EP3850112A1 - Procédé d'identification de la prédisposition à l'infertilité masculine basé sur des polymorphismes du gène du récepteur de la vitamine d - Google Patents

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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Publication number
EP3850112A1
EP3850112A1 EP19758729.8A EP19758729A EP3850112A1 EP 3850112 A1 EP3850112 A1 EP 3850112A1 EP 19758729 A EP19758729 A EP 19758729A EP 3850112 A1 EP3850112 A1 EP 3850112A1
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EP
European Patent Office
Prior art keywords
infertility
vdr
fokl
predisposition
vitamin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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EP19758729.8A
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German (de)
English (en)
Inventor
Milena Grazia Rita Sinigaglia
Luigia TRABACE
Vito Angelo GIAGULLI
Matteo Domenico CARBONE
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Universita' Degli Studi di Foggia
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Universita' Degli Studi di Foggia
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Publication of EP3850112A1 publication Critical patent/EP3850112A1/fr
Pending legal-status Critical Current

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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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to the sector of diagnostics and more in particular to an in vitro method for detecting the predisposition of a male individual to infertility based on the identification of genetic polymorphisms of the vitamin D receptor gene.
  • Idiopathic male infertility is defined as the infertility condition that cannot be clearly attributed to hormonal factors or environmental elements and that involves over 40% of the population of infertile males (Olesen IA, Andersson AM, Aksglaede L, Skakkebaek NE, Rajpert-de Meyts E, Joergensen N, Juul A. Clinical, genetic, biochemical, and testicular biopsy findings among 1,213 men evaluated for infertility. Fertil Steril. 2017 Jan; 107 (1 ): 74-82. e7 ) .
  • AR-CAG Polymorphism of the androgen receptor (AR) CAG (AR-CAG) is also known, which is characterized by the presence of a variable number of repeats of the CAG triplet in the gene that codifies for the androgen receptor; in particular it has been highlighted that the length of the repeat characterizes different subclasses of infertile patients (Giagulli VA, Carbone MD, De Pergola G, Guastamacchia E, Resta F, Licchelli B, Sabba C, Triggiani V.
  • Giagulli VA Carbone MD
  • De Pergola G De Pergola G
  • Guastamacchia E Resta F
  • Licchelli B Sabba C
  • Triggiani V Triggiani V.
  • IMI idiopathic male infertility
  • Canadian patent No. CA2865424 describes a method for improving the capacity of sperm to fertilize an egg in vitro and/or in vivo that comprises a means containing active vitamin D (calcitriol) or analogs thereof with which sperm samples are incubated .
  • Chinese patent No. CN107603982 describes a method for the diagnosis of male infertility caused by the mutation of the Piwill gene, implied in the function of the germ cell- intrinsic regulator auto-regenerative capacity.
  • Chinese patent No. CN105492631 describes a method for the diagnosis of idiopathic azoospermia using as genetic markers different mutations of the androgen receptor gene.
  • Chinese patent No. CN104928385 describes a diagnostic method based on the detection of mutation sites of the promoter region of the UBE2B gene (UBE2B ubiquitin conjugating enzyme) involved in the repair of DNA damage.
  • WO2013068486 describes miR-29, involved in the regulation of gene expression, as a blood biomarker for determining apoptosis in germ cells.
  • US patent application No. US2012283126 describes a method for the diagnosis of infertility based on the identification of mutations of the DPY19L2 gene, involved in the extension of the head of the sperm and the formation of acrosomes during spermatogenesis .
  • Canadian patent No. CA2789941 describes a method for determining the fertility status of a male individual based on the deletion polymorphism of the DEFB-126 gene that codifies for Beta-defensin 126, which causes globozoospermia .
  • Korean patent No. KR20080082562 describes a diagnostic kit for evaluating male infertility based on the detection of micro deletions of different genes of the Y chromosome selected from the group consisting of TMSB4Y, AMELY, RPS4Y2 , VCY, ZFY, TBL1Y, SMCY, XKRY, CYorf15A, NLGN4Y, ElFIAY, UTY, RBM1 , CYorfl5B, TTTY5 , TTTY6, TTTY17 and DDX3Y .
  • US patent application No. US2005130202 describes a diagnostic method for identifying male infertility which is based on the presence or absence of at least one mutation or mutations of the POLG gene that codifies for mitochondrial DNA polymerase.
