Method for diagnosing endometriosis

Abstract

The present invention relates to an in vitro method for diagnosing endometriosis in an individual, comprising: - a step of determining the level of methylation of circulating DNA encoding gene DNMT1 and at least one of the genes selected from the group consisting of DIP2C, RRP1 and USP1, in a biological sample from the individual; - a step of deducing from the determined levels of methylation whether the individual is afflicted with endometriosis.

Description

[0001] METHOD FOR DIAGNOSING ENDOMETRIOSIS

[0002] Field of the invention

[0003] The present invention relates to a method for diagnosing endometriosis.

[0004] Technical background

[0005] Endometriosis is a condition in which cells similar to those found inside the uterus grow in places where they should not. Individuals, in particular women, who have endometriosis may experience significant pain, infertility or both.

[0006] Endometriosis is characterized by a wide range of non-specific symptoms which makes it difficult to diagnose. Usually endometriosis symptoms occur at regular times that are often before, during or after monthly periods. Symptoms may include: painful menstrual cramps, pelvic pain that can radiate to the back, down the legs or into the vagina or rectum, bloating, constipation and / or diarrhea, pain with urination, pain with a bowel movement, or pain during or after sexual intercourse.

[0007] As such, the only way to currently diagnose endometriosis is through laparoscopy, a surgical procedure that is done under general anesthesia. Using a laparoscope the surgeon looks inside the pelvic cavity and other areas to see if endometrial cells / lesions are present.

[0008] Although laparoscopy is only not highly invasive, there is still a need for non- invasive diagnosis method of endometriosis.

[0009] In this regard, Zachariah et al. (2009) Reproductive BioMedicine Online 18: 407- 41 1 have shown that the concentration of circulating cell free (ccf) nuclear DNA (nDNA) is significantly elevated in individuals with endometriosis as compared with a healthy control group and that ccf plasma nDNA might be a potential biomarker to develop non-invasive test for endometriosis. However, more recently, Alonso ef al. (2022) Reproductive BioMedicine Online 44:495-503 did not find significant differences in any of the circulating DNA (cirDNA) parameters between women with and without endometriosis. The authors note that the low statistical power and heterogenous pelvic pathology in their control group render it difficult to determine whether the negative results reflect a true lack of increase in circulating DNA in endometriosis.

[0010] It is therefore an object of the invention to improve methods for diagnosing endometriosis which are based on circulating nucleic acids, in particular DNA. Summary of the invention

[0011] The present invention arises from the unexpected finding, by the present inventors, that the genic regions DNMT1 , DIP2C, RRP1 and USP1 of circulating DNA of individuals afflicted with endometriosis is significantly less methylated than that of control individuals.

[0012] Accordingly, the present invention relates to an in vitro method for diagnosing endometriosis in an individual, comprising:

[0013] - a step of determining the level of methylation of circulating DNA encoding gene DNMT1 and at least one of the genes selected from the group consisting of DIP2C, RRP1 and USP1 , in a biological sample from the individual;

[0014] - a step of deducing from the determined levels of methylation whether the individual is afflicted with endometriosis.

[0015] In a preferred embodiment of the invention, the in vitro method as defined above further comprises a step of determining the level of circulating DNA in the biological sample from the individual and the step of deducing whether the individual is afflicted with endometriosis is based on the determined levels of methylation and on the level of circulating DNA.

[0016] In a preferred embodiment of the invention, the in vitro method as defined above further comprises a step of determining that the level of circulating DNA in the biological sample from the individual is above a predetermined value, in particular before the step of determining the level of methylation of circulating DNA.

[0017] The present invention also relates to a kit for diagnosing endometriosis comprising PCR primer pairs for amplifying all or portions of gene DNMT1 and of at least one of the genes selected from the group consisting of DIP2C, RRP1 and USP1 .

[0018] The present invention also relates to a DNase for use in a method of treatment of endometriosis in an individual, wherein the individual has been diagnosed as being afflicted with endometriosis by implementing the in vitro method for diagnosing endometriosis as defined above. of the invention

[0019] Definitions

[0020] As a preliminary remark, it should be noted that the term "consisting of" means "constituted by", i.e. when an object "consists of" an element or several elements, the object cannot include other elements than those mentioned. In contrast, the term "comprising" means "including", "containing" or "encompassing", / .e. when an object "comprises" an element or elements, other elements than those mentioned can also be included in the object. In other words, when an object "comprises" an element or elements, it consists of the element(s) and possibly of other elements than these.

