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WO2020234072A1 - Diagnostic du syndrome pcos - Google Patents

Diagnostic du syndrome pcos Download PDF

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Publication number
WO2020234072A1
WO2020234072A1 PCT/EP2020/063278 EP2020063278W WO2020234072A1 WO 2020234072 A1 WO2020234072 A1 WO 2020234072A1 EP 2020063278 W EP2020063278 W EP 2020063278W WO 2020234072 A1 WO2020234072 A1 WO 2020234072A1
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mir
pcos
treatment
adolescent
expression level
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Francis De Zegher
Lourdes IBAÑEZ
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Katholieke Universiteit Leuven
Hospital Sant Joan De Deu
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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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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 methods of diagnosing PolyCystic Ovary Syndrome (PCOS), methods of detecting responders and non-responders to a PCOS therapy and methods to monitor the efficacy of such therapy.
  • PCOS PolyCystic Ovary Syndrome
  • the invention further relates to miRNA based asays in performing the above methods.
  • PCOS Polycystic ovary syndrome
  • PCOS is thought to result from the interaction of many factors, the latest evidence pointing to a key role for hepato-visceral fat excess which is usually preceded by a mismatch between (reduced) prenatal and (augmented) postnatal weight gain [de Zegher et at. (2016) Trends Endocrinol Metab 29, 815-818].
  • lifestyle measures and medications that reduce hepato-visceral adiposity for example, a low-dose combination of spironolactone, pioglitazone and metformin (SPIOMET), normalizes the PCOS phenotype more than does a treatment with an oral contraceptive (OC) [Ibanez et a/. (2017) J Ado/esc Health 61, 446-453].
  • the sequence of events leading to PCOS may be subject to genetic and epigenetic modulations influencing both the phenotype and possibly the outcome during and after intervention. Indeed, different loci related to PCOS have been identified by genome-wide association studies (GWAS) but together only explain ⁇ 10% of heritability. For this reason, there is mounting interest on the effects of epigenetic regulation - and in particular on the effects of microRNAS (miRNAs) - in the phenotypic variation of PCOS. miRNAs are single-stranded, small non-coding RNAs aproximately 19-25 nucleotides in length that bind to the 3'UTR region of mRNAs inhibiting their translation, and thus working as negative posttranscriptional regulators of gene expression.
  • GWAS genome-wide association studies
  • miRNAs play pivotal roles in numerous biological processes, and have emerged as diagnostic biomarkers in different diseases.
  • miRNA expression has been reported to be altered in serum, adipose tissue, follicular fluid, and in granulosa and theca cells [Long et al. (2014) Cell Physiol Biochem 33, 1304-1315; Song et al. (2016) PLoS One 11, e0163756; Chen et al. (2013) Diabetes 62, 2278-2286; Naji et a/. (2017) Sci. Rep. 7, 14671; Xue et at. (2016) J Cell Biochem 119, 3913-3921].
  • miR- 106 ameliorates hyperglycemia and vascular endothelial cell dysfunction and promotes adipogenesis
  • miR-652 purportedly increases insulin-stimulated lipogenesis in adipocytes
  • miR-451a inhibits hepatic gluconeogenesis by down-regulating glycerol-kinase.
  • miR-451a A reduced presence of miR-451a has been documented in the fatty liver of ob/ob mice [a type 2 diabetes animal model with spontaneous non-alcoholic fatty liver disease (NAFDL) development], in mice with high fat diet (HFD)-induced non-alcoholic steatohepatitis (NASH), in rats receiving a HFD or high-fructose diet or a combination of both, and also in humans with NASH.
  • HFD high fat diet
  • NASH non-alcoholic steatohepatitis
  • overexpression of miR-451a is able to decrease triglyceride accumulation in the liver of mice, and also in cultured hepatic cells [Zeng et at. (2016) Mol Cell Endocrinol 474, 260-271].
  • the present invention discloses a miRNA profile of adolescent girls with PCOS, distinct from that of healthy control girls, and reflecting the [perpetuating vs normalizing] effects of interventions [respectively with an oral contraceptive (OC) vs with a low- dose combination of spironolactone-pioglitazone-metformin (SPIOMET) for 1 year] on the PCOS-underpinning pathophysiology.
