the probes and primers are "specific" to the miRNAs they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC.
  • Tm melting temperature
  • SCC is a 0.15 M NaCl, 0.015 M Na-citrate
  • miRNA arrays or miRNA probe arrays which are macroarrays or microarrays of nucleic acid molecules (probes) that are fully or nearly complementary or identical to a plurality of miRNA molecules positioned on a support or support material in a spatially separated organization.
  • Macroarrays are typically sheets of nitrocellulose or nylon upon which probes have been spotted.
  • Microarrays position the nucleic acid probes more densely such that up to 10,000 nucleic acid molecules can be fit into a region typically 1 to 4 square centimeters.
  • Microarrays can be fabricated by spotting nucleic acid molecules, e.g., genes, oligonucleotides, etc., onto substrates or fabricating oligonucleotide sequences in situ on a substrate. Spotted or fabricated nucleic acid molecules can be applied in a high density matrix pattern of up to about 30 non- identical nucleic acid molecules per square centimeter or higher, e.g. up to about 100 or even 1000 per square centimeter. Microarrays typically use coated glass as the solid support, in contrast to the nitrocellulose-based material of filter arrays. By having an ordered array of miRNA-complementing nucleic acid samples, the position of each sample can be tracked and linked to the original sample.
  • nucleic acid molecules e.g., genes, oligonucleotides, etc.
  • array devices in which a plurality of distinct nucleic acid probes are stably associated with the surface of a solid support are known to those of skill in the art.
  • Useful substrates for arrays include nylon, glass, metal, plastic, latex, and silicon.
  • Such arrays may vary in a number of different ways, including average probe length, sequence or types of probes, nature of bond between the probe and the array surface, e.g. covalent or non-covalent, and the like.
  • the population of target nucleic acids is contacted with the array or probes under hybridization conditions, where such conditions can be adjusted, as desired, to provide for an optimum level of specificity in view of the particular assay being performed.
  • Suitable hybridization conditions are well known to those of skill in the art and reviewed in Sambrook et al. (2001). Of particular interest in many embodiments is the use of stringent conditions during hybridization. Stringent conditions are known to those of skill in the art.
  • miRNAs quantification method may be performed by using stem-loop primers for reverse transcription (RT) followed by a real-time TaqMan® probe.
  • said method comprises a first step wherein the stem-loop primers are annealed to miRNA targets and extended in the presence of reverse transcriptase. Then miRNA-specific forward primer, TaqMan® probe, and reverse primer are used for PCR reactions. Quantitation of miRNAs is estimated based on measured CT values.
  • Expression level of a miRNA may be expressed as absolute expression level or normalized expression level.
  • expression levels are normalized by correcting the absolute expression level of a miRNA by comparing its expression to the expression of a mRNA that is not a relevant for determining patient having or at risk of having or developing an atherosclerosis, e.g., a housekeeping mRNA that is constitutively expressed.
  • Suitable mRNA for normalization includes housekeeping mRNAs such as the U6, U24, U48 and SI 8. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, or between samples from different sources.
  • kits suitable for performing the methods of the invention comprising means for measuring the expression level of at least one member of the miR-29 family in the sample obtained from the patient.
  • kits may include probes, primers macroarrays or microarrays as described above.
  • the kit may comprise a set of miRNA probes as above defined, usually made of DNA, and that may be pre-labelled. Alternatively, probes may be unlabelled and the ingredients for labelling may be included in the kit in separate containers.
  • the kit may further comprise hybridization reagents or other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards.
  • kits of the invention may comprise amplification primers (e.g. stem- loop primers) that may be pre-labelled or may contain an affinity purification or attachment moiety.
  • the kit may further comprise amplification reagents and also other suitably packaged reagents and materials needed for the particular amplification protocol.
  • the kit of the invention relates to a kit suitable for identifying whether a patient has, or is at risk of having or developing male infertility, comprising means for measuring in a sample obtained from said patient, at least at least one member of the miR-29 family.
  • the kit of the invention relates to a kit suitable for a method of stratifying a patient having, or being at risk of having or developing male infertility, comprising means for measuring, in a sample obtained from said patient, at least at least one member of the miR-29 family.