[0021] ‘‘Endometriosis” is described in the 1 1thversion of the International Classification of Diseases (ICD-1 1 ) of the World Health Organization (WHO) under code GA10 as a a condition of the uterus that is frequently idiopathic. This condition is characterised by ectopic growth and function of endometrial tissue outside the uterine cavity. This condition may be associated with remaining vestigial tissue from the wolffian or mullerian duct, or fragments endometrium refluxed backward into the peritoneal cavity during menstruation. This condition may also present with dysmenorrhoea, dyspareunia, non-menstrual pelvic pain, infertility, alteration of menses, or may be asymptomatic. Confirmation is by laparoscopy and histological identification of ectopic fragments. Endometriosis notably encompasses:

[0022] Endometriosis of the reproductive system (GA10.B), such as endometriosis of the uterosacral ligaments (GA10.B0), endometriosis of the pelvic side wall (GA10.B1 ), endometriosis of rectovaginal septum or vagina (GA10.B2), endometriosis of fallopian tube (GA10.B3), superficial ovarian endometriosis (GA10.B4), or deep ovarian endometriosis (GA10.B5);

[0023] Endometriosis of the digestive system (GA10.C), such as endometriosis of the gallbladder (GAI 0. CO), endometriosis of intestine (GAI 0. Cl ), endometriosis of pelvic peritoneum (GA10.C2), or Peritoneal pockets due to endometriosis (GA10.C3);

[0024] Endometriosis of urinary system (GA10.D), such as endometriosis of the bladder (GA10.D0);

[0025] Endometriosis of the circulatory system (GA10.E);

[0026] Endometriosis of the nervous system (GAI 0.F);

[0027] Thoracic endometriosis (GA 10. G);

[0028] Endometriosis in cutaneous scar (GA10.H); or

[0029] Endometriosis-related adhesions (GA10.J).

[0030] Endometriosis presents in different severity stages. The American Society for Reproductive Medicine (ASRM) thus defines four stages or grades according to the number of lesions and depth of infiltration: minimal or stage I (few superficial implants), mild or stage II (more and deeper implants), moderate or stage III (many deep implants, small cysts on one or both ovaries, presence of filmy adhesions), and severe or stage IV (many deep implants, large cysts on one or both ovaries, many dense adhesions).

[0031] As intended herein, a “biological sample” is a sample obtained from an individual that contains biological material, such as a bodily fluid.

[0032] As intended herein, an “individual” is a mammal, in particular a human. It can be a male or a female. More particularly, the individual is a human female.

[0033] As intended herein, an in vitro method for diagnosing a disease is not a diagnostic method practiced on the human or animal body. It is carried out on biological samples that have been obtained from the human or animal body in a step that precedes, and that is not part of, the in vitro method as defined above.

[0034] As intended herein, “circulating DNA” is considered synonym to “acellular DNA” and to “circulating cell-free DNA”. It relates to small fragments of DNA, in particular double-stranded DNA, with a distribution peak of about 170 bp, that can be found in bodily fluids or biological samples, in particular cell-free bodily fluids or biological samples, such as plasma or serum.

[0035] As intended herein, the “level of methylation” of circulating DNA relates to the quantity of methyl-cytosine nucleotides that can be found in the circulating DNA.

[0036] Cytosine Methylated

[0037] Cytosine

[0038] The DNMT1 gene encodes the DNA (cytosine-5)-methyltransferase 1 enzyme that transfers methyl groups to cytosine nucleotides of genomic DNA. Its human sequence can notably be accessed under NCBI Reference Sequence NG_028016.

[0039] The DIP2C gene encodes the disco interacting protein 2 homolog C. Its human sequence can notably be accessed under NCBI Reference Sequence NM_014974.

[0040] The RRP1 gene encodes ribosomal RNA processing 1 . Its human sequence can notably be accessed under NCBI Reference Sequence NG_046959.

