Method for stratifying patients with symptoms ascribable to vulvodynia

Abstract

A method for stratifying an individual with vulvodynia or genetically predisposed to one or more phenotypic traits associated with vulvodynia.

Description

[0001] METHOD FOR STRATIFYING PATIENTS WITH SYMPTOMS ASCRIBABLE TO VULVODYNIA

[0002] The present invention relates to a method for stratifying an individual with vulvodynia or genetically predisposed to one or more phenotypic traits associated with vulvodynia.

[0003] Vulvodynia is a multifactorial and complex syndrome that manifests itself by persistent genital and sexual pain and is characterized by burning, pain, irritation, swelling, and dryness (Bornstein J et al. (2015) “ISSVD, ISSWSH, and IPPS Consensus Terminology and Classification of Persistent Vulvar Pain and Vulvodynia.” J Sex Med. 2016;13(4):607-12). The actual incidence of this condition is not fully known and it is estimated that it may affect between 7 and 16% of women of reproductive age in the general population (Harlow WT et al., (2014) “Prevalence of symptoms consistent with a diagnosis of vulvodynia: population-based estimates from 2 geographic regions”. Am J Obstet Gynecol. 2014; 210(l):40.el-8; Puckall CF et al., (2016), “Vulvodynia: Definition, prevalence, impact, and pathophysiological factors.” J Sex Med. 2016;13(3):291-304; Graziottin A et al. (2020) “Vulvar pain: The revealing scenario of leading comorbidities in 1183 cases.” Eur J Obstet Gynecol Reprod Biol. 2020;252:50-55). The etiopathogenesis of vulvodynia is not yet clearly characterized, although several studies have correlated it with chronic inflammation, increased nerve fiber proliferation, and increased frequency of vaginal and bladder bacterial or fungal infections (Pukall CF et al. (2016) “Vulvodynia: Definition, prevalence, impact, and pathophysiological factors.” J Sex Med 2016;13(3):291-304). To date, only a few studies have been conducted on the genetic basis of vulvodynia (Witkin et al. (2020) “Genetic Factors in Vulvodynia” Female Sexual Pain Disorders: Evaluation and Management pages 69-74). Some single nucleotide polymorphisms (SNPs) have been identified as risk factors for this condition because they show a higher incidence in women with vulvodynia. These polymorphisms have been observed in some genes involved in inflammatory and immune processes, increasing susceptibility to prolonged inflammation or bladder and vaginal infections (Witkin et al. (2020) "Genetic Factors in Vulvodynia” Female Sexual Pain Disorders: Evaluation and Management, pages 69-74). These genetic variants have been classified as risk factors for the development of vulvodynia, thus suggesting a polygenic nature of this complex condition, in which a woman's risk is largely defined by a large number of SNPs, whose combined effect can be a determining factor not only for diagnosis but also for guiding personalized therapeutic treatment.

[0004] Given the heterogeneity of its etiopathogenesis and symptoms, vulvodynia often remains undiagnosed and untreated for years. The reason for this lies in the intrinsic complexity of the condition. As it is a condition with a strong multifactorial component, to date there are few studies that have directly associated vulvodynia with a genetic basis. This is because none of the studies conducted to date have approached vulvodynia as a multifactorial and polygenic disease. Therefore, there is a lack of genomewide association studies (GWAS) on large cohorts of subjects with vulvodynia symptoms and controls. For these reasons, diagnosis and, above all, choice of treatment still follow subjective and empirical approaches, often proceeding by trial and error based on the patient's response.

[0005] In view of the limitations described above, the aim of the present invention is to provide a method for stratifying patients with vulvodynia or patients at risk of developing symptoms related to phenotypic traits associated with vulvodynia.

[0006] Another object of the invention is to provide a method for determining an individual's genetic predisposition to vulvodynia.

[0007] Finally, an object of the present invention is to provide a method that makes it possible to better understand the etiopathogenesis of the disease and inform a personalized therapeutic approach.

