WO2016042015A1

WO2016042015A1 - Method for evaluating developmental competence of an oocyte

Info

Publication number: WO2016042015A1
Application number: PCT/EP2015/071190
Authority: WO
Inventors: Vincent Puard; Thomas BOURQUARD; Dominique Royere; Eric Reiter
Original assignee: Centre National De La Recherche Scientifique (Cnrs); Institut National De La Recherche Agronomique; Centre Hospitalier Regional Universitaire De Tours; Universite Francois Rabelais De Tours
Priority date: 2014-09-16
Filing date: 2015-09-16
Publication date: 2016-03-24

Abstract

The present invention is relative to an in vitro method for evaluating developmental competence of an oocyte or an embryo from a subject, comprising the step of determining the expression level of a combination of at least 2 biological markers in a biological sample, wherein said biological markers are chosen in the list consisting in PTX3, FOS, PLCB1, POMT2, RPL7, S100A13, MERTK, POLR3K and POPDC2.

Description

METHOD FOR EVALUATING DEVELOPMENTAL COMPETENCE OF AN OOCYTE

The present invention relates to methods for evaluating developmental competence of a mammalian oocyte or embryo.

Defining the developmental ability of an embryo during in vitro fertilization remains a major challenge both in humans and domestic mammals. Morphological criteria are most frequently used to evaluate the developmental and implantation ability of the embryos in human assisted reproductive technology (ART). However such morphological criteria (zygote scoring, early cleavage and embryo morphology at day 2 or 3) remain poorly predictive of developmental or implantation ability (Guerif F. et al. : Does early morphology provide additional selection power 330 to blastocyst selection for transfer? Reproductive Biomedicine Online 2010, 21 :510-519). Direct studies on embryos, such as genomic or proteomic analyses are difficult in human, since such approach remains invasive and might alter the embryo integrity (Jones GM et al. , Novel strategy with potential to identify developmentally competent IVF, Hum Reprod 2008, 23: 1748- 1759). Therefore ART laboratories need some indirect and non-invasive selection criteria, to easily and reproducibly select competent oocyte.

Various studies reported on molecules inside the follicle or the embryo microenvironment through proteomic and metabolomic analysis of oocytes or embryos (Royere D. et al. , Non invasive assessment of embryo quality: proteomics, metabolomics and oocyte-cumulus dialogue, Gynecol Obstet Fertil 2009, 37:917-920) and have identify potential biomarkers of oocyte or embryo quality. Other studies focused on the somatic cells (cumulus [CCs] and/or granulosa cells [GCs]) surrounding the oocyte since their interactions are involved in the acquisition of oocyte meiotic and developmental competence (Gilchrist RB et al. , Oocyte-secreted factors: regulators of cumulus cell 340 function and oocyte quality, Human Reproduction Update 2008, 14:159-177). Potential biomarkers of oocyte competence in humans and animals were proposed, based on specific genes expression in cumulus cells (Assou S. et al. ; Human cumulus cells as biomarkers for embryo and pregnancy outcomes, Molecular Human Reproduction 2010, 16:531 -538). Indeed, the fact that protein translation is a highly regulated process implies that mRNA abundance does not always correlate with the level of the corresponding proteins. Such discrepancy might be explained by differences in mRNA stability, degradation/synthesis rates or post-translational modification of the proteins. Therefore, the corresponding protein could lead to increased robustness and predictive value of biomarkers. Despite the fact that the ultimate effectors in cells remain the proteins, few studies addressed cumulus cells proteome analysis mainly because of a lack of sensitivity of the techniques. Global proteins expression pattern of human individual cumulus cells was investigated by two-dimensional polyacrylamide gel electrophoresis according to ovarian stimulation protocol without identification of specific proteins linked to oocyte competence (Hamamah S. , Comparative protein expression profiling in human cumulus cells in relation to oocyte fertilization and ovarian stimulation protocol, Reprod Biomed Online 2006, 13:807-814). Cumulus cells proteome was explored by mass spectrometry and Western Blot on pooled Cumulus cells and specific proteins implicated in fatty acid metabolism and pre-mRNA splicing according to maternal age were highlighted (McReynolds S. et al. , Impact of maternal aging on the molecular signature of human cumulus cells, Fertility and Sterility 2012, 98: 1574-1580. e1575). However, to date no study investigating individual cumulus cells proteome and specific proteins according to oocyte competence has been reported.

The inventors have determined that, among the numerous genes expressed in cumulus cells and known to be somewhat correlated with oocyte competency, a specific set of markers (i.e. the biological markers PTX3, FOS, PLCB1 , POMT2, RPL7, S100A13, MERTK, POLR3K and POPDC2) could be used to efficiently determine the developmental competency of an oocyte or an embryo before implantation.