[0041] The USP1 gene encodes ubiquitin specific peptidase 1. Its human sequence can notably be accessed under NCBI Reference Sequence NM_003368. As intended herein, a “CpG island” is a region of DNA characterized by a high frequency of the cytosine (C) and guanine (G) dinucleotide sequence ("CpG"). These regions are often found near gene promoters and are typically defined as being at least 200 base pairs in length with a GC content greater than 50% and an observed- to-expected ratio of CpGs greater than 0.60. CpG islands are commonly associated with the regulation of gene expression due to their potential for differential methylation.

[0042] "DNase” relates to deoxyribonuclease.

[0043] Method for diagnosing endometriosis

[0044] Where a step of determining the level of circulating DNA in the biological sample from the individual is implemented, the circulating DNA can be quantified as indicated in the experimental part below, or by means of other methods described in the scientific literature, in particular fluorometric or spectrophotometric methods such as QUBIT® (Life Technologies) or NANODROP™ (Thermo Scientific). Recently, the analysis of cell-free DNA has been abundantly described in methods for diagnosing certain cancers or in the prenatal diagnosis of chromosome anomalies. As such, several technologies for isolating and analyzing cell-free DNA have been described, both in scientific publications, such as Huebner ef al. (2021 ) Reprod Biomed Online 43:788-798, and in patent literature.

[0045] Determining the level of methylation of circulating DNA can be carried out by any method described in the scientific literature.

[0046] In particular, three major molecular methods, based on enzymatic, immunological or chemical detection, make it possible to quantify methylated cytosines in DNA. In the context of the present invention, these various methods can be combined with methods of hybridization on chips or of high-rate sequencing for more detailed resolution. The four techniques most usually used for determining the methylation level of DNA are MeDIP-seq, WGBS, RRBS and 450K Bead Array. These various techniques can easily be implemented by a person skilled in the art, thanks to the availability of detailed protocols in the literature, of commercial kits and of specialized laboratories. These various techniques produce concordant results, possibly with variable sensitivities of detection of differentially methylated regions between samples. Thus, in the context of the present invention, the "control” level of methylation for the analyzed genes is preferably calibrated using the technique that will be used for measuring the methylation level of the genes in the biological sample.

[0047] According to the immunological detection method, antibodies specifically binding to the methylated cytosines allow detection by immunoprecipitation. This method is called MeDIP (Methylated DNA ImmunoPrecipitation). Usually, DNA is fragmented by sonication, and the most methylated fragments will be the most favorably precipitated in the presence of the antibody, making it possible to obtain a fraction of the genome enriched with methylation. In the context of the present invention, the sonication step is not essential, given the fragmented character of cell- free DNA. Coupled with the high-rate sequencing (MeDIP-seq), this method makes it possible to measure a local methylation density, with a resolution of approximately 200 nucleotides corresponding to the mean size of cell-free DNA fragments, at a reasonable cost. It allows the complete coverage of the gene, with however a bias for the regions most rich in CpG units, / .e so-called CpG islands.

[0048] According to the chemical detection methods, the determination of the level of methylation of cytosines is based on bisulfite. In the presence of this chemical compound, cytosine is converted into uracil, whereas the methylated cytosines are not affected. This method thus enables the methylation to be read by analyzing the single nucleotide polymorphisms (SNPs), in which a T corresponds to a non-modified cytosine and a C to a methylated cytosine on the reference genome before conversion.

[0049] The complete sequencing of DNA after treatment with bisulfite, e.g. WGBS (whole-Genome Bisulfite Sequencing), makes it possible to access the methylation status of all the cytosines, which allows mapping methylation on the whole genome.

[0050] RRBS (Reduced Representation Bisulfite Sequencing) is a technique derived from WGBS, based on the prior selection of the genome regions rich in CpG, i.e. CpG islands, by the use of restriction enzymes. By reducing the number of fragments to be sequenced, the cost and the depth of the sequencing are greatly improved thereby, in the same order of magnitude as MeDIP-seq.

[0051] Finally, the DNA converted with bisulfite can also be hybridized on an oligonucleotide chip, comprising specific oligonucleotides of the differentially methylated genes in individuals having endometriosis.