[0008] This aim, as well as the objects mentioned and others that will become more apparent hereinafter, are achieved by a method for stratifying an individual with vulvodynia or genetically predisposed to one or more phenotypic traits associated with vulvodynia, the method comprising: a) obtaining the genotype of an individual; b) accessing a database comprising one or more of the following genotype / phenotype correlation data groups:

[0009] - group 1 : polymorphisms correlated with levels of component 1 of the membrane progesterone receptor;

[0010] - group 2: polymorphisms correlated with levels of estrogen receptors;

[0011] - group 3: polymorphisms correlated with levels of total testosterone;

[0012] - group 4: polymorphisms correlated with prolactin levels;

[0013] - group 5: polymorphisms correlated with levels of component 2 of the membrane progesterone receptor; and

[0014] - group 6: polymorphisms correlated with mineralcorticoid receptor levels; c) calculating, for at least one of groups 1 to 6, a polygenic risk score (PRS) for the respective phenotypic trait in the individual by comparing the genotype of step a) with the database of step b); d) comparing the PRS obtained in step c) with the distribution of PRSs in the general population for the same phenotypic trait and determining whether said PRS is lower than the 25th quantile, comprised between the 25th and 75th quantiles, or higher than the 75th quantile compared with the distribution of PRSs in the general population; e) establishing the individual's genetic predisposition to the phenotypic trait associated with vulvodynia, where:

[0015] - a PRS of the individual lower than the 25th quantile indicates the individual’s genetic predisposition for low levels of the correlated phenotypic trait;

[0016] - a PRS of the individual comprised between the 25th and 75th quantiles indicates the individual's genetic predisposition for normal levels of the correlated phenotypic trait;

[0017] - a PRS of the individual greater than the 75th quantile indicates the individual's genetic predisposition for high levels of the correlated phenotypic trait; f) stratifying the individual with vulvodynia or genetically predisposed to one or more phenotypic traits associated with vulvodynia based on the genetic predisposition to high or low levels of one or more of the phenotypic traits associated with vulvodynia, established in step e).

[0018] The first step of the method according to the invention provides for obtaining the genotype of the individual. The starting sample may be a buccal swab or peripheral blood. Various techniques known to the person skilled in the art may be used to obtain the genotype, including the application of SNP arrays and whole genome sequencing with either standard coverage (for example, 3 OX) or low coverage (for example, 2X).

[0019] In a preferred embodiment, the genotype of the individual is obtained by means of whole genome sequencing (WGS).

[0020] In step c), the method according to the invention provides for calculating the polygenic risk score (PRS), i.e., calculating an individual score resulting from the weighted sum of the estimated effects of millions of SNPs on the phenotype of interest.

[0021] In recent decades, genome-wide association studies (GWAS) have identified numerous significant associations between genetic variants and many complex human diseases. Recent computational advances have supported the application of thousands of genetic variants in the calculation of polygenic risk scores, which are weighted sums of the estimated effects of genetic variants on the phenotype (without however considering the impact that the environment or lifestyle may have on the investigated trait). In particular, PRS is a genetic measure of an individual's predictive risk of developing a specific phenotype, such as common complex diseases. The application of PRS could significantly improve disease prevention by anticipating diagnosis and / or better directing therapeutic treatment. PRS can be applied to both qualitative and quantitative phenotypic traits, i.e., to investigate genetic predisposition to a specific disease or to altered levels of continuous traits (such as hormone levels).

[0022] In one embodiment of the method according to the invention, the individual is European and the distribution of PRS in the general population is a normal distribution calculated on the basis of the data of the 1000 Genome Project (1000 Genomes Project Consortium, Auton A et al., (2015) “A global reference for human genetic variation”. Nature. 2015;526(7571):68-74).