The inventors have thus developed a method for evaluating developmental competence of oocytes or of embryos, which is based on assessing the level of those markers in cumulus cells or samples comprising thereof, from the subject. It is therefore to be understood that the method of the invention does not require nor the use neither the destruction of embryos, and in particular of human embryos. By "evaluating the developmental competence of an oocyte", it herein means assessing the competence of an oocyte to complete maturation, undergo successful fertilization, and reach the blastocyst stage.

By "evaluating the developmental competence of an embryo", it herein means assessing the competence of an embryo to reach the blastocyst stage.

According to the invention, an oocyte or an embryo are considered competent if it can be assessed by the method of the invention that they are likely to yield a blastocyst at 5 or 6 days after fertilization, independently of whether said blastocyst may further give rise to a diagnosed pregnancy.

Thus, the present invention provides an in vitro method for evaluating developmental competence of an oocyte or an embryo from a subject, comprising the step of determining the expression level of a combination of at least 2 biological markers in a biological sample, wherein said biological markers are chosen in the list consisting in PTX3, FOS, PLCB1 , POMT2, RPL7, S100A13, MERTK, POLR3K and POPDC2.

By "biological sample", it is herein referred to any solid or liquid sample that is taken from a subject. Preferably, the sample comprises follicular fluid, cumulus cells, polar bodies, oocytes, or culture media in which the oocytes, cumulus cells, are cultured. More preferably, the sample comprises cumulus cells, and even more preferably consists in cumulus cells. By "cumulus cells", it is herein referred to cells from the cumulus oophorus, a tissue which surrounds the oocyte both in the ovarian follicle and after ovulation.

By "oocyte", it should be understood a female gametocyte or germ cell involved in reproduction. In other words, "oocyte" refers to immature ovum, or egg cell. Oocytes are produced from cells called "oogonia" in the ovary during female gametogenesis, in a process called "oogenesis". The female germ cells produce a primordial germ cell (PGC), which then undergoes mitosis, forming oogonia. During oogenesis, the oogonia become primary oocytes. Oogenesis results in the formation of both primary oocytes before birth, and of secondary oocytes after it as part of ovulation. In the context of the invention the term "oocyte" preferably refers to secondary oocytes. Samples comprising cumulus cells may easily be prepared according to usual protocols known in the field. A non-limiting example of a method to prepare samples comprising cumulus cells is disclosed herein, in the experimental part.

In the meaning of the present invention, the term "subject" refers to an animal, preferably a mammal and more preferably a human. More preferably, the subject is a female human (a woman) in need of or receiving in vitro fertilization treatment.

In some cases, the methods according to the invention may further comprise a preliminary step of taking a biological sample from the subject. In addition, the methods according to the invention may comprise another preliminary step, prior to the step of determining the expression level of a combination of at least 2 biological markers, corresponding to the transformation of the biological sample into a DNA sample or into a protein sample, which is then ready to use for in vitro detection. Preparation or extraction of DNA or proteins from a biological sample is only routine procedure well known to those skilled in the art.

The inventors have established, through thorough analysis of data collected among several sets of samples from human female patients who had been, that among the markers PTX3, FOS, PLCB1 , POMT2, RPL7, S100A13, MERTK, POLR3K and POPDC2, some specific combinations of markers are particularly significant for evaluating developmental competence of a given oocyte or embryo. Indeed, they found that individual biological makers were not significant for evaluating developmental competence of a given oocyte or embryo in contrast to combinations of specific biological markers according to the present invention. They also found that combinations of biological markers that correlate with the higher AUC include the biological marker FOS. Those specific combinations of biological markers can advantageously be used for instance in the context of establishing a Preimplantation diagnosis.

Preferably, the combination of biological markers comprise FOS and at least one additional biological marker chosen in the list consisting in PTX3, PLCB1 , POMT2, RPL7, S100A13, MERTK, POLR3K and POPDC2. Preferably, in the method according to the present invention, the expression level of a biological marker is determined by measuring the level of the corresponding peptide, or by measuring the level of the mRNA from said gene or a fragment thereof. Techniques appropriate for such measuring are further detailed herein. In the context of the present invention, "peptide" means a molecule comprising at least two amino acids, and the terms "polypeptide", "peptide" and "protein" may be used interchangeably.

In the context of the present invention, "mRNA" or "RNA" means a molecule of at least 50 ribonucleic acids, for example at least 100 or 150 ribonucleic acids, preferably at least 200 ribonucleic acids, for example at least 250 or 350 ribonucleic acids, and in a particularly preferred manner, of at least 400 ribonucleic acids.

By the peptide corresponding to the biological marker FOS, it is herein referred to the peptide encoded by the gene FOS. Preferably, it is herein referred to the human peptide of sequence the sequence SEQ ID No.1.

By the mRNA corresponding to the biological marker FOS, it is herein referred to the mRNA transcript encoded by the gene FOS. Preferably, it is herein referred to the human mRNA of sequence the sequence SEQ ID No.2.