  • OC oral contraceptive
  • SPIOMET spironolactone-pioglitazone-metformin
  • the miRNA profiling was performed by RNA sequencing, differentially expressed miRNAs being validated by qRT-PCR in 13 control and 31 PCOS girls.
  • miR-451a could diagnose PCOS (versus a healthy condition) with 100% sensitivity and 100% specificity.
  • SPIOMET but not OC
  • was accompanied by on-treatment normalization of the miRNA profile in PCOS girls; miR-451a concentrations after 1 year on OC or SPIOMET treatment associated closely (r 0.66; P ⁇ 0.0001) with post-treatment ovulation rates (assessed by salivary progesterone).
  • Circulating miR-451a is a biomarker suitable to guide diagnosis and treatment of PCOS in adolescent girls.
  • the invention relates to the use miRNA expression as a marker for diagnosing PCOS and monitoring the efficacy of a PCOS treatment.
  • the use implies the use of reagents such as probes and PCR primers for detecting the expression level of miRNA in a biological sample such as blood.
  • PCOS treatment is a treatment with with oestro-progestagen contraceptive such spironolactone, pioglitazone and metformin (SPIOMET).
  • the present invention allows thus to monitor the efficacy whereby a treatment normalizes the underpinning PCOS pathophysiology in an adolescent girl with PCOS.
  • the methods of the invention thus allow the miRNA based monitoring independently of other assessments such as determining the normalisation of the ovulatory functione.
  • the method can be equally performed when the first measurement is made when the treatment is already ongoing, for example after 1, 2 or 3 weeks after the start of the treatment, or for example after 1, 2, or 3 months after the start of the treatment.
  • a change in miRNA compared with a later measurement caan then be equally determined.
  • the treatment is a treatment with oestro-progestagen contraceptive such as spironolactone, pioglitazone and metformin (SPIOMET).
  • oestro-progestagen contraceptive such as spironolactone, pioglitazone and metformin (SPIOMET).
  • SPIOMET oestro-progestagen contraceptive
  • the step of determining the second level is performed between 1 and 12 months ((eg 1, 2, 4, 6 9 or 12 months), after the onset of said treatment or after the first measurement.
  • Panel B Correlations between the circulating concentrations of differentially expressed miRNAs and a selection of clinical, biochemical and imaging markers across the study population of 13 healthy controls and 31 girls with PCOS. Correlations with P ⁇ 0.001 are shown in bold.
  • OC oral contraceptive
  • SPIOMET low-dose spironolactone
  • Panel B miR-106-5p Z-score increased significantly in the SPIOMET subgroup after
  • Panel C miR-206 Z-score decreased significantly in the OC subgroup over 2 years. *p ⁇ 0.0001 by one-way ANOVA; #P ⁇ 0.0001 between subgroups after 1 and 2 years, by two-sided t-test.
  • FIG. 7 Receiver operating characteristics (ROC) curves (panels A to D) and summary (Panel E) of validated miRNAs for discriminating girls with PCOS from healthy controls.
  • PCOS Polycystic ovary syndrome
  • Alescence refers to the age range between 10 and 24 years more typically between 10 and 19 years.
  • Non-obese refers to a body mass index below 30, typically below 27,5 and more typically below 25.
  • Responder refers to a PCOS patient wherein a treatment is accompanied by a rise of the circulating concentrations of a particular miR, or of a particular combination of miRs, typically above the -2 SD limit of a reference range.
  • expression level may be determined by measuring the amount of microRNA in the sample fluid using a variety of suitable reagents.
  • the expression level of the microRNA can be determined, for example, with an assay for global gene expression in a biological fluid (e.g. using a microarray assay for microRNA expression profiling analysis, or a ready-to-use microRNA qPCR plate), or by specific detection assays, such as quantitative PCR, quantitative reverse-transcription (real-time) PCR (qRT- PCR), locked nucleic acid (LNA) real-time PCR, or northern blotting.
  • the measurement of the expression level of a microRNA in a biological fluid may be carried out with an oligonucleotide probe specific for the detection of said microRNA.
  • Said oligonucleotide probe may bind directly and specifically to the microRNA, or may specifically reverse transcribe said microRNA. Alternatively, said oligonucleotide probe may bind a cDNA obtained from said microRNA. Said oligonucleotide probe may also amplify a cDNA obtained from said microRNA.