[0023] In one embodiment, the method according to the invention makes it possible to identify a genetic predisposition to vulvodynia. In particular, in one embodiment where the individual is European, in step c) the GWAS ID: prot-a-2259 database is used, obtaining the PRS correlated to the level of component 1 of the progesterone membrane receptor. A PRS correlated to the level of component 1 of the progesterone membrane receptor greater than the 75th quantile, calculated on the basis of the data of the 1000 Genome Project, is indicative of an increased risk of developing symptoms ascribable to vulvodynia. In particular, genetic predisposition to increased levels of component 1 of the progesterone membrane receptor is indicative of a reduced progesterone level, which causes increased sensitivity to pain and reduced trophism of the vestibule.

[0024] In another embodiment, where the individual is European, in step c) the GWAS ID: prot-a-2260 database is used, obtaining the PRS correlated to the level of component 2 of the progesterone membrane receptor. A PRS greater than the 75th quantile, calculated on the basis of the data of the 1000 Genome Project, is indicative of increased susceptibility of the vestibule to prolonged pain, preferably measured by means of electrostimulation of C- fibers with a Neurometer® CPT® device according to the manufacturer's instructions (Neurotron Incorporated, USA) (Murina, F. et al., (2010) “Electrodiagnostic Functional Sensory Evaluation of Patients With Generalized Vulvodynia: A Pilot Study” JLGTD 14 (3) 221-224).

[0025] In another embodiment, where the individual is European, in step c) the GWAS ID: prot-a-991 database is used, obtaining the PRS correlated to the level of estrogen receptors. A PRS lower than the 25th quantile, calculated on the basis of the data of the 1000 Genome Project, is indicative of increased sensitivity to pain due to reduced trophism of the vestibular mucosa.

[0026] In another embodiment, where the individual is European, in step c) the GWAS ID: ieu-b-4864 database is used, obtaining the PRS correlated to the total testosterone level. A PRS lower than the 25th quantile, calculated on the basis of the data of the 1000 Genome Project, is indicative of increased sensitivity to pain due to reduced trophism of the vestibular mucosa.

[0027] In another embodiment, where the individual is European, in step c) the GWAS ID: prot-a-2375 database is used, obtaining the PRS correlated to the prolactin level. A PRS greater than the 75th quantile, calculated on the basis of the data from the 1000 Genome Project, is indicative of increased sensitivity of the vestibule to acute and prolonged pain, preferably measured by means of electrostimulation of A3 fibers and C-fibers with a Neurometer® CPT® instrument according to the manufacturer's instructions (Neurotron Incorporated, USA).

[0028] In another embodiment, where the individual is European, in step c) the GWAS ID: prot-a-2089 database is used, obtaining the PRS correlated to the level of mineralocorticoid receptors. A PRS greater than the 75th quantile calculated on the basis of the data of the 1000 Genome Project is indicative of increased sensitivity of the vestibule to prolonged pain, preferably measured by means of electrostimulation of C-fibers with a Neurometer® CPT® instrument according to the manufacturer's instructions (Neurotron Incorporated, USA).

[0029] In a particularly preferred embodiment of the invention, the PRS is obtained for each of the mentioned phenotypic traits associated with vulvodynia, and in particular:

[0030] - the levels of component 1 of the progesterone membrane receptor,

[0031] - the levels of component 2 of the progesterone membrane receptor,

[0032] - the levels of estrogen receptors,

[0033] - the total testosterone levels,

[0034] - the prolactin levels, and

[0035] - the mineralocorticoid receptor levels.

[0036] The invention therefore makes it possible to identify PRS values as markers that describe genetic predisposition to altered levels of hormones and their receptors and allow women to be stratified into three macrogroups:

[0037] 1. women at greater risk of developing vulvodynia, allowing to accelerate diagnosis in women with a genetic predisposition to altered levels of hormones and their receptors;

[0038] 2. women with vulvodynia whose symptoms are more closely linked to reduced trophism of the vestibular mucosa and who may have a genetic predisposition to altered levels of total testosterone and / or altered levels of estrogen receptors; based on this distinction, it is possible to guide the therapeutic choice towards the use or non-use of active ingredients based on total testosterone or estrogen.