[0052] As will be clear to the person of skill in the art, the circulating DNA encoding the gene DNMT1 , DIP2C, RRP1 or USP1 need not comprise the totality of the sequence said genes. As such, preferably, the step of determining the level of methylation of circulating DNA encoding gene DNMT1 and at least one of the genes selected from the group consisting of DIP2C, RRP1 and USP1 is conducted on a portion of the circulating DNA encoding DNMT1 , DIP2C, RRP1 or USP1 , in particular a portion that includes CpG islands. It well within the ordinary skills of the person skilled in the art to identify CpG islands. As will be clear to the person of skill in the art. CpG islands are preferably targeted because of their high potential for methylation.

[0053] By way of example, a CpG island can be found from about nucleotide 41 100 to about nucleotide 42000 in NCBI Reference Sequence NG_028016.3 of DNMTI1 .

[0054] Similarly, a CpG island can be found from about nucleotide 1 to about nucleotide 470 in NCBI Reference Sequence NM_014974.3 of DIP2C.

[0055] A CpG island can be found from about nucleotide 4800 to about nucleotide 5450 in NCBI Reference Sequence NG_046959.1 of RRP1 .

[0056] A CpG island can be found from about nucleotide 1 to about nucleotide 340 in NCBI Reference Sequence NM_003368.5 of USP1 .

[0057] Preferably, in the in vitro method as defined above, determining the level of methylation is carried out by submitting circulating DNA to bisulfite treatment followed by polymerase chain reaction.

[0058] The level of methylation can then be determined by various methods. The level of methylation of the amplicon obtained by the polymerase chain reaction can be sequenced. Probes can also be used that either bind or do not bind to a target sequence on the amplicon obtained by the polymerase chain reaction depending on its methylation status. Besides, sets of PCR primers can be used that either bind or do not bind to a target sequence depending on its methylation status and thereby yield or do not yield an amplicon as a result of the PCR. This latter technique is called Methylation-Specific PCR (MSP).

[0059] According to an embodiment of the in vitro method as defined above, a biological sample is treated by bisulfite. A PCR with primers amplifying a CG-rich portion of the DNMT1 gene and of at least one of the genes selected from the group consisting of DIP2C, RRP1 and USP1 is then conducted on the bisulfite-treated biological sample to yield amplicons. The amplicons are sequenced. The mean percentage / level of methylation of the amplicons is then calculated by comparison with a reference sequence corresponding to the amplicon or with an amplicon obtained from a bisulfite-treated biological sample of a healthy or non-endometriosis individual. The endometriosis / non-endometriosis status of the individual from whom the biological sample was taken can then be deduced from the percentages / levels of methylation of each of the amplicons / genes.

[0060] Preferably, in the in vitro method as defined above, the step of deducing from the determined levels of methylation whether the individual is afflicted with endometriosis is computer implemented.

[0061] Preferably, in the in vitro method as defined above, the step of deducing from the determined levels of methylation whether the individual is afflicted with endometriosis is implemented by calculating the result of a parametric equation comprising as variables the levels of methylation and optionally the level of circulating DNA.

[0062] Preferably, in the in vitro method as defined above, wherein the level of methylation is determined by comparison with a predetermined value obtained from the determination of the level of methylation of circulating DNA in biological samples from individuals not afflicted by endometriosis.

[0063] Preferably, the in vitro method as defined above comprises a step of determining the level of methylation of circulating DNA encoding DNMT1 , DIP2C, RRP1 and USP1 genes.

[0064] Preferably, the in vitro method as defined above further comprises a step of determining the level of methylation of circulating DNA encoding at least one gene, in particular all the genes, selected from the group consisting of CALD1 , FN1 , HDAC1 , RMI2, and TDRD5.

[0065] CALD1 relates to Caldesmon 1 (Testis Secretory Sperm-Binding Protein Li 227n).

[0066] FN1 relates to Epididymis Secretory Sperm Binding Protein.

[0067] HDAC1 relates to Histone Deacetylase 1.

[0068] RMI2 relates to RecQ-Mediated Genome Instability 2.

[0069] TRD5 relates to Tudor Domain-Containing 5.

[0070] Preferably, the in vitro method according as defined above comprises determining the level of methylation of circulating DNA of less than 15 genes.