  • Kit refers to any combination of reagents or apparatus that can be used to perform a method of the invention.
  • Kits for use in the present invention comprise probes for detection of a limited amount of dedicated miRNA (up to 10, 15 or 20 miRNA) to distinguish over arrays containing probes for more than 100 or 1000 miRNA.
  • the dedicated miRNA may comprise housekeeping miRNA or exogenous miRNA from other species that serve as control.
  • the kit may also comprise instructions for use to diagnose whether a subject classifies as a responder to the IL10, PINS, anti-CD3 diabetes therapy.
  • oligonucleotide probe refers to a short sequence of nucleotides that match a specific region of a microRNA or a cDNA obtained from said microRNA, or fragments thereof, and then used as a molecular probe to detect said microRNA or cDNA sequence.
  • oligonucleotide probe specifically for the detection of a microRNA
  • said oligonucleotide probe may bind specifically to a cDNA obtained from said microRNA.
  • Said oligonucleotide probe may also specifically amplify a cDNA obtained from said microRNA.
  • control miRNA refers to one or more miRNA which expression is determined using a similar, preferably identical, methodology, as the experimental miRNA. As illustrated in the examples, the expression of numerous miRNA does not differ between responders and non-responders. One or more of these miRNA can be used as internal control. Alternatively, an miRNA (e.g. a non-human) is added to a sample and acts as an external control.
  • a reference is obtained by using a sample or a pool of samples from a population of validated responders or by using a sample or a pool of samples from a population of validated non-responders.
  • Experimental data can be compared with control data and classified as belonging to the responder or non-responder group. Compared with a control group of non-responders a non-responder will have about equal expression levels, and a responder will have lower expression levels. Equally, compared with a control group of responders a responder will have about equal expression levels, and a non-responder will have higher expression levels.
  • MicroRNA Expression Detection Methods Wang Zhiguo, Yang Baofeng, 2010, XX; “Circulating MicroRNAs Methods and Protocols”, series: Methods in Molecular Biology, Vol. 1024 Ochiya, Takahiro (Ed.) 2013.
  • Determination of expression values obtained by qPCR can be expressed as ⁇ -dct values as explained for example in Livak 8i Schmittgen (2001) Methods 25, 402- 408. This value represents the fold change in expression of the target miRNA relative to a control miRNA.
  • the term "statistically significant" differences between the groups studied relates to condition when using the appropriate statistical analysis (e.g. Chi-square test, t- test) the probability of the groups being the same is less than 5%, e.g. p ⁇ 0,05. In other words, the probability of obtaining the same results on a completely random basis is less than 5 out of 100 attempts.
  • RNA expression levels can be used in accordance with the methods described herein.
  • Non limiting examples of such techniques include microarray analysis, Northern blotting, nuclease protection assays, RNA fingerprinting, polymerase chain reaction, ligase chain reaction, Qbeta replicase, isothermal amplification method, strand displacement amplification, transcription based amplification systems, quantitative nucleic acid amplification assays (e.g., polymerase chain reaction assays), combined reverse transcription/nucleic acid amplification, nuclease protection (SI nuclease or RNAse protection assays), Serial Analysis Gene Expression (SAGE), next generation sequencing, gene expression microarray, as well as other methods.
  • SAGE Serial Analysis Gene Expression
  • the probe can be labelled by any of the many different methods known to those skilled in this art.
  • the labels most commonly employed for these studies are radioactive elements, enzymes, chemicals that fluoresce when exposed to ultraviolet light, and others.
  • a number of fluorescent materials are known and can be utilized as labels. These include, but are not limited to, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow.
  • the radioactive label can be detected by any of the currently available counting procedures.
  • isotopes include 3 H, 14 C, 32 P, 35 S, 51 Cr, 57 Co, 58 Co, 59Fe, 90 Y, 125 I, 131 I, and 186 Re.
  • the present invention discloses a serum miRNA profile of girls with PCOS versus healthy controls, the longitudinal changes of the differentially expressed miRNAs in PCOS girls during randomized interventions, and their association with endocrine- metabolic and imaging markers.
  • the miRNA signature of non-obese adolescents with PCOS was reveals a down-regulation of miR-106b-5p, miR-206, miR-652-3p, and particularly of miR-451a, which distinguished most between healthy controls and girls with PCOS.