[0039] 3. women with vulvodynia whose symptoms are mainly linked to hormonal alterations that cause direct changes in nociception and who have a genetic predisposition to altered levels of prolactin, mineralocorticoid receptors, and component 2 of the progesterone membrane receptor; based on this distinction, it is possible to guide the therapeutic choice towards the use or non-use of active ingredients with antiprolactinemic activity or active ingredients based on progesterone, corticosteroids, or active ingredients that are antagonists of mineralocorticoid receptors.

[0040] The invention will now be further described with reference to the following non-limiting example.

[0041] EXAMPLE:

[0042] The genetic predisposition to altered hormone levels was investigated by analyzing 30 European subjects presenting symptoms of vulvodynia such as persistent vulvar pain, burning, localized irritation in the vestibule when stimulated, with a duration of these symptoms of at least 3 months and not attributable to other causes, and 30 controls, i.e., asymptomatic women with no vulvovaginal conditions, in order to identify statistically significant differences between subjects with vulvodynia and controls, as well as genetic markers that can stratify subjects with vulvodynia and provide guidance on the most appropriate and personalized therapeutic approach.

[0043] Genetic predisposition was investigated by calculating the PRS.

[0044] In detail, the polygenic risk calculation was performed by means of the following steps:

[0045] 1) collection of a biological sample from each study participant by peripheral blood sampling.

[0046] 2) isolation of genomic DNA from the biological sample using an automatic method based on the use of magnetic beads. Specifically, the MagCore Super automatic extractor (RBC-Bioscience, New Taipei City, Taiwan) was used following the protocol of the MagCore® Genomic DNA Tissue Kit (RBC-Bioscience, New Taipei City, Taiwan).

[0047] 3) The genomic DNA was then tagmented on microspheres using the Illumina DNA PCR-Free Prep protocol before proceeding with whole genome sequencing (WGS) using Illumina technology.

[0048] 4) The libraries were then quantified and validated by qPCR following the KAPA Library Quantification Kit protocol (Roche).

[0049] 5) Genome sequencing was performed with 2x150 bp sequences and low coverage (on average 2X) using Illumina technology, obtaining a number of sequences for each sample of at least 20 million.

[0050] 6) The quality of the raw data was checked using the FastQC vO.l 1.9 program (Andrews, S. (2010). FastQC: a quality control tool for high throughput sequence data. Available online at: http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc). The raw data were filtered based on quality using the Trimmomatic 0.39 program (Bolger et al., 2014 Trimmomatic: A flexible trimmer for Illumina Sequence Data. Bioinformatics, Aug l;30(15):2114-20. doi:

[0051] 10.1093 / bioinformatics / btul 70).

[0052] 7) The sequences were aligned to the human reference genome (version GRCh37 / hgl9) using the bwa-mem (Burrows-Wheeler Aligner) v0.7.17-rl l88 program (Li H, et al. (2009) “Fast and accurate short read alignment with Burrows-Wheeler transform.” Bioinformatics. 25(14): 1754- 60). The samtools vl .9 program (Li H et al. (2009) “The Sequence Alignment / Map format and SAMtools.” Bioinformatics; 25(16):2078-9) was then used to calculate the average coverage of the samples and manipulate the alignment files.

[0053] 8) The genotype of the participants was then imputed using the GLIMPSE2 software (Rubinacci et al. (2021), “Efficient phasing and imputation of low-coverage sequencing data using large reference panels”. Nature Genetics 53.1 (2021): 120-126; Rubinacci et al. (2023), “Imputation of low-coverage sequencing data from 150,119 UK Biobank genomes”. Nat Genet 55, 1088-1090 (2023). https: / / doi.org / 10.1038 / s41588-023-01438-3) and using the 1000 Genome Project dataset as a reference.

[0054] 9) Data of the 1000 Genome Project were used to confirm the European origin of the study participants through principal component analysis.