  • the kit of the invention relates to a kit which further comprise means for comparing the expression level of at least one member of the miR-29 family in the sample with a control, wherein detecting differential in the expression level of at least one member of the miR-29 family between the sample and the control is indicative of a risk of having or developing male infertility.
  • the control may consist in sample associated with a healthy patient not afflicted with male infertility or in a sample associated with a patient afflicted with male infertility.
  • a further aspect of the invention relates to a method for monitoring the efficacy of a treatment of male infertility, comprising a step of measuring in a sample obtained from said patient the expression level of at least one member of the miR-29 family.
  • Methods of the invention can be applied for monitoring the treatment (e.g., drug compounds) of the patient.
  • the effectiveness of an agent to affect the expression level of at least one member of the miR-29 family (as hereinafter described) according to the invention can be monitored during treatments of patients receiving male infertility treatments.
  • the "male infertility treatment” that is referred to any type of male infertility therapy undergone by the male infertile patients, including hormones such as testosterone or diet food.
  • the invention relates to a method for monitoring the efficacy of a treatment of male infertility, said method comprising the steps consisting of:
  • the invention also relates to a compound that inhibits the expression level of at least one member of the miR-29 family for use in the prevention or the treatment of male infertility.
  • the invention relates to a compound that inhibits the expression level of at least one miRNA selected from the group consisting of miR-29a, miR-29b and miR-29c for use in the prevention or the treatment of male infertility.
  • inhibiting the expression level of a miRNA or “inhibiting miRNA expression” mean that the production of miRNA in a sample (e.g. plasma or seminal plasma) after treatment is less than the amount produced prior to treatment.
  • One skilled in the art can readily determine whether miRNA expression has been inhibited in a sample, using for example the techniques for determining miRNA transcript level discussed above for the diagnostic methods.
  • a "compound that inhibits the expression level of a miRNA” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of said miRNA. Such compounds include but are not limited to nucleic acids or small organic molecules.
  • Suitable compounds for inhibiting miRNA expression includes miRNA inhibitory nucleic acids selected from the group consisting of antagomirs, antisense nucleic acids, double-stranded RNA (such as short- or small-interfering RNA or "siRNA”) and enzymatic R A molecules such as ribozymes.
  • miRNA inhibitory nucleic acids can be designed according to methods known in the art. These miRNA inhibitory nucleic acids encompassing oligonucleotides composed of naturally occurring nucleobases, sugars, and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally- occurring portions that function similarly.
  • modified or substituted oligonucleotides are may be used over native forms because of desirable properties such as, for example, enhanced affinity for nucleic acid target, and/or increased stability in the presence of nucleases.
  • the miRNA inhibitory nucleic acids include oligomers or polymers of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or both, or modifications thereof.
  • the miRNA inhibitory nucleic acid can be a single-stranded, double stranded, partially double stranded or hairpin oligonucleotide. It preferably consists of, consists essentially of, or comprises at least 12 or more contiguous nucleotides substantially complementary to an endogenous miRNA or a pre- miRNA.
  • partially double stranded refers to double stranded structures that contain fewer nucleotides on one strand. In general, such partial double stranded agents will have less than 75% double stranded structure, less than 50%, or less than 25%, 20% or 15% double stranded structure.
  • An miRNA inhibitory nucleic acid comprises a region sufficient complementary to the target nucleic acid (e.g., target miRNA, pre-miRNA), and is of sufficient length, such that the miRNA inhibitory nucleic acid forms a duplex with the target nucleic acid.
  • the miRNA inhibitory nucleic acid can modulate the function of the targeted molecule.
  • the target molecule is an miRNA, such as a member of the miR-29 family
  • the miRNA inhibitory nucleic acid can inhibit the gene silencing activity of the target miRNA, which action will up-regulate expression of the mRNA targeted by the target miRNA.
  • An miRNA inhibitory nucleic acid can be partially or fully complementary to the target miRNA.
  • the miRNA inhibitory nucleic acid can be further stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification.
  • the miRNA inhibitory nucleic acid includes a phosphorothioate at least the first, second, or third internucleotide linkage at the 5' or 3' end of the nucleotide sequence.