  • Randomized interventions over one year disclosed that treatment with SPIOMET normalized those strikingly low miRNA concentrations, but that treatment with an OC failed to do so. More normal on-treatment miR-451a levels were followed by more normal post-treatment ovulation rates. SPIOMET-related increments in miR- 106b-5p, miR-652-3p and miR-451a concentrations were partly maintained after treatment discontinuation.
  • the differentially expressed miRNAs in particular miR-451a and miR-652-3p, were closely and inversely related to androgen excess, and to the degree of mismatch between (reduced) prenatal and (augmented) postnatal weight gain. Given that these miRNAs did not differ between healthy women and PCOS women, the present findings corroborate the concept that adolescent PCOS and adult PCOS may have different features [Ibanez et a/.
  • the present invention shows that miR-451a is the most down-regulated miR in PCOS girls, and is the most differentiating between healthy controls and PCOS girls, and is a biomarker contributing to guide PCOS diagnosis.
  • circulating miR-451a associated closely with hepato-visceral fat, which is thought to be a key PCOS driver, and is shown in the present invention to be the most up-regulated miRNA after SPIOMET treatment, increasing almost 4-fold.
  • Relatively elevated levels of miR-451a were observed after 1 year on SPIOMET treatment (and even longer), and these levels associated with higher post-treatment ovulation rates.
  • the present invention discloses that adolescent girls with PCOS display a distinct miRNA profile as compared to healthy control girls; SPIOMET treatment over one year normalizes the expression of several down-regulated miRNAs related to glucose homeostasis, energy metabolism, and the control of the cell cycle; miR-451a was the most up-regulated miR after SPIOMET treatment and showed the highest sensitivity and specificity to differentiate girls with PCOS from healthy controls, indicating its use as a biomarker for PCOS diagnosis and treatment outcome.
  • the original study population consisted of 36 non-obese adolescent girls with PCOS who participated in a randomized, open-label, controlled trial (ISRCTN29234515), whose primary endpoint was ovulation rate after OC or SPIOMET intervention [Ibanez et al. (2017) J Adolesc Health 61, 446-453] at the Adolescent Endocrinology Unit of Sant Joan de Deu University Hospital, Barcelona, Spain. Longitudinal measurement of circulating miRNAs was a secondary endpoint, and was performed in spare serum samples available from a total of 31 girls (mean age, 15.7 yr; BMI, 23.1 Kg/m 2 ; flow chart, Figure 4).
  • the inclusion criteria were: hirsutism (score of 8 or more on modified Ferriman 8i Gallwey scale); oligomenorrhea (menstrual intervals spanning more than 45 days); menarche at least 2 years before study onset; no need for contraception (no sexual activity).
  • Exclusion criteria were congenital adrenal hyperplasia due to 21 -hydroxylase deficiency; glucose intolerance or diabetes mellitus; thyroid, liver, or kidney dysfunction; hyperprolactinemia; use of medications affecting gonadal or adrenal function, or carbohydrate or lipid metabolism.
  • the girls were randomized to receive for 1 year an OC containing 20 pg of ethinylestradiol plus 100 mg of levonorgestrel for 21/28 days (and placebo for 7/28 days) or SPIOMET, a low-dose combination of spironolactone 50 mg/d, pioglitazone 7.5 mg/d, and metformin 850 mg/d, and were followed in the subsequent year off treatment. All girls were asked to adhere to a Mediterranean diet, and to perform exercise regularly.
  • HOMA-insulin resistance was calculated as [fasting insulin in mU/L] x [fasting glucose in mg/dl_]/405. Ovulations were derived from salivary progesterone measurements, combined with the information contained in the menstrual diaries kept by the girls.
  • Salivas were obtained weekly during the second and fourth trimesters of the post-treatment year over 12 consecutive weeks in each trimester, as reported in Ibanez et al. (2017).
  • Progesterone was measured by ELISA (Novatec, Inmundiagnostica, Dietzenbach, Germany); intra- and inter-assay coefficients of variation were 5.5% and 6.0%.