[0055] 10) The imputed genetic variants of each participant were used to calculate the individual polygenic risk for the following phenotypic traits: levels of component 1 and component 2 of the progesterone membrane receptor (GWAS ID: prot-a-2259 and prot-a-2260, respectively), levels of estrogen receptors (GWAS ID: prot-a-991), total testosterone levels (GWAS ID: ieu-b-4864), prolactin levels (GWAS ID: prot-a-2375) and mineralocorticoid receptor levels (GWAS ID: prot-a-2089). The reference results (i.e., summary statistics) of the various GWAS are available in the IEU Open GWAS database (https: / / gwas.mrcieu.ac.uk / ; Hemani et al. (2018), “The MR-Base Collaboration. The MR-Base platform supports systematic causal inference across the human phenome.” eLife 2018;7:e34408. doi: 10.7554 / eLife.34408; Elsworth et al. (2020), “The MRC IEU OpenGWAS data infrastructure.” bioRxiv 2020.08.10.244293vl. doi: 10.1101 / 2020.08.10.2442932020; Lyon et al., (2020) “The variant call format provides efficient and robust storage of GWAS summary statistics”. bioRxiv 2020.05.29.115824; doi: https: / / doi.org / 10.1101 / 2020.05.29.115824).

[0056] The PRS was calculated using the Plink 1.9 program (www.cog- genomics.org / plink / L9 / ; Chang et al. (2015), “second-generation PLINK: rising to the challenge of larger and richer datasets” Gigascience 2015 Feb 25:4:7. doi: 10.1186 / sl3742-015-0047-8.) individually on each sample combined with the European dataset from the 1000 Genome Project.

[0057] 11) To assess whether the sample under analysis has a genetic predisposition to low, normal or high levels of the phenotypic trait investigated, the PRS was compared with the distribution of PRS in the European population of the 1000 Genome Project, dividing this distribution into quantiles.

[0058] • If the PRS of the sample is less than the 25th quantile, its genetic predisposition is to low levels of the trait being considered;

[0059] • If the PRS of the sample is comprised between the 25th and 75th quantiles, the predisposition is at normal levels of the trait being considered;

[0060] Finally, if the PRS of the sample is greater than the 75th quantile, then the genetic predisposition is for high levels of the trait investigated.

[0061] This biostatistical analysis is performed using the “stats” v4.3.0 software from R (R Core Team (2023). R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria).

[0062] Once the PRS for each phenotypic trait was calculated, linear regression models were applied to:

[0063] - assess whether the PRSs vary significantly between subjects with vulvodynia and controls;

[0064] - investigate linear relationships between PRS and clinical endophenotypes within the group of subjects with vulvodynia and the control group, separately. The clinical endophenotypes considered are thickness of the vestibular mucosa and Neurometer® CPT® data (2000 Hz, 250 Hz, and 5 Hz), which are mainly influenced by hormone activity.

[0065] In both groups, individual body mass index (BMI), contraceptive use (C), recurrent vaginitis (RV), and first and second principal components (PCA1 and PCA2) were added as covariates to improve the predictive ability of the model. In particular, covariates C and RV were used as binary variables (0 = No or 1 = Yes).

[0066] Disease duration (DD) was further included in these covariates exclusively for the model applied to subjects with vulvodynia, in order to investigate linear relationships of PRS and clinical endophenotypes.

[0067] To monitor the inflation of parameter estimate variances, the variance inflation factor (VIF) was measured.

[0068] All statistical analyses were performed using the R “stats” and “olsrr” software (R Core Team (2023). R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria).

[0069] The results of the comparison of PRSs between subjects with vulvodynia and controls are shown in Table 1:

[0070] Table 1: Results of the linear regression model to determine whether PRSs vary significantly between subjects with vulvodynia and controls; * indicates significant variability with a P-value < 0.05.

[0071] The results of the comparison of PRSs between subjects with vulvodynia and controls show that:

[0072] - PRS values indicating a genetic predisposition to low levels of component 1 of the progesterone membrane receptor are more commonly observed in the control group (protective effect of this genetic profile).

[0073] Subjects with vulvodynia, on the other hand, tend to have PRS values indicating a predisposition to higher levels of this receptor with respect to controls. A genetic predisposition to higher levels of this receptor is indicative of a vulnerability to reduced progesterone levels, which can cause increased pain sensitivity and altered vestibular trophism.