[0071] Preferably, it is determined that the circulating DNA encoding the gene DNMT1 and the at least one of the genes selected from the group consisting of DIP2C, RRP1 and USP1 are hypomethylated.

[0072] Preferably, it is deduced that the individual is afflicted with endometriosis if the level of methylation of the circulating DNA encoding the gene DNMT1 and of at least one of the genes selected from the group consisting of DIP2C, RRP1 and USP1 , more preferably of all of genes DIP2C, RRP1 and USP1 , is lower than the level of methylation of the circulating DNA encoding the gene DNMT1 and of at least one of the genes selected from the group consisting of DIP2C, RRP1 and USP1 , more preferably of all of genes DIP2C, RRP1 and USP1 , of a control individual or an individual not afflicted with endometriosis.

[0073] Preferably, it is deduced that the individual is afflicted with endometriosis if the level of methylation of the circulating DNA encoding the gene DNMT1 and of at least one of the genes selected from the group consisting of DIP2C, RRP1 and USP1 , more preferably of all of genes DIP2C, RRP1 and USP1 , is lower than pre-determined or referenced levels of methylation of circulating DNA encoding the gene DNMT1 and of at least one of the genes selected from the group consisting of DIP2C, RRP1 and USP1 , more preferably of all of genes DIP2C, RRP1 and USP1 .

[0074] Preferably, the biological sample is selected from the group consisting of a whole blood sample, a serum sample or a plasma sample.

[0075] Preferably, in the in vitro method as defined above, stage I or II endometriosis is diagnosed.

[0076] Kit

[0077] Preferably, the above-defined kit, comprises PCR primer pairs and optionally probes for amplifying and detecting all or portions of genes DNMT1 , DIP2C, RRP1 and USP1.

[0078] Preferably, the above-defined kit further comprises reagents for bisulfite treatment of DNA.

[0079] DNase

[0080] Advantageously, a clear clinical improvement can be observed in individuals afflicted by endometriosis who have been treated during 1 month by intramuscular injection of DNase I at a dose of 2500 IU every two days. In particular, treated individuals experience an improvement of their quality of life which is associated to a decrease of pain and of dyspareunia. Biologically, an important reduction of the quantity of circulating DNA can be observed. As will be clear to the person of skill in the art, the above-defined in vitro method is useful as a companion diagnostic in the frame of treatment of endometriosis, in particular with DNase.

[0081] Preferably, the above-defined DNase a recombinant DNase, more preferably a human recombinant DNase. Preferably also, the DNase is a DNase I, more preferably a recombinant DNase I, such as dornase alfa (Pulmozyme®).

[0082] The DNase can be administered by any suitable. However, it is preferentially administered intramuscularly.

[0083] Preferably, the DNase is administered at a dose of at least 2500 Ul every two days, for at least two weeks, preferably at least three weeks, and even more preferably for one month or more. A dose two times greater can also administered to the individual. For example, a dose of 5000 Ul can be administered every two days, or the DNase can be administered at a daily dose of 2500 Ul.

[0084] Preferably also, a dose of 2500 Ul every day or every two days can be administered, until a significant improvement of the symptoms is observed (for example, until the pain has significantly decreased) and / or until the level of circulating DNA has dropped below a predetermined threshold (corresponding, for example, to a level close to the level observed on average in individuals not suffering from endometriosis.

[0085] Preferably also, a dose of 2500 Ul every day or every two days is administered for an initially determined period, for example for one, two or three months. The person skilled in the art will obviously be able to adjust this period according to the level of gravity of the endometriosis and / or other parameters such as, for example, the level of cell-free DNA in the individual at the start of treatment.

[0086] Various therapeutic protocols are envisaged in the context of the present invention, dependent in particular on the level of seriousness of the endometriosis. Thus, for the less serious stages (stage I or II endometriosis), endometriosis can be treated solely by administering DNase as described above, while for the more severe forms (stage III or IV endometriosis), treatment by DNase will advantageously be able to be supplemented by surgery.