  • Body composition was assessed by dual X-ray absorptiometry with a Lunar Prodigy and Lunar software (version 3.4/3.5, Lunar Corp, WI); abdominal fat (subcutaneous and visceral) and hepatic fat were assessed by magnetic resonance imaging (MRI) with a multiple- slice MRI 1.5 Tesla scan (Signa LX Echo Speed Plus Excite, General Electric, Milwaukee, WI).
  • Central fat was arbitrarily defined as the sum of visceral fat (in cm 2 ) and hepatic fat (in %).
  • Small cDNA libraries were generated from 200 ng of total RNA using a TruSeq Small RNA Library Prep Kit (Illumina, San Diego, USA), according to the manufacturer's instructions. Briefly, small RNA fractions consisting of 16-26 nucleotides were purified from total RNA and enriched using denaturing polyacrylamide gel electrophoresis (PAGE).
  • Adapters were ligated at 3' and 5' ends of the enriched fragments using T4 ligase and then amplified by RT-PCR.
  • cDNA libraries were purified with an automated agarose gel separation system (Labchip XT, PerkinElmer, Waltham, MA, USA) and sequenced on a Hiseq 2500 system (Illumina, San Diego, CA, USA), using default parameters (single read lx50bp).
  • the expression of miRNAs in each library was extracted from the RNA-seq data using featureCounts function available in Bioconductor R package Rsubread [Liao et at. (2013) Nucleic Acids Res 41, el08; Liao et a/.
  • RNA extraction and differential miRNA expression analysis were performed using the eBayes moderated t-statistic by limma package and individual miRNAs P values were derived [Ritchie et a/. (2015) Nucleic Acids Res 43, e47].
  • FC log2 fold-change
  • the results are expressed as the ratio of the average expression in girls with PCOS versus the average expression in control girls (Z-score).
  • the resulting P values were adjusted using the Benjamini-Hochberg procedure and a False Discovery Rate (FDR) cut-off of 0.05 was used as statistical significant threshold.
  • a miRNA was considered to be differentially expressed if its adjusted P value was ⁇ 0.01.
  • the most differentially expressed miRNAs were validated in the entire study population (13 controls, 31 girls with PCOS; Figure 4) by RT-qPCR (see below for details).
  • miRNAs were reverse-transcribed using the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA), and the associated miRNAs- specific stem-loop primers (TaqMan microRNA assay kit, #4427975), with minor modifications.
  • a customized RT primer pool was prepared by pooling all miRNAs- specific primers of interest.
  • miRNA-specific primers were pooled and diluted in lX Tris-EDTA (TE) buffer to obtain a final dilution of 0.05X each; 6 mI of this mixture was added to the reaction mix containing 0.3 mI 100 mM dNTP, 3 mI enzyme (50 U/mI), 1.5 mI 10X RT buffer, 0.19 mI RNase inhibitor (20u/mI) and 3 mI of serum RNA.
  • a final volume of 15 mI was reverse-transcribed under the following conditions: 30 min at 16°C to anneal primers, 30 min at 42°C for the extension phase, and 5 min at 85°C to stop the reaction. cDNA was then stored at -20°C.
  • Pre-amp PCR conditions consisted in 10 min at 95°C, 2 min at 55°C, and 2 min at 72°C, followed by 12 cycles of 15 s at 95°C, 4 min at 60°C and a final step of 10 min at 99.9°C.
  • the pre-amp products were diluted 4X in 0.1X TE buffer pH 8.0, and stored at -20°C.
  • a pre-amp primer pool was built targeting the same miRNAs that were reverse-transcribed, and containing 10 mI of each individual 20X TaqMan small RNA assay (part of #4427975), diluted in 1000 mI IX TE.
  • the reaction mix was prepared by combining 3.75 mI of pre-amp primer pool with 2.5 mI of RT product, 12.5 mI of TaqMan PreAmp Master Mix (2X) (#4391128) and 6.25 mI Nuclease-free water.
  • RT-qPCR were performed on an ABI PRISM 7500 thermal cycler from Applied Biosystems with the following conditions: one denaturing step at 95°C for 10 min, followed by 45 cycles consisting of denaturing at 95°C for 15 s, and annealing and elongation at 60°C for 60 s, followed by an inactivation step of 10 min at 99.9°C.
  • Real-time PCR reactions with multiple cDNAs were performed in a 20 pi final volume.