  • the miRNA inhibitory nucleic acid includes a 2'-modified nucleotide, e.g., a 2'- deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'-0-aminopropyl (2 * -0-AP), 2 * -0-dimethylamino ethyl (2 * -0-DMAOE), 2 * -0-dimethylaminopropyl (2 * -0- DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0-N-methylacetamido (2'-0-NMA).
  • a 2'-modified nucleotide e.g., a 2'- deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'-0-amin
  • the miRNA inhibitory nucleic acid includes at least one 2'-0-methyl-modified nucleotide, and in some embodiments, all of the nucleotides of the miRNA inhibitory nucleic acid include a 2'-0-methyl modification.
  • the miRNA inhibitory nucleic acid can be modified so as to be attached to a ligand that is selected to improve stability, distribution or cellular uptake of the agent, e.g., cholesterol.
  • the oligonucleotide miRNA inhibitory nucleic acid can further be in isolated form or can be part of a pharmaceutical composition used for the methods described herein, particularly as a pharmaceutical composition formulated for parental administration.
  • the pharmaceutical compositions can contain one or more oligonucleotide agents, and in some embodiments, will contain two or more oligonucleotide agents, each one directed to a different miRNA (e.g. directed to at least two members of the miR-29 family).
  • An miRNA inhibitory nucleic acid can be constructed using chemical synthesis and/or enzymatic ligation reactions using procedures known in the art.
  • the miRNA inhibitory nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned.
  • the expression vectors can be DNA plasmids or viral vectors.
  • the expression vectors capable of expressing the miRNA inhibitory nucleic acids can be delivered as described herein, and can persist in target cells.
  • viral vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary.
  • Viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus (lentivirus), adenovirus, or alphavirus.
  • Pol II or Pol Ill-based constructs are used to express nucleic acid molecules of the invention (see for example Thompson, U.S. Pats. Nos. 5,902,880 and 6,146,886). Once expressed, the miRNA inhibitory nucleic acid interacts with the target RNA (e.g., miRNA or pre-miRNA) and inhibits miRNA activity.
  • target RNA e.g., miRNA or pre-miRNA
  • liposomes may be used to deliver miRNA inhibitory nucleic acid to a patient.
  • Liposomes can also increase the blood half-life of the gene products or nucleic acids.
  • Liposomes suitable for use in the invention can be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half-life of the liposomes in the blood stream.
  • the miRNA inhibitory nucleic acid is an antagomir, which is a chemically-modified, single-stranded RNA that is antisense to the miRNA sequence.
  • An antagomir is from about 12 to about 33 nucleotides in length, preferably at least about 15 nucleotides in length.
  • a preferred modification is 2'-0-methylation of the ribose. Additional or alternative modifications can include phosphorothioate linkage near 5' and 3' ends or a cholesterol moiety conjugated to the 3' end.
  • Antagomirs form highly stable, sequence-specific duplexes with their corresponding target miRNAs and potently attenuate miRNA activity.
  • antagomirs act as competitive inhibitors of endogenous target mRNA binding to the miRNA, resulting in suppression of miRNA function.
  • Antagomirs can also induce degradation of target miRNAs.
  • Antagomirs bind to their target miRNAs through sequence- specific base pairing.
  • a morpholino is an example of an antagomir.
  • a locked nucleic acid (LNA) antisense oligonucleotide is also an example of antagomir.
  • Antagomirs for use in the present invention preferably inhibit miR-29a, miR-29b and miR-29c.
  • sequence of the antagomir against miR-29a is represented by SEQ ID NO: 1.
  • sequence of the antagomir against miR-29b is represented by SEQ ID NO: 2.
  • Antagomirs can be designed according to methods known in the art. See Krutzfeldt et al. (2005) and U.S. Publication No. 2009/0092980, incorporated herein by reference. Antagomirs are commercially available, for example, from Ambion, Inc. (Austin, Tex.). Another preferred miRNA inhibitory nucleic acid is a miRNA sponge (Ebert et al.,
  • Small organic molecule refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e.g., proteins, nucleic acids, etc.) Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da. Such small organic molecules may be for instance FDA-approved drugs.
  • the small organic molecule is a peroxisome proliferator- activated receptor gamma (PPAR)-y agonist.