[0087] Preferably, the treatment of endometriosis with DNase includes a plurality of phases. In a first phase, referred to as “aggressive phase”, the individual will receive DNase at a fairly high dose, for example 2500 Ul per day. This aggressive phase can have a fixed duration (for example from 1 to 3 months), or a duration determined by the clinical development of the illness or by a biological marker (such as the level of plasmatic cell-free DNA). At the end of this aggressive phase, and in order to improve the result and / or to avoid a relapse, the treatment will be continued by a maintenance treatment, which will be selected by the practitioner according to various factors. For example, for an individual having a pregnancy plan, the treatment with DNase will be continued, where applicable by selecting a dose or a frequency that is lower than during the aggressive treatment. By way of example, an administration of 2500 Ul once or twice per week can be envisaged, or even every two weeks or even once a month. For a individual not having a pregnancy plan, the maintenance treatment may be either continuation of the treatment by Dnase (under the same conditions as for a individual having a wish for pregnancy), or a hormone treatment intended to suppress periods (continuous single-phase oestroprogestative or progestative contraceptives, Danazol or GnRH analogues or antagonist).

[0088] The invention will be further described by way of the following non-limiting example and figure.

[0089] Description of the figure

[0090] Figure 1 represents the RPP30 copy number (vertical axis) in circulating DNA obtained from respectively endometriosis-afflicted individuals or control individuals.

[0091] EXAMPLE

[0092] A study was conducted to determine that the methylation level of the portions of circulating DNA which harbor certain genes is a diagnostic marker of endometriosis.

[0093] Study cohort

[0094] A cohort of 78 individuals comprising 38 individuals diagnosed as being afflicted with endometriosis (“endometriosis group”) and 40 control individuals (“control group”), / .e. not afflicted with endometriosis, was used.

[0095] Circulating DNA extraction

[0096] Circulating DNA was extracted from plasma samples obtained from the individuals of the cohort using QIAamp DNA mini Blood kit (Qiagen, ref. 51 104), following the manufacturer’s protocol. DNA concentration and purity was measured using a NanoDrop ND-1000 (Thermo Scientific) spectrophotometer. DNA concentrations above 1 ng / L were obtained.

[0097] Determination of the number of copies of circulating DNA

[0098] The absolute amount circulating DNA was determined by determining the number of copies of the RRP30 gene per mL of plasma sample. Quantification was conducted by real Real-time PCR (qPCR) in 20pil final reaction volume using LightCycler 480 System II apparatus (Roche) using the plasmid Hs_RPP30 positive control (IDT, ref.10006626) as a calibrator.

[0099] The results are shown in the following Table 1 and in Figure 1 .

[0100] Table 1 - RRP30 copy number in the endometriosis and control group There is a statistically significant difference in RPP30 copy number between the endometriosis group and the control group (Anova Test, p=0.0000763), which would appear to confirm that a higher amount of circulating DNA is indicative of endometriosis.

[0101] However, it can be observed that some individuals of the endometriosis group have amounts of circulating DNA that are lower than the mean level of circulating DNA in the control group. Conversely, some individuals of the control group have amounts of circulating DNA that are higher than the median level of circulating DNA in the endometriosis group.

[0102] Accordingly, the level of circulating DNA is not a completely satisfactory diagnostic marker of endometriosis and both its sensitivity and its specificity can be improved.

[0103] Bisulfite treatment of circulating DNA

[0104] Bisulfite treatment of DNA is a preliminary step for DNA methylation analysis. This step consists in converting non-methylated cytosine (C) into uracil (U). Thus, DNA that has been treated with bisulfite retains only methylated cytosines (mC). After sequencing, the methylated cytosine (mC) loci appears like a cytosine (C), and the unmethylated loci appears like a thymine (T). As such, the higher the number of C in a sequence, the higher its methylation level.

[0105] The circulating DNA samples were submitted to bisulfite treatment using EZ DNA Methylation Kit (Zymo Research) following the manufacturer’s recommendations. According to initial concentrations, 35pi I of DNA sample were used for the reaction. At the end of the treatment, DNA was eluted with 25pi I of elution Buffer, then diluted using l O I H2O.

[0106] Determination of the methylation level of circulating DNA

[0107] The inventors then sought to determine whether differential methylation levels of circulating DNA between the endometriosis and control groups could be evidenced.