  • Vitamin D plays an important role in regulating cell growth in many tissues including the testicle and the prostate (Gruber BM, Anuszewska EL. Studies on the influence of vitamin D3 metabolites on apoptosis induction in human neoplastic cells. Acta Pol Pharm. 2003 Sep-Oct ; 60 ( 5 ) : 363- 6 ; Foresta C,
  • the variant of Fokl polymorphism of the start codon in the N- terminal region of the VDR gene is known, which comes from a deletion within the translation start site of exon 2, which leads to proteins that differ in terms of 3 amino acids (Gene ID: 7421, rsl0735810, https://www.ncbi.nlm.nih.gOv/gene/7421#summary).
  • Alleles are a shorter protein of 424 amino acids (variant F) and a longer protein composed of 427 amino acids (variant f) where the variant F is more effective in interacting with other transcription factors, co-regulation of different genes (Arai H, Miyamoto K, Taketani Y, Yamamoto H, Iemori Y, Morita K, Tonai T, Nishisho T, Mori S, Takeda E.
  • a vitamin D receptor gene polymorphism in the translation start codon effect on protein activity and relation to bone mineral density in Japanese women. J Bone Miner Res.
  • the diagnostic methods available to date are based on particularly invasive testicular biopsy and on the evaluation of different biological parameters that are not specifically predictive of idiopathic conditions.
  • the technical problem that the present invention sets out to solve is providing a diagnostic method that overcomes the technical disadvantages that methods known to date present and that, therefore, is not at all invasive and has no interaction with the body of the subject, unlike testicular biopsies, and that is one-factorial, i.e. it is based on the univocal evaluation of a single parameter, hence simplifying the predictivity and preventing any causal and/or systematic errors that can arise in multi-factorial analyses.
  • the technical problem is solved by providing a method for determining the predisposition to infertility of a male individual comprising the identification and quantification of the variants of polymorphism of the start codon in exon 2 in the N-terminal region of the vitamin D receptor gene (VDR fokl) wherein the variants in heterozygosis and in recessive homozygosis are indicative of infertility.
  • VDR fokl vitamin D receptor gene
  • a further object of the present invention is a diagnostic kit for determining the predisposition to infertility of a male individual comprising the elements necessary for the identification and quantification of the variants in homozygosis, recessive and wild-type, and in heterozygosis, of polymorphism of the start codon in exon 2 in the N-terminal region of the vitamin D receptor gene (VDR fokl), for the combined and/or sequential use thereof.
  • VDR fokl vitamin D receptor gene
  • Figure 1 shows a graph of the significant differences between the frequencies of the three forms of VDR-Fok I in fertile subjects (FM) and those with idiopathic oligozoospermia (IIO), idiopathic azoospermia (IIA) or ex-cryptorchidism (CrypM) .
  • Fokl polymorphism of the vitamin D receptor gene D VDR consists of a nucleotide substitution from thymine T to cytosine C in the translation start codon of the of the VDR gene, which from ATG mutates into ACG; said polymorphism leads to the translation of three amino acids from the translation start site of the gene with the consequent alteration of the relative protein, missing three amino acids.
  • the codon ACG defines the F allele, while the ATG codon defines the f allele.
  • the combination of these alleles can produce the genotypes ff, Ff and FF.
  • the F variant corresponds to a shorter protein of 424 amino acids
  • the f variant corresponds to a longer protein of 427 amino acids.
  • genotype FF (short form) causes an increase of the transcription activation.
  • polymorphism means genetic polymorphism, i.e. the presence of a genetic variation can be determined by substitutions, deletions or insertions of bases in the DNA.
  • start codon means the sequence of 3 nucleotides (triplet) that initiates the translation on the mRNA.
  • the Fokl restriction enzyme is a bacterial endonuclease that recognizes the palindromic sequence 5'-GGATG-3' operating a cut to 9/13 nucleotides downstream of the recognition site (Sugisaki H, Kanazawa S. New restriction endonucleases from Flavobacterium okeanokoites (Fokl) and Micrococcus luteus (Mlul). Gene, 1981; 16(1-3) : 73-8) .
  • idiopathic male infertility means the infertility condition that cannot be attributed to hormonal factors or environmental elements and that is not associated with spermiographic and clinical abnormalities, but is characterized by modification of motility and/or morphology modification and/or sperm concentration.