  • PPAR- ⁇ agonists include but are not limitated to pioglitazone, 15 deoxy-delta-12,14-PGJ2 or a thiazolidinedione (TZD also known as glitazone) including thiazolidine-2,4-dione PPAR- ⁇ agonist.
  • the PPAR- ⁇ agonist is a thiazolidinedione (TZD) selected from the group consisting of pioglitazone, rosiglitazone, troglitazone, ciglitazone, DRF-2593 (balaglitazone), CS-011 (rivoglitazone), englitazone and darglitazone.
  • ZD thiazolidinedione
  • PPAR- ⁇ agonist that can be used according to the present invention may be the RS)-5-(4-[2-(5-ethylpyridin-2-yl)ethoxy]benzyl)thiazolidine-2,4-dione (pioglitazone also known as ACTOS®; Takeda) (US Patent Publications No. US 4,687,777 and US 5,995,584, which are hereby incorporated by reference in their entireties).
  • Pioglitazone has the stru ture of the Formula:
  • PPAR- ⁇ agonists include: JTT-501 , GI 262570, R- 483, DRL-17564, INT-131, T-2384.
  • Other exemplary PPAR- ⁇ agonists that are contemplated by the invention include but are not limited to, for example, those as described in the following international patent applications which are hereby incorporated by reference in their entireties: WO2005086904, WO2007060992, WO2007100027, WO2007103252, WO2007122970, WO2007138485, WO2008006319, WO2008006969, WO2008010238, WO2008017398, WO2008028188, WO2008066356, WO2008084303, WO2008089461, WO2008089464, WO2008093639, WO2008096769, WO2008096820, WO2008096829, US2008194617, WO2008099944, WO2008108602, WO2008109334, WO2008
  • ⁇ - ⁇ agonist activity may be determined by a cell line transiently transfected with ⁇ - ⁇ bound to a DNA binding domain that controls luciferase expression.
  • the degree of receptor agonism is measured by the amount of luciferase activity after compound treatment.
  • the invention relates to a method for the prevention or the treatment of male infertility in a patient in need thereof comprising administering to said patient a therapeutically effective amount of at least one compound that inhibits the expression level of member of the miR-29 family.
  • a "therapeutically effective amount" of a compound of the invention is an amount sufficient to prevent or to treat infertility in a patient at a reasonable benefit/risk ratio applicable to any medical treatment.
  • a therapeutically effective amount of said compound to be administered to a given patient can be determined by taking into account factors such as the size and weight of the patient; the extent of disease penetration; the age, health and sex of the patient; the route of administration; and whether the administration is regional or systemic.
  • a therapeutically effective amount of said compound can be based on the approximate or estimated body weight of a patient to be treated. Preferably, such effective amounts are administered parenterally or enterally, as described herein.
  • a therapeutically effective amount of the compound is administered to a patient can range from about 5-3000 micrograms/kg of body weight, and is preferably between about 700-1000 micrograms/kg of body weight, and is more preferably greater than about 1000 micrograms/kg of body weight.
  • the compound can be administered to the patient once (e.g., as a single injection or deposition).
  • said compound can be administered once or twice daily to a patient for a period of from about three to about twenty-eight days, more preferably from about seven to about ten days.
  • the compound is administered once a day for seven days.
  • a dosage regimen comprises multiple administrations, it is understood that the effective amount of the compound administered to the patient can comprise the total amount of gene product administered over the entire dosage regimen.
  • a further aspect of the invention thus relates to a method for screening a compound that inhibits the expression level of at least one member of the miR-29 family for use in the prevention or the treatment of male infertility.
  • the screening method may involve measuring or, qualitatively or quantitatively, ability of said candidate compound to inhibit, reduce or suppress the expression level of at least one member of the miR-29 family in miR-29-expressing cells (including miR-29a, miR-29b or miR-29c-expressing cells), and efficiently protects and treats against male infertility by inhibiting, reducing or suppressing apoptosis of testicular germ cells.
  • miR-29-expressing cells including miR-29a, miR-29b or miR-29c-expressing cells
  • the screening method of a compound that inhibits the expression level of at least one member of the miR-29 family for use in the prevention or the treatment of male infertility comprises the steps consisting of: a) providing a plurality of cells expressing at least one member of the miR-29 family; b) incubating said cells with a candidate compound;
  • screening methods involve providing appropriate cells which express at least one member of the miR-29 family.