[0108] The methylation level of circulating DNA was thus determined at the CpG islands of 9 genes:

[0109] - CALD1

[0110] - DIP2C - DNMT1

[0111] - FN1

[0112] - HDAC1

[0113] - RMI2

[0114] - RRP1

[0115] - TDRD5

[0116] - USP1

[0117] Briefly, the bisulfite-treated samples of circulating DNA were submitted to PCR amplification using Taq’Ozyme HS Mix (Ozyme) with primers amplifying regions of the above genes which comprise the CpG islands. The amplimers were then subjected to sequencing on a NextSeq Illumina Platform.

[0118] The sequences of the 9 amplimers were then analyzed to determine the number of C and T nucleotides for each individual and the mean number of C and T nucleotides for each gene were determined respectively for the endometriosis group and the control group. Besides, a chi square test of homogeneity was applied to determine for each gene if the distribution of C and T nucleotide is the same or different when comparing the control group and the endometriosis group (p value type 1 error alpha set to 0.05).

[0119] The results are shown in the following Table 2:

[0120] Table 2 - Methylation level in the control and endometriosis group On average, the methylation level appears to be higher in the control group than in the endometriosis group, / .e. circulating DNA of endometriosis individuals appears to be hypomethylated as compared to that of control individuals. However, the difference in methylation level is statistically significant only when considering the DIP2C, DNMT1, RRP1 and USP1 regions of circulating DNA.

Claims

CLAIMS1. An in vitro method for diagnosing endometriosis in an individual, comprising:- a step of determining the level of methylation of circulating DNA encoding gene DNMT1 and at least one of the genes selected from the group consisting of DIP2C, RRP1 and USP1 , in a biological sample from the individual;- a step of deducing from the determined levels of methylation whether the individual is afflicted with endometriosis.

2. The in vitro method according to claim 1 , further comprising a step of determining that the level of circulating DNA in the biological sample from the individual is above a predetermined value, in particular before the step of determining the level of methylation of circulating DNA.

3. The in vitro method according to claim 1 or 2, wherein it is determined that the circulating DNA encoding the gene DNMT1 and the at least one of the genes selected from the group consisting of DIP2C, RRP1 and USP1 are hypomethylated.

4. The in vitro method according to anyone of claims 1 to 3, comprising a step of determining the level of methylation of circulating DNA encoding DNMT1 , DIP2C, RRP1 and USP1 genes.

5. The in vitro method according to anyone of claims 1 to 4, comprising determining the level of methylation of circulating DNA of less than 15 genes.

6. The in vitro method according to anyone of claims 1 to 5, wherein the step of deducing from the determined levels of methylation whether the individual is afflicted with endometriosis is computer implemented.

7. The in vitro method according to anyone of claims 1 to 6, wherein the step of deducing from the determined levels of methylation whether the individual is afflicted with endometriosis is implemented by calculating the result of a parametric equation comprising as variables the levels of methylation.

8. The in vitro method according to anyone of claims 1 to 7, wherein the sample is selected from the group consisting of a whole blood sample, a serum sample or a plasma sample.

9. The in vitro method according to anyone of claims 1 to 8, wherein determining the level of methylation is carried out by submitting circulating DNA to bisulfite treatment followed by polymerase chain reaction.

10. The in vitro method according to anyone of claim 1 to 9, wherein the level of methylation is determined by comparison with a predetermined value obtained from the determination of the level of methylation of circulating DNA in biological samples from individuals not afflicted by endometriosis.

11. The in vitro method according to anyone of claim 1 to 10, wherein stage I or II endometriosis is diagnosed.

12. A kit for diagnosing endometriosis comprising PCR primer pairs for amplifying all or portions of gene DNMT1 and of at least one of the genes selected from the group consisting of DIP2C, RRP1 and USP1.

13. The kit according to claim 12, comprising PCR primer pairs and probes for amplifying and detecting all or portions of genes DNMT1 , DIP2C, RRP1 and USP1 .

14. The kit according to claim 12 or 13, further comprising reagents for bisulfite treatment of DNA.

15. DNase for use in a method of treatment of endometriosis in an individual, wherein the individual has been diagnosed as being afflicted with endometriosis by implementing the in vitro method for diagnosing endometriosis according to anyone of claims 1 to 1 1 .

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