[0074] PRSs indicating a genetic predisposition to low levels of component 1 of the progesterone membrane receptor correspond to values below the 25th quantile of the PRS distribution in the general European population (distribution obtained using data from the 1000 Genome Project):

[0075] 25th quantile threshold: -2.6E-05

[0076] PRSs indicating a genetic predisposition to high levels of component 1 of the progesterone membrane receptor correspond to values above the 75th quantile of the PRS distribution in the general European population (distribution obtained using data of the 1000 Genome Project):

[0077] 75th quantile threshold: 1.89E-05

[0078] All PRS values comprised between the 25th and 75th quantiles indicate a genetic predisposition to normal levels of component 1 of the progesterone membrane receptor.

[0079] Linear regression analysis of the PRSs with clinical endophenotypes performed within the control group alone, as described above, did not reveal any significant correlation.

[0080] The results of linear regression analysis of PRSs with clinical endophenotypes performed within the group of vulvodynia subjects alone are shown in Table 2:

[0081] Table 2: List of continuous phenotypic traits for which a significant correlation was observed between the PRS of subjects with vulvodynia and the clinical variables investigated (thickness of vestibular mucosa and Neurometer® CPT® data). * indicates significant variability with a P- value < 0.05.

[0082] The results show: a) a positive correlation between PRS values of subjects with vulvodynia at certain estrogen receptor levels and thickness of the vestibular mucosa. A PRS indicating a genetic predisposition of subjects with vulvodynia to low estrogen receptor levels corresponds to lower values of the thickness of the vestibular mucosa; conversely, a PRS indicating a predisposition to high estrogen receptor levels corresponds to higher vestibular mucosa thickness values. PRSs indicating a genetic predisposition to low estrogen receptor levels correspond to values below the 25th quantile of the PRS distribution in the general European population (distribution obtained using data of the 1000 Genome Project):

[0083] 25th quantile threshold: -2.2E-05

[0084] PRSs indicating a genetic predisposition to high levels of estrogen receptors correspond to values above the 75th quantile of the PRS distribution in the general European population (distribution obtained using data of the 1000 Genome Project):

[0085] 75th quantile threshold: 1.9E-05

[0086] All PRS values comprised between the 25th and 75th quantile indicate a genetic predisposition to normal levels of estrogen receptors. b) A positive correlation between PRS values indicating a genetic predisposition of subjects with vulvodynia to certain levels of total testosterone and vestibular mucosa thickness.

[0087] A PRS indicating a genetic predisposition of subjects with vulvodynia to low total testosterone levels corresponds to lower values of vestibular mucosa thickness; conversely, a PRS indicating a predisposition to high total testosterone levels corresponds to higher values of vestibular mucosa thickness.

[0088] PRSs indicating a genetic predisposition to low total testosterone levels correspond to values below the 25th quantile of the PRS distribution in the general European population (distribution obtained using data of the 1000 Genome Project):

[0089] 25th quantile threshold: -6.2E-06

[0090] PRSs indicating a genetic predisposition to high levels of total testosterone correspond to values above the 75th quantile of the PRS distribution in the general European population (distribution obtained using data of the 1000 Genome Project):

[0091] 75th quantile threshold: 1.31E-06 All PRS values comprised between the 25th and 75th quantiles indicate a genetic predisposition to normal levels of total testosterone. c) A negative correlation between PRS values indicating genetic predisposition of subjects with vulvodynia to certain prolactin levels and Neurometer® CPT® values at baseline at 250Hz (electrostimulation of A3 fibers and assessment of acute pain sensitivity) and Neurometer® CPT® values at baseline at 5Hz (electrostimulation of A3 fibers and assessment of acute pain sensitivity) and Neurometer® CPT® values at baseline at 5Hz (electrostimulation of C-fibers and assessment of prolonged pain sensitivity).

[0092] A PRS indicating a genetic predisposition of subjects with vulvodynia to high prolactin levels corresponds to lower Neurometer® CPT® values at baseline at 250Hz and 5Hz, indicating greater susceptibility to acute pain and prolonged pain in the vestibule.