  • said cells may be selected from the group consisting of the mammal cells reported yet to express at least one member of the miR-29 family (e.g. human prostatic cancer cells VCaP cell line in response to androgen as described in Waltering et al, 2011).
  • the miR-29 family e.g. human prostatic cancer cells VCaP cell line in response to androgen as described in Waltering et al, 2011.
  • apoptotic process in this cell line transfected with "apoptomir” may be visualized with annexinV approaches (immunofluorescence, flux cytometry).
  • a nucleic acid encoding at least one member of the miR-29 family may be employed to transfect cells to thereby express at least one member of the miR family of the invention.
  • Such a transfection may be accomplished by methods well known in the art.
  • said cells may be the CG61 spg (CG-1) mouse spermatagonia type B- spermatocyte cells lines transfected with miR-29a, miR-29b or miR-29c microRNAS as described above in Materials & Methods.
  • the screening method of the invention may further comprising a step consisting of administering the candidate compound selected at step d) to an animal model of male infertility to validate the therapeutic and/or protective effects of said candidate compound on male infertility.
  • said animal model of male infertility may be the animal model described above (i.e. the early (PND1 to 5) postnatal exposure of male rats to xenoestrogen compounds such as the estradiol benzoate EB) as described above in Materials & Methods.
  • the candidate compounds may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo or natural compounds.
  • the candidate compound may be selected from the group of (a) proteins or peptides, (b) nucleic acids and (c) small organic molecules (natural or not).
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound that inhibits the expression level of at least one member of the miR-29 family or a vector encoding thereof for use in the prevention or the treatment of male infertility wherein said miRNA is selected from the group consisting of miR-29a, miR-29b and miR-29c.
  • compositions of the present invention are preferably formulated as pharmaceutical compositions, prior to administering to a patient, according to techniques known in the art.
  • Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen- free.
  • pharmaceutical formulations include formulations for human and veterinary use. Methods for preparing pharmaceutical compositions of the invention are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference.
  • the pharmaceutical formulations comprise at least one compound that inhibits the expression level of at least one member of the miR-29 family (e.g., 0.1 to 90% by weight), or a physiologically acceptable salt thereof, mixed with a pharmaceutically-acceptable carrier.
  • the pharmaceutical formulations of the invention can also comprise at least one compound of the invention which are encapsulated by liposomes and a pharmaceutically-acceptable carrier.
  • Preferred pharmaceutically-acceptable carriers are water, buffered water, normal saline, 0.4% saline, 0.3%> glycine, hyaluronic acid and the like.
  • the pharmaceutical compositions of the invention comprise at least one compound of the invention which is resistant to degradation by nucleases.
  • nucleases One skilled in the art can readily synthesize nucleic acids which are nuclease resistant, for example by incorporating one or more ribonucleotides that are modified at the 2'-position into the miRNAs. Suitable 2'-modified ribonucleotides include those modified at the 2'-position with fluoro, amino, alkyl, alkoxy, and O-allyl.
  • compositions of the invention can also comprise conventional pharmaceutical excipients and/or additives.
  • Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents.
  • Suitable additives include, e.g., physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (such as, for example, calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
  • Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized.
  • solid pharmaceutically acceptable carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a solid pharmaceutical composition for oral administration can comprise any of the carriers and excipients listed above and 10- 95%, preferably 25%-75%, of the at least one compound of the invention.
  • a pharmaceutical composition for aerosol (inhalational) administration can comprise 0.0120% by weight, preferably 1%-10% by weight, of one compound of the invention encapsulated in a liposome as described above, and a propellant.
  • a carrier can also be included as desired; e.g., lecithin for intranasal delivery.
  • FIGURES Figure 1: Neonatal exposure to EB induces a long-term alteration in miR-29s levels.
  • Mature miR-29a (A), miR-29b (B), and miR-29c (C) levels were determined by real-time RT- PCR in adult rat testes from unexposed (0) or 0.75, 1.25, and 2.5 ⁇ g/day EB-exposed animals.