  • OC and SPIOMET treatment had no detectable effects on BMI, bone mineral density, lean mass or total fat, and both treatments attenuated the markers of androgen excess comparably. However, they had divergent effects on cardio-metabolic risk markers, as judged by the reduction in HOMA-IR, C-reactive protein, cIMT, hepato- visceral fat, and by the increase in HDL-cholesterol, and high-molecular-weight- adiponectin levels with SPIOMET but not with OC intervention, as reported in Ibanez et al. (2017) J Adolesc Health 61, 446-453.
  • LC-MS body mass index
  • SHBG sex hormone-binding globulin
  • FAI free androgen index
  • HOMA-IR homeostasis model assessment insulin resistance
  • OGTT oral glucose tolerance test
  • cIMT carotid intima-media thickness
  • DXA dual X-ray absorptiometry
  • MRI magnetic resonance imaging.
  • Glycemia and insulinemia Z- scores were derived as described (8). Values are mean + SEM.
  • miRNA deep sequencing detected a total of 320 miRNAs in serum samples from healthy controls and from untreated PCOS girls (Figure 5). Among those, 196 miRNAs were shared by the two subgroups. Sixteen miRNAs were differentially expressed in PCOS girls (FDR ⁇ 5%), as compared to control girls (miR-451a, miR- 652-3p, miR-106b-5p, miR-206, miR-22-3p, miR-92a-3p, miR-4732, miR-25-3p, miR-16-5p, miR-16-2-3p, miR-194-5p, miR-130b-5p, miR-15a-5p, miR-10b-5p, miR-584-5p, and miR-151a-3p) ( Figure 1).
  • EXAMPLE 4 Correlation between circulating miRNAs expression and clinical endocrine-metabolic and imaging variables in girls with PCOS and in control girls
  • a ROC curve was performed for each of the four validated miRNAs and calculated the AUC in each case (Figure 7).
  • sensitivity + specificity -1 a cut-off value was identified of 0.06, 0.31, 0.08 and 0.78 respectively for miR-451a, miR-652-3p, miR-106b-5p and miR-206.
  • the capacity of circulating miR-451a, miR-106b-5p and miR-652-3p to differentiate was remarkable, with a sensitivity and specificity of 100% and 100%, 100% and 85%, and 81% and 100%, respectively (Figure 7).
  • the miRSystem web tool was used to identify the gene targets and the signaling pathways regulated by miR-451a, miR-652-3p, miR-106b-5p and miR-206.
  • KEGG and Reactome pathway enrichment analysis showed that genes targeted by those miRNAs, were all part of a complex regulatory network involved in glucose and lipid metabolism as well as in inflammation (Table 2).

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Abstract

L'invention concerne l'utilisation in vitro de réactifs pour mesurer le niveau d'expression d'au moins miR-451a dans un échantillon corporel, dans le diagnostic du syndrome des ovaires polykystiques (PCOS) chez une adolescente âgée entre 10 et 24 ans.
PCT/EP2020/063278 2019-05-17 2020-05-13 Diagnostic du syndrome pcos WO2020234072A1 (fr)

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Non-Patent Citations (32)

* Cited by examiner, † Cited by third party
Title
"Methods in Molecular Biology", vol. 1024, 2013, article "Circulating MicroRNAs Methods and Protocols"
BO XU ET AL: "Characterization of microRNA profile in human cumulus granulosa cells: Identification of microRNAs that regulate Notch signaling and are associated with PCOS", MOLECULAR AND CELLULAR ENDOCRINOLOGY., vol. 404, 1 March 2015 (2015-03-01), IE, pages 26 - 36, XP055626901, ISSN: 0303-7207, DOI: 10.1016/j.mce.2015.01.030 *