  • Idiopathic male infertility can be associated with oligoozospermia, azoospermia or cryptorchidism
  • DNA extraction from the biological sample means all the techniques known in the state of the art which are used to isolate DNA from the biological sample taken from the patient, which are appropriately selected by a person skilled in the art.
  • DNA amplification means any technique able to increase the number of copies of DNA available from a sample. It tends to be PCR, a polymerase chain reaction, whose protocols can be suitably selected by a person skilled in the art.
  • DNA selection means any technique used for selecting a certain sequence or group of sequences within a DNA sample, the technique and protocols can be suitably selected by a person skilled in the art .
  • electrophoresis means the analytical and separation technique based on the movement of electrically charged particles immersed in a fluid due to the effect of an electric field applied through a pair of electrodes to the fluid itself, in the case of DNA electrophoresis on agarose gel is commonly used and the most appropriate protocol can be selected by a person skilled in the art .
  • the object of the present invention is a method for determining the predisposition of a male individual to infertility comprising the identification and quantification of the variants in homozygosis, recessive and wild-type, and in heterozygosis, of polymorphism of the start codon in exon 2 in the N-terminal region of the gene of the vitamin D receptor (VDR fokl), wherein the variants in heterozygosis and in recessive homozygosis are indicative of infertility.
  • VDR fokl vitamin D receptor
  • the variants of polymorphism of the start codon in exon 2 in the N-terminal region of the vitamin D receptor gene (VDR fokl) produce the genotypes: recessive homozygote ff which is positive to digestion with restriction enzyme fokl in both alleles, wild-type homozygosis FF which is negative to digestion with restriction enzyme, fF positive to digestion with restriction enzyme fokl in a single allele.
  • the identification and quantification of the variants in homozygosis is performed by genetic analysis of biological samples.
  • the biological samples are semen or blood, more preferably peripheral blood.
  • the method for determining the predisposition of a male individual to infertility comprises the following steps: a) extraction of DNA from the biological sample;
  • step b) amplification of the DNA extracted in step a) ;
  • step b) selection from the DNA amplified in step b) of the DNA corresponding to the vitamin D receptor gene (VDR fokl) by the comparison with standard molecular marker to identify a band of 265 base pairs corresponding to the genotype VDR-FokI F;
  • step a) the extraction of DNA from the biological sample is carried out by standard procedures that can be appropriately selected by a person skilled in the art, the extraction of DNA can be carried out by filtration and/or centrifugation; filtration and/or centrifugation protocols can be appropriately selected by a person skilled in the art.
  • step b) the amplification is carried out by PCR.
  • step b) in the PCR the following primers are used : PRIMER VDR-FORWARD
  • the amplification reaction by PCR consists of one cycle at 94°C for 5 minutes, 35 cycles at 94°C for 30 seconds, 62°C for 30 seconds and 72°C for 1 minute, one cycle at 72°C for 10 minutes.
  • step c) the selection is performed by electrophoresis followed by a comparison with a known parameter of a known molecular weight.
  • the electrophoresis is agarose gel electrophoresis .
  • step f) the analysis is carried out with a UV transilluminator .
  • the infertility is idiopathic male infertility ( IMI ) .
  • the idiopathic male infertility is idiopathic male infertility associated with oligoozospermia, azoospermia or cryptorchidism.
  • the method for determining the predisposition of a male individual to infertility by the identification and quantification of the variants in homozygosis, recessive and wild-type, and in heterozygosis, of polymorphism of the start codon in exon 2 in the N-terminal region of the gene of the vitamin D receptor (VDR fokl), wherein the variants in heterozygosis and in recessive homozygosis are indicative of infertility comprises that the blood samples are collected in EDTA test tubes, from which the DNA is extracted using standard procedures.