  • Mature miR-29a (D), miR-29b (E), and miR-29c (F) levels were determined by real-time RT- PCR in immature (PND 6) or adult (PND 90) rat testes from unexposed (-EB) or exposed (2.5 ⁇ g/day, +EB) animals. Histograms represent relative microRNA levels normalized to U87 and SnoRNA levels. The values represent the mean ⁇ SEM determined from 7-8 different animals (* /? ⁇ 0.05).
  • FIG. 2 Effects of knock-down of miR-29 family on DNMT and MCL-1 protein levels in germ cells.
  • Protein levels for DNMT3A (A), DNMT3B (B), DNMT1 (C) and MCL- 1 (D) were determined by western blotting analysis in the GC-1 germ cell line transiently transfected (48 h) by miR-29a, miR-29b, and miR-29c. Histograms represent protein levels expressed as the percentage of the ratio (target protein/actin protein) detected in the scramble- treated control cells. The values represent the mean ⁇ SEM (* /? ⁇ 0.05).
  • Figure 3 Correlation between miR29a plasma levels and sperm count or FSH plasma levels.
  • Mature miR29a levels were determined by real-time RT-PCR in plasma from fertile ( ⁇ ) or oligospermic ( ⁇ ) patients. The miR29a levels were normalized to RNU44. The miR29 a ratio were represented according to the sperm count (A) or the FSH plasma levels (B) of each patient.
  • FIG. 4 MiR-29a expression in plasma from fertile and infertile men.
  • Patients were grouped according to the spermatozoa number in sperm (azoospermia, cryptozoospermia, oligospermia or fertile -normal number-) .
  • C Patients were grouped according to the mobility of spermatozoa (asthenospermia or normal mobility -fertile-).
  • D Patients were grouped as fertile or infertile.
  • FIG. 5 MiR-29b expression in plasma from fertile and infertile men.
  • a & B Patients were grouped according to the spermatozoa number in sperm (azoospermia, cryptozoospermia, oligospermia or fertile -normal number-) .
  • C Patients were grouped according to the mobility of spermatozoa (asthenospermia or normal mobility -fertile-).
  • D Patients were grouped as fertile or infertile.
  • Figure 6 MiR-29c expression in plasma from fertile and infertile men.
  • Patients were grouped according to the spermatozoa number in sperm (azoospermia, cryptozoospermia, oligospermia or fertile -normal number-) .
  • C Patients were grouped according to the mobility of spermatozoa (asthenospermia or normal mobility -fertile-).
  • D Patients were grouped as fertile or infertile.
  • EXAMPLE 1 MODEL OF MALE INFERTLITY: MALE RATS EXPOSED TO XENOESTROGENS
  • TUNEL and Immunofluorescence Paraffin sections (5 ⁇ ) of Bouin-fixed testes were mounted onto Superfrost Plus slides. The slides were used for either TUNEL or immunohistochemistry approaches. TUNEL experiments were performed as previously described (Bozec et al. 2004), using terminal deoxynucleotidyl transferase (Euromedex, Mundolsheim, France), biotin-11-dUTP (Roche Diagnostics, Meylan, France), Streptavidin- fluorescein conjugate (Merck KGaA, Darmstadt, Germany).
  • the sections were counterstained with 4,'6'-diaminido-2-phenylindole (DAPI), mounted with coverslips using mounting medium and incubated overnight at 37°C .
  • DAPI 4,'6'-diaminido-2-phenylindole
  • the results were expressed as the number of TUNEL-positive or PCNA-positive cells per 100 random round seminiferous tubules.
  • sections were incubated 10 min in PBS 0.1% Triton X-100 at room temperature, then in PBS-5% SVF- 1% protease inhibitor cocktail (Sigma) for 30 min.
  • the antibodies used in this study were Dnmt3A (1/1 ,000; Cell Signaling, #2160), Dnmt3B (1/1,000; Cell Signaling, #2161), Dnmtl (1/1,000; Stressgen, KAM-TF040), Cleaved Caspase-3 (1/500; Cell Signaling, #9661), Mcl-1 (1/5,000; Rockland), and Actin (1/20,000; Jackson's Laboratory).