[0093] Conversely, a PRS indicating a genetic predisposition of subjects with vulvodynia to low prolactin levels corresponds to higher values of Neurometer® CPT® at baseline at 250Hz and 5Hz, indicating lower susceptibility to acute and prolonged pain in the vestibule.

[0094] PRSs indicating a genetic predisposition to low prolactin levels correspond to values below the 25th quantile of the PRS distribution in the general European population (distribution obtained using data of the 1000 Genome Project):

[0095] 25th quantile threshold: 1.92-05

[0096] PRSs indicating a genetic predisposition to high prolactin levels correspond to values above the 75th quantile of the PRS distribution in the general European population (distribution obtained using data of the 1000 Genome Project):

[0097] 75th quantile threshold: 6.1E-05

[0098] All PRS values comprised between the 25th and 75th quantile indicate a genetic predisposition to normal prolactin levels. d) A negative correlation between PRS values indicating a genetic predisposition to certain levels of component 2 of the progesterone membrane receptor and Neurometer® CPT® values at baseline at 5Hz (electrostimulation of C-fibers and assessment of sensitivity to prolonged pain).

[0099] A PRS value corresponding to a genetic predisposition to high levels of said receptor is correlated with low Neurometer® CPT® values at baseline at 5Hz, indicating increased susceptibility of the vestibule to prolonged pain. Conversely, a PRS indicating a genetic predisposition to low levels of component 2 of the progesterone membrane receptor is correlated with higher levels of Neurometer® CPT® at baseline at 5Hz, i.e., with a lower susceptibility of the vestibule to prolonged pain.

[0100] PRSs indicating a genetic predisposition to low levels of component 2 of the progesterone membrane receptor correspond to values below the 25th quantile of the PRS distribution in the general European population (distribution obtained using data of the 1000 Genome Project):

[0101] 25th quantile threshold: -3.1E-05

[0102] PRSs indicating a genetic predisposition to high levels of component 2 of the progesterone membrane receptor correspond to values above the 75th quantile of the PRS distribution in the general European population (distribution obtained using data of the 1000 Genome Project):

[0103] 75th quantile threshold: 1.96E-05

[0104] All PRS values comprised between the 25th and 75th quantile indicate a genetic predisposition to normal levels of component 2 of the progesterone membrane receptor. e) A negative correlation between PRS values indicating a genetic predisposition to certain levels of mineralcorticoid receptors and Neurometer® CPT® values at baseline at 5Hz (electrostimulation of C-fibers and assessment of sensitivity to prolonged pain).

[0105] A PRS indicating a genetic predisposition to high levels of said receptor is correlated with low values of Neurometer® CPT® at baseline at 5Hz, which correspond to a greater susceptibility of the vestibule to prolonged pain. Conversely, a PRS indicating a genetic predisposition to low levels of mineralcorticoid receptors is correlated with higher Neurometer® CPT® baseline values at 5Hz, which correspond to a lower susceptibility of the vestibule to prolonged pain.

[0106] PRSs indicating a genetic predisposition to low levels of mineralcorticoid receptors correspond to values below the 25th quantile of the PRS distribution in the general European population (distribution obtained using data of the 1000 Genome Project):

[0107] 25th quantile threshold: -2.5E-05

[0108] PRSs indicating a genetic predisposition to high levels of mineralcorticoid receptors correspond to values above the 75th quantile of the PRS distribution in the general European population (distribution obtained using data of the 1000 Genome Project):

[0109] 75th quantile threshold: 2.44E-05

[0110] All PRS values comprised between the 25th and 75th quantiles indicate a genetic predisposition to normal levels of mineralcorticoid receptors.

[0111] The disclosures in Italian Patent Application No. 102024000017425, from which this application claims priority, are incorporated herein by reference.