  • VDR- Fokl genotypes were analysed through PCR (Polymerase chain reaction), performed in buffer 10X (10 nMol trisHCl, pH 8,3), MgCl 2 , dNTP, PRIMER VDR-FORWARD SEQ.ID.N.l: GAT GCC AGC TGG CCC TGG CAG TG, and PRIMER VDR-REVERSE SEQ.ID.N.2: ATG GAA ACA CCT TGC TTCTTC TCC CTC, H20, Super Taq, mineral oil; the amplification reaction performed in Thermal Cycling consisting of a cycle at 94°C for 5 minutes, 35 cycles at 94°C for 30 seconds, 62°C for 30 seconds and 72°C for 1 minute, one cycle at 72°C for 10 minutes; the product of PCR mixed with loading buffer for electrophoresis Blu 6X and loaded into agarose gel wells at 3% in buffer TAE IX with the addition of ethidium bromide, the electrophoretic separation performed at
  • Fokl F genotype defined by the sample that has a band of 265 bp-base pairs, for the identification of the VDR F polymorphism after performing an enzymatic digestion of the product previously amplified with restriction enzyme fok I, buffer 10X, sterile water and mineral oil, all incubated overnight at 37°C; the restriction product mixed with Blu 6X and subjected to electrophoretic separation on agarose gel at 3% in buffer TAE IX and ethidium bromide for 45 min at 80-100 V and analysed on a UV transilluminator.
  • a further object of the present invention is a diagnostic kit for determining the predisposition of a male subject to infertility, comprising the necessary elements for the identification and quantification of the variants in homozygosis, recessive and wild-type, and in heterozygosis of polymorphism of the start codon in exon 2 in the N-terminal region of the vitamin D receptor gene (VDR fokl), for the combined and/or sequential use thereof.
  • the diagnostic kit comprises the primers that amplify the region that comprises the start codon of exon 2 in the N-terminal region of the vitamin D receptor gene (VDR fokl ) :
  • the diagnostic kit comprises a standard molecular marker for identifying a band of 265 base pairs corresponding to the genotype VDR-FokI F.
  • the diagnostic kit comprises a Folk I endonuclease restriction enzyme.
  • the diagnostic kit comprises, for the combined and/or sequential use thereof: a PRIMER VDR-FORWARD
  • SEQ.ID.N.l GAT GCC AGC TGG CCC TGG CAG TG; a PRIMER VDR-REVERSE
  • SEQ.ID.N.2 ATG GAA ACA CCT TGC TTCTTC TCC CTC; a standard molecular marker for identifying a band of 265 base pairs corresponding to the genotype VDR-FokI F; a Fok I endonuclease restriction enzyme.
  • IMI idiopathic infertility
  • IMA azoospermia
  • IOI idiopathic oligozoosperm
  • vitamin D serum levels 25 hydroxy-vitamin D3
  • a Mediterranean diet 50% carbohydrates, 20% protein and 30% fat
  • smoking and drinking alcohol the prevalence of smoking in the sterile and in the fertile group was not significantly different, being 14% and 12.5% respectively of infertile and fertile subjects, whereas both groups declared that they drank alcohol moderately ( ⁇ 24 grams/day).
  • the assessment was performed at the secondary reference Endocrinology centre (Unita ambulatoriale di Endocrinologia e Malattie Metaboliche, Presidio di Assistenza Territoriale "F Jaia” di Conversano, Bari) .
  • the testicular volume (TV) was evaluated through the Prader orchidometer and reported as the average volume of both testicles. None of the subjects was obese [body mass index (BMI) ⁇ 30 Kg/m 2 ] or affected by dysmetabolic disorders, such as altered glucose tolerance or confirmed diabetes mellitus, and had normal kidney, heart and liver function. 70% of the infertile men had never taken infertility therapy, whereas the remaining 30%, who were having therapy, had stopped at least 3 months before the start of the evaluation. The evaluation was performed in accordance with the year 2000 version of the Declaration of Helsinki and according to good clinical practice. All the voluntary subjects read and understood the informed consent and signed it during the clinical evaluation and investigations. No medical therapy was performed; on the contrary, the fact of having resorting to therapy, even for different reasons from fertility, was a reason for the exclusion of the volunteer from the evaluation.
  • Peripheral blood was collected in test tubes made with EDTA.
  • the DNA was extracted using standard procedures.
  • VDR-FokI genotypes (rs 2228570) SNP were analysed through PCR (Polymerase chain reaction) .
  • the PCR was performed in 24 m ⁇ of total volume containing: 2.5 m ⁇ of buffer 10X (10 nMol trisHCl, pH 8.3), 0.75 m ⁇ di MgCl2,
  • a negative control was always amplified (test without DNA) and a positive control (known DNA, previously evaluated) .