  • Membranes were scanned using a Luminescent Image Analyzer 3000 CCD Camera (Fujifilm, Dusseldorf, Germany) and quantified using MultiGauge useful (Fujifilm).
  • Complementary DNA was synthesized from total RNA (1 ⁇ g) with MMLV (10 units ⁇ L) (Invitrogen, Cergy Pontoise, France) and random hexamer primers (5 ⁇ ) (Invitrogen, Cergy Pontoise, France) in a final volume of 20 according to the manufacturer's instructions. A 1/20 dilution of each reverse transcription product was used for the real-time RT-PCR analyses.
  • the real-time RT-PCR measurement of individual cDNA (2 ⁇ ⁇ of 1/20 dilution) was performed using Quantitect SYBR Master Mix (4 ⁇ ) (Qiagen), PCR primers (2 ⁇ ⁇ of 10 ⁇ solution), and ultrapure water (2 ⁇ ) to measure the duplex DNA formation with the Roche Lightcycler system.
  • Gene amplification was carried out as follows: initial activation of HotStarTaq DNA polymerase at 95°C for 10 min; 45 cycles in three steps: 95 °C for 15 sec, 60°C for 15 sec, and 72°C for 15 sec.
  • melting temperature analysis was carried out by a slow increase in temperature (0.1°C/sec) up to 95°C.
  • Standard curves were generated with testicular cDNA pools from animals with different treatments. Normalized expression was calculated using the Ct method. The data were normalized to ⁇ -actin levels.
  • EB estradiol benzoate
  • testicular weight was observed between untreated (3.23 ⁇ 0.13) and EB-exposed rats to 0.75 (3.21 ⁇ 0.1), 1.25 (3.03 ⁇ 0.13) and 2.5 (3.06 ⁇ 0.07) ⁇ g per day EB.
  • PND90 a significant increase was observed (30%, p ⁇ 0.05) in the TUNEL- positive cell number and active CASP3 levels.
  • Active CASP3 protein levels were significantly increased in testes from 1.25 ⁇ g/day-(twofold, p ⁇ 0.05) and 2.5 ⁇ g/day-treated animals (1.8-fold, p ⁇ 0.05).
  • the TUNEL-positive cells were mainly germ cells (spermatocytes and/or spermatids), but not somatic Sertoli and Leydig cells. With regards to the rate of proliferation, although adult spermatogonia and preleptotene spermatocytes specifically displayed PCNA staining, no significant change in the accumulation of PCNA was observed between untreated and EB-exposed male rats.
  • Neonatal exposure to EB induces long-term alteration of apoptomirs miR-29 in adult rat testes: Early postnatal exposure to EB induced a dose-dependent increase in miR-29 (a, b and c) levels in adult (PND 90) rat testicular tissue. Indeed, miR-29a displayed a significant increase at 1.25 (1.4-fold, p ⁇ 0.01) and 2.5 (1.3-fold, p ⁇ 0.05) ⁇ g/day of EB (Fig. 1A). A highly significant increase in the miR-29b transcript was observed at 1.25 (1.6-fold, p ⁇ 0.05) and 2.5 (2.6-fold, p ⁇ 0.01) ⁇ g/day of EB (Fig. IB).
  • miR-29c levels were significantly increased at 0.75 (1.35-fold, p ⁇ 0.05), 1.25 (1.6-fold, p ⁇ 0.01), and 2.5 (1.4-fold, p ⁇ 0.05) ⁇ g/day of EB (Fig. 1C).
  • MiR-29a (Fig. ID), -29b (Fig. IE), and -29c (Fig. IF) were affected at PND 90 but not at PND 6 (immature testes) or PND30 (pubertal testes). Indeed, no induction of miR-29a (Fig. ID), miR-29b (Fig. IE) or miR-29c (Fig. IF) expression was detected after neonatal EB exposure at PND6 and PND 30.
  • Mcl-1 as an anti-apoptotic factor and DNMTs, because their alterations induce germ cell death (Doerksen et al, 2000).
  • Mcl-1 protein levels in adult testes from rats exposed during early postnatal life to EB, Mcl-1 protein levels (but not mRNA, data not shown) were significantly reduced at a dose of 2.5 (41 % decrease, p ⁇ 0.05) ⁇ g/day of EB. Mcl-1 protein levels were affected at PND 90 but not at PND 6 (data not shown).