CHEN ET AL., DIABETES, vol. 62, 2013, pages 2278 - 2286
CHUN ZHAO ET AL: "Role of Serum miRNAs in the Prediction of Ovarian Hyperstimulation Syndrome in Polycystic Ovarian Syndrome Patients", CELLULAR PHYSIOLOGY AND BIOCHEMISTRY., vol. 35, no. 3, 1 January 2015 (2015-01-01), CH, pages 1086 - 1094, XP055626845, ISSN: 1015-8987, DOI: 10.1159/000373934 *
DE ZEGHER ET AL., OBESITY (SILVER SPRING, vol. 25, 2017, pages 1486 - 1489
DE ZEGHER ET AL., TRENDS ENDOCRINOL METAB, vol. 29, 2018, pages 815 - 818
DING ET AL., BIOSCIBIOTECHNOLBIOCHEM, vol. 80, 2016, pages 461 - 465
EISENBERG ET AL., FERTIL STERIL, vol. 107, 2017, pages 269 - 275
IBANEZ ET AL., HORM RES PAEDIATR, vol. 88, 2017, pages 371 - 395
IBANEZ ET AL., HORM RES PEDIATR, vol. 88, 2017, pages 371 - 395
IBANEZ ET AL., J ADOLESC HEALTH, vol. 61, 2017, pages 446 - 453
IBANEZ ET AL., NAT REV ENDOCRINOL, vol. 10, 2014, pages 499 - 508
JIANG ET AL., ENDOCRINE, vol. 53, 2016, pages 280 - 290
KONG ET AL., MOL CELL ENDOCRINOL, vol. 486, 2019, pages 18 - 24
LAUREN W. ROTH ET AL: "Altered microRNA and gene expression in the follicular fluid of women with polycystic ovary syndrome", JOURNAL OF ASSISTED REPRODUCTION AND GENETICS, vol. 31, no. 3, 4 January 2014 (2014-01-04), pages 355 - 362, XP055210738, ISSN: 1058-0468, DOI: 10.1007/s10815-013-0161-4 *
LI ET AL., MOL HUM REPROD, vol. 25, 2019, pages 638 - 646
LIAO ET AL., BIOINFORMATICS, vol. 30, 2014, pages 923 - 930
LIAO ET AL., NUCLEIC ACIDS RES, vol. 41, 2013, pages el08
LIVAKSCHMITTGEN, METHODS, vol. 25, 2001, pages 402 - 408
LONG ET AL., CELL PHYSIOL BIOCHEM, vol. 33, 2014, pages 1304 - 1315
MATHA DIAZ: "Circulating miR-451a: a Biomarker to Guide Diagnosis and Treatment of Polycystic Ovary Syndrome in Adolescent Girls", 19 September 2019 (2019-09-19), pages 1, XP055626864, Retrieved from the Internet <URL:https://www.postersessiononline.eu/173580348_eu/congresos/58ESPE/aula/-T_1_58ESPE.pdf> [retrieved on 20190927], DOI: 10.3252/pso.eu.58ESPE.2019 *
MCCARTHY ET AL., NUCLEIC ACIDS RES, vol. 40, 2012, pages 4288 - 4297
MORA MURRI ET AL: "Effects of Polycystic Ovary Syndrome (PCOS), Sex Hormones, and Obesity on Circulating miRNA-21, miRNA-27b, miRNA-103, and miRNA-155 Expression", JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM, vol. 98, no. 11, 1 November 2013 (2013-11-01), pages E1835 - E1844, XP055156024, ISSN: 0021-972X, DOI: 10.1210/jc.2013-2218 *
MURRI MORA ET AL: "Non-targeted profiling of circulating microRNAs in women with polycystic ovary syndrome (PCOS): effects of obesity and sex hormones", METABOLISM, CLINICAL AND EXPERIMENTAL, W.B. SAUNDERS CO., PHILADELPHIA, PA, US, vol. 86, 2 February 2018 (2018-02-02), pages 49 - 60, XP085438148, ISSN: 0026-0495, DOI: 10.1016/J.METABOL.2018.01.011 *
NAJI ET AL., SCI. REP., vol. 7, 2017, pages 14671
RITCHIE ET AL., NUCLEIC ACIDS RES, vol. 43, 2015, pages e47
ROBINSON ET AL., BIOINFORMATICS, vol. 26, 2010, pages 139 - 140
SONG ET AL., PLOS ONE, vol. 11, 2016, pages e0163756
SORENSEN ET AL., CHEM BIOL INTERACT, vol. 259, 2016, pages 8 - 16
WANG ZHIGUOYANG BAOFENG, MICRORNA EXPRESSION DETECTION METHODS, 2010, pages XX
XUE ET AL., J CELL BIOCHEM, vol. 119, 2018, pages 3913 - 3921
ZENG ET AL., MOL CELL ENDOCRINOL, vol. 474, 2018, pages 260 - 271

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