  • the amplification was performed in Thermal Cycling (PTC-100, MJ Research) and consisted of a cycle at 94°C for 5 minutes; 35 cycles at: 94°C for 30 seconds, 62°C for 30 seconds and 72°C for 1 minute; one cycle at 72 °C for 10 minutes.
  • VDR-FokI F genotype is defined when the sample has a band of 265 bp- base pairs.
  • VDR F The digestion of VDR F was performed with 10 m ⁇ of amplified product VDR F, 2m1 of restriction enzyme, 3 m ⁇ of buffer 10X, 15 m ⁇ of sterile water, 2 drops of mineral oil. Everything was incubated overnight at 37° C.
  • the genotypes were identified according to the enzymatic digestion pattern as "FF" (Fokl negative in both alleles) (265 bp-base pairs) , ff (Fokl positive in both alleles) and Ff (heterozygote) .
  • the measurements of VDR polymorphism were repeated twice in two different determinations.
  • the amplified products ff (Fokl positive in both alleles) and Ff (heterozygote) were sequenced on ABI PRISM 3130 Genetic Analyzer.
  • Vitamin D deficiency is defined by circulating Vitamin D levels lower than 30 ng/ml (Holick MF, Binkley NC, Bischoff-Ferrari HA, Gordon CM, Hanley DA, Heaney RP, Murad MH, Weaver CM. Endocrine Society. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2011 Jul; 96 (7) : 1911-30) .
  • the semen analysis was performed following recent World Health Organization criteria (World Health Organization. WHO laboratory manual for the examination and processing of human semen 5th ed. Switzerland: WHO; 2010) .
  • test c2 The chi-squared test (test c2) was used to determine the differences in frequency distribution between the VDR-FokI polymorphism both in fertile and in infertile subjects. A 5% "a priori” was considered statistically significant. The analyses were performed using the Statistica software for Windows, version 12.0 (StatSoft, Tulsa, Okhla) .
  • Vit D receptor in the testicular function of fertile men and those with idiopathic infertility (IMI) who had oligoozospermia, azoospermia or cryptorchidism was evaluated, in particular the role of the variant of polymorphism of the start codon Fokl in the N-terminal region of the VDR gene (VDR-FokI) which comes from a deletion within the start site for the translation of exon 2 that leads to proteins that differ by just 3 amino acids.
  • VDR-FokI the variant of polymorphism of the start codon Fokl in the N-terminal region of the VDR gene
  • IB/FSH ratios were significantly different between the different groups of participants.
  • the Vit. D levels which were in the ranges considered to be sufficient in all the groups studied, were not different between normal and fertile subjects and infertile ones.
  • Figure 1 shows a graph of the distribution between the three forms (FF,ff and Ff) of VDR-FokI polymorphism in the different groups evaluated.
  • FM fertile and healthy subjects
  • the frequency of the FF polymorphism (wild-type) was significantly lower in infertile subjects [especially in the azoospermia group (IAI)] (P 0.01)
  • the Ff heterozygote form was significantly increased in the ex-cryptorchidism group (CrypM) (P 0.01) and in the IOI (P 0.01) .
  • Table 2 shows the statistical differences between the clinical (TV and BMI ) , seminal (SC, SV, IM and PM), hormonal ( FSH, LH, T, IB, Vit D) characteristics and the IB/T and T/LH ratios in all the subjects included in the evaluation, regardless of whether they belong to infertile or fertile subjects, split into three groups according to the three forms of VDR-FokI polymorphism: FF, Ff and ff.
  • Table 3 shows the correlations (Sperm rank test; P ⁇ 0.05 in bold) between the different clinical characteristics (TV and BMI), the sperm ones (SV, SC, IM and PM) and the plasma levels of the hormones (FSH, LH, T and IB) and of their ratios, of the serum levels of Vit. D in the subgroups of infertile subjects and of fertile men considered in a single group.
  • the TV was significantly and directly correlated with the serum values of T and IB, with the T/LH and IB/FSH ratios and with the sperm parameters (SV and SC) , whereas it was negatively correlated with both the gonadotropins (FSH and LH) (P 0.05) .
  • the BMI was significantly and positively correlated both with SV and IM (both P 0.05) .