  • DNMT3A2 mRNA levels were unchanged in adult rat testes.
  • antibodies rising against DNMT3A2 protein lack specificity, we were unable to quantify the potential changes in these protein levels.
  • DNMT3A, DNMT3B, and DNMT1 protein levels were significantly (p ⁇ 0.05) decreased in adult (PND 90) testes from rats exposed during early postnatal life to EB, DNMT protein levels were unchanged in immature (PND 6) rat and pubertal (PND30) testes following immediate exposure to EB.
  • EXAMPLE 2 TRANSFECTION MIR29 FAMILY IN GC-1 LINES
  • GC-1 spg mouse spermatogonia type B- spermatocyte cell lines, provided by Pr Chambon (IGBMC, France), were maintained in Dulbecco's modified Eagle's Medium/Glutamax medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen) at 37°C in a humidified, C02-controlled (5%) incubator.
  • miR- 29a, -b, or -c microRNAs cells were transfected in 12-well plates using Hiperfect Transfection Reagent (Qiagen) according to the manufacturer's protocol, with 50 nM (final concentration) scrambled negative control miRNA or miR-29a, miR-29b, and miR-29c analogs (Applied Biosystems) or 25 nM (final concentration) negative control siRNA (NC siRNA, Eurogentec) or DNMT1, DNMT3b siRNA (Applied Biosystems. Cells were left 48 h with miRNA or siRNA treatment and harvested for protein or RNA isolation.
  • Hiperfect Transfection Reagent Qiagen
  • mice receive via intravenous injection specific antagomiR. Analysis of lesions sizes and locations are performed. Two antagomirs against miR-29a and miR-29b, respectively representing by SEQ ID NO: 1 and SEQ ID NO: 2 are used. It should be further noted that for these two antagomirs all nucleotides are 2'-OMe modified and the two first nucleotides and the three last nucleotides are phosphothioate modified.
  • EXAMPLE 4 CLINICAL DATA OBTAINED FROM PATIENTS SUFFERING WITH MALE INFERTILITY
  • Plasma and seminal plasma and testicular tissues were obtained from men consulting for artificial reproductive technologies (ART) (Centre Hospitalier Universitaire [CHU] de Nice, France) and from fertile men. The study was conducted according to the rules of Declaration of Helsinki and the Ethic Committee of the Medical Faculty and the State Medical Board (CHU de Nice) agreed to these investigations. An informed consent was obtained from all patients. Shortly after punction, plasma and leucocytes were separated by centrifugation (3000 rpm, 10 min). Plasma (blood and seminal) and testicular tissues were frozen at -80°C until use. R A from plasma and plasma seminal were extracted with TRIzol reagent. After precipitation, pellet was resuspended in sterile-water and submitted to a colunm purification (High Pure miR A Isolation kit Roche). Results

Abstract

La présente invention concerne le diagnostic et le traitement de l'infertilité masculine, en particulier l'implication de la famille des miARN 29 dans l'infertilité masculine.
PCT/EP2012/072171 2011-11-08 2012-11-08 Méthodes pour le diagnostic et le traitement de l'infertilité masculine WO2013068486A1 (fr)

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WO2014180981A1 (fr) * 2013-05-10 2014-11-13 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédé pour diagnostiquer et traiter la stérilité mâle
WO2020052946A1 (fr) 2018-09-11 2020-03-19 Universita' Degli Studi Di Foggia Procédé d'identification de la prédisposition à l'infertilité masculine basé sur des polymorphismes du gène du récepteur de la vitamine d
CN111378760A (zh) * 2018-12-28 2020-07-07 上海市计划生育科学研究所 精子特异miRNAs在辅助生殖体外受精诊断中的应用
CN111876497A (zh) * 2020-08-31 2020-11-03 中国农业大学 一种鉴定或辅助鉴定动物精液品质的方法

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WO2020052946A1 (fr) 2018-09-11 2020-03-19 Universita' Degli Studi Di Foggia Procédé d'identification de la prédisposition à l'infertilité masculine basé sur des polymorphismes du gène du récepteur de la vitamine d
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