Claims

CLAIMS1. A method for stratifying an individual with vulvodynia or genetically predisposed to one or more phenotypic traits associated with vulvodynia, the method comprising: a) obtaining the genotype of an individual; b) accessing a database comprising one or more of the following genotype / phenotype correlation data groups:- group 1 : polymorphisms correlated with levels of component 1 of the membrane progesterone receptor;- group 2: polymorphisms correlated with levels of estrogen receptors;- group 3: polymorphisms correlated with levels of total testosterone;- group 4: polymorphisms correlated with prolactin levels;- group 5: polymorphisms correlated with levels of component 2 of the membrane progesterone receptor; and- group 6: polymorphisms correlated with mineralcorticoid receptor levels; c) calculating, for at least one of groups 1 to 6, a polygenic risk score (PRS) for the respective phenotypic trait in the individual by comparing the genotype of step a) with the database of step b); d) comparing the PRS obtained in step c) with the distribution of PRSs in the general population for the same phenotypic trait and determining whether said PRS is lower than the 25th quantile, comprised between the 25th and 75th quantiles, or higher than the 75th quantile compared with the distribution of PRSs in the general population; e) establishing the individual's genetic predisposition to the phenotypic trait associated with vulvodynia, where:- a PRS of the individual lower than the 25th quantile indicates the individual’s genetic predisposition for low levels of the correlated phenotypic trait;- a PRS of the individual comprised between the 25th and 75th quantiles indicates the individual's genetic predisposition for normal levels of the correlated phenotypic trait;- a PRS of the individual greater than the 75th quantile indicates the individual's genetic predisposition for high levels of the correlated phenotypic trait; f) stratifying the individual with vulvodynia or genetically predisposed to one or more phenotypic traits associated with vulvodynia based on the genetic predisposition to high or low levels of one or more of the phenotypic traits associated with vulvodynia, established in step e).

2. The method according to claim 1, wherein the individual is European and the distribution of PRSs in the general population is a normal distribution calculated from 1000 Genome Project data (1000 Genome Project Consortium, 2015).

3. The method according to claim 2, wherein in step c) the GWAS ID: prot-a-2259 database is employed, obtaining the PRS correlated with the level of component 1 of the membrane progesterone receptor, where a PRS higher than the 75th quantile is indicative of a genetic predisposition to vulvodynia.

4. The method according to claim 2, wherein in step c) the GWAS ID: prot-a-2260 database is employed, obtaining the PRS correlated with the level of component 2 of the membrane progesterone receptor, where a PRS higher than the 75th quantile is indicative of a higher susceptibility of the vestibule to prolonged pain, measured by means of C- fiber electrostimulation.

5. The method according to claim 2, wherein in step c) the GWAS ID: prot-a-991 database is employed, obtaining the PRS correlated with the estrogen receptor level, where a PRS lower than the 25th quantile is indicative of a higher pain sensitivity due to reduced trophism of the vestibular mucosa.

6. The method according to claim 2, wherein in step c) the GWAS ID: ieu-b-4864 database is used, obtaining the PRS correlated with the total testosterone level, where a PRS lower than the 25th quantile is indicative of increased pain sensitivity due to reduced trophism of the vestibular mucosa.

7. The method according to claim 2, where in step c) the GWAS ID: prot-a-2375 database is used, obtaining the PRS correlated with the prolactin level, where a PRS higher than the 75th quantile is indicative of increased vestibular sensitivity to acute and prolonged pain measured by electrostimulation of A3 fibers and C-fibers.

8. The method according to claim 2, where in step c) the GWAS ID: prot-a-2089 database is used, obtaining the PRS correlated with the level of mineralcorticoid receptors, where a PRS higher than the 75th quantile is indicative of increased vestibular sensitivity to prolonged pain, measured by means of C-fiber electrostimulation.

9. The method according to any one of the preceding claims, wherein the PRS is obtained for the levels of components 1 and 2 of the membrane progesterone receptor, for estrogen receptor levels, for total testosterone levels, for prolactin levels, and for mineralcorticoid receptor levels.

10. The method according to any one of the preceding claims, wherein the genotype of the individual is obtained by whole genome sequencing (WGS).

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