  • Both SC and SV were positively correlated with the serum levels of T and IB and the T/LH and IB/FSH ratios (P 0.05), whereas they were inversely correlated with gonadotropins (FSH and LH) (P 0.05) .
  • the Vit. D plasma levels were positively correlated both with the circulating gonadotropins and the levels of IB (P 0.05) .
  • Vitamin D enzymes involved in the synthesis thereof and/or the metabolism thereof and the finding of VDR in germ cells, sperm, Leydig cells, epididymis, seminal vesicles and prostate, both during foetal life and in adult life highlights a possible direct role of Vit. D in male reproduction, during the whole of reproductive life (Yang B, Sun H, Wan Y, Wang H, Qin W, Yang L, Zhao H, Yuan J, Yao B. Associations between testosterone, bone mineral density, vitamin D and semen quality in fertile and infertile Chinese men. Int J Androl. 2012 Dec; 35 ( 6) : 783-92.
  • VDR-Fokl polymorphism but not the serum levels of Vitamin D, had a significantly different distribution between healthy men with proven fertility and those who complained of idiopathic infertility, the FF form being the most frequent in fertile subjects with respect to those with idiopathic infertility.
  • IAI idiopathic azoospermia
  • subjects with idiopathic azoospermia (IAI) almost exclusively had the mutated form in homozygosis (ff) , whereas the Ff form was significantly expressed in IOI and CrypM.
  • Vitamin D receptor rather than Vitamin D itself, can have a key role in the modulation of the testicular functions and in particular spermatogenesis, Vit D receptor action independent, (Pike JW, Meyer MB, Benkusky
  • VDR-Fokl polymorphism is therefore a predictive factor in the deterioration of spermatogenesis in men with idiopathic infertility .

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Abstract

L'invention concerne un procédé de détection de la prédisposition d'un individu mâle à l'infertilité fondé sur de l'identification des polymorphismes du gène du récepteur de la vitamine D, comprenant l'identification et la quantification des variants dans l'homozygosité, récessive et de type sauvage, et dans l'hétérzygosité, du polymorphisme du codon de départ de l'exon 2 dans La région N-terminale du gène du récepteur de la vitamine D (VDR fokI), les variants de l'hétérozygosité et de l'homozygosité récessive étant indicatifs de l'infertilité.
EP19758729.8A 2018-09-11 2019-08-26 Procédé d'identification de la prédisposition à l'infertilité masculine basé sur des polymorphismes du gène du récepteur de la vitamine d Pending EP3850112A1 (fr)

Applications Claiming Priority (2)

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IT201800008506 2018-09-11
PCT/EP2019/072692 WO2020052946A1 (fr) 2018-09-11 2019-08-26 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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Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FI990380A (fi) 1999-02-22 2000-08-23 Ancobio Ltd Diagnostinen ja seulontamenetelmä
KR100926255B1 (ko) 2008-07-04 2009-11-12 (주)지노첵 남성 불임의 진단 방법 및 ｙ 염색체 미세결실 검출을 위한남성 불임 진단용 키트
EP2401407A4 (fr) 2009-02-26 2012-10-03 Univ California Polymorphisme de délétion du defb-126 associé à l'infertilité
FR2952373B1 (fr) 2009-11-12 2013-05-17 Univ Grenoble 1 Controle de la fertilite humaine via dpy19l2
US20140100419A1 (en) 2011-02-28 2014-04-10 Rigshospitalet Copenhagen University Hospital Active vitamin d or analogs thereof for use in in vivo or in vitro fertilization
WO2013068486A1 (fr) 2011-11-08 2013-05-16 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthodes pour le diagnostic et le traitement de l'infertilité masculine
CN104928384A (zh) 2014-09-02 2015-09-23 深圳市第二人民医院 一种检测与男性不育症相关的遗传标记的引物对
WO2017000289A1 (fr) 2015-07-01 2017-01-05 深圳市第二人民医院 Marqueur génétique associé à l'azoospermie idiopathique
CN107603982B (zh) 2016-07-06 2021-01-05 中国科学院分子细胞科学卓越创新中心 诊断Piwil1基因突变导致的男性不育的方法及试剂盒
IT201600115036A1 (it) 2016-11-15 2018-05-15 Lta Biotech S R L Metodo per la diagnosi in vitro di infertilità maschile.

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