By the peptide corresponding to the biological marker PTX3, it is herein referred to the peptide encoded by the gene PTX3. Preferably, it is herein referred to the human peptide of sequence the sequence SEQ ID No.3.

By the mRNA corresponding to the biological marker PTX3, it is herein referred to the mRNA transcript encoded by the gene PTX3. Preferably, it is herein referred to the human mRNA of sequence the sequence SEQ ID No. . By the peptide corresponding to the biological marker PLCB1 , it is herein referred to the peptide encoded by the gene PLCB1 . Preferably, it is herein referred to the human peptide of sequence the sequence SEQ ID No.5.

By the mRNA corresponding to the biological marker PLCB1 , it is herein referred to the mRNA transcript encoded by the gene PLCB1 . Preferably, it is herein referred to the human mRNA of sequence the sequence SEQ ID No.6. By the peptide corresponding to the biological marker POMT2, it is herein referred to the peptide encoded by the gene POMT2. Preferably, it is herein referred to the human peptide of sequence the sequence SEQ ID No.7.

By the mRNA corresponding to the biological marker POMT2, it is herein referred to the mRNA transcript encoded by the gene POMT2. Preferably, it is herein referred to the human mRNA of sequence the sequence SEQ ID No.8.

By the peptide corresponding to the biological marker RPL7, it is herein referred to the peptide encoded by the gene RPL7. Preferably, it is herein referred to the human peptide of sequence the sequence SEQ ID No.9. By the mRNA corresponding to the biological marker RPL7, it is herein referred to the mRNA transcript encoded by the gene RPL7. Preferably, it is herein referred to the human mRNA of sequence the sequence SEQ ID No.10.

By the peptide corresponding to the biological marker S100A13, it is herein referred to the peptide encoded by the gene S100A13. Preferably, it is herein referred to the human peptide of sequence the sequence SEQ ID No.11.

By the mRNA corresponding to the biological marker S100A13, it is herein referred to the mRNA transcript encoded by the gene S100A13. Preferably, it is herein referred to the human mRNA of sequence the sequence SEQ ID No.12.

By the peptide corresponding to the biological marker MERTK, it is herein referred to the peptide encoded by the gene MERTK. Preferably, it is herein referred to the human peptide of sequence the sequence SEQ ID No.13.

By the mRNA corresponding to the biological marker MERTK, it is herein referred to the mRNA transcript encoded by the gene MERTK. Preferably, it is herein referred to the human mRNA of sequence the sequence SEQ ID No.14. By the peptide corresponding to the biological marker POLR3K, it is herein referred to the peptide encoded by the gene POLR3K. Preferably, it is herein referred to the human peptide of sequence the sequence SEQ ID No.15. By the mRNA corresponding to the biological marker POLR3K, it is herein referred to the mRNA transcript encoded by the gene POLR3K. Preferably, it is herein referred to the human mRNA of sequence the sequence SEQ ID No.16.

By the peptide corresponding to the biological marker POPDC2, it is herein referred to the peptide encoded by the gene POPDC2. Preferably, it is herein referred to the human peptide of sequence the sequence SEQ ID No.17.

By the mRNA corresponding to the biological marker POPDC2, it is herein referred to the mRNA transcript encoded by the gene POPDC2. Preferably, it is herein referred to the human mRNA of sequence the sequence SEQ ID No.18. Yet, as it is well known in the art, the expression of a gene requires first its transcription into a mRNA transcript, then the translation of said mRNA into protein. Both transcription and translation are steps that are susceptible to be highly regulated in vivo, which is one of the main reasons why a high level of mRNA expression of a given gene does not always correlate with a high level of protein expression from the very same gene. As a result, for a specific gene, the mRNA expression may correlate with a specific phenotype while the protein expression does not. Conversely, for another gene, the protein expression may correlate with a specific phenotype while the mRNA expression does not. It is possible for a person skilled in the art to measure biological markers both at the RNA level and at the protein level. However, the current molecular and biochemical techniques do not enable to do so without running separate experiments. Moreover, in the field of preimplantation diagnosis, this strategy is rarely advisable, because of the scarcity of available biological sample to analyse.

Hence, for simplicity of use, the inventors have determined, among the specific set of markers of the invention, the best combination of biological markers to be used when measuring mRNA expression and the best combination of biological markers to be used when measuring protein expression.

The specific combinations of markers of the invention are particularly useful, since they can be tested in a multiplex experiment, thus limiting the amount of sample utilized. Moreover, the combinations of the invention have been specifically selected by the inventors for their significant correlation with developmental competence. Hence, the inventors have determined that the specific combinations of markers i) PTX3, FOS, PLCB1 , POMT2, RPL7 and S100A13 and ii) FOS, MERTK, POLR3K and POPDC2 are associated with the highest AUCs.

The sensitivity of a method is the proportion of actual positives which are correctly identified as such, and can be estimated by the area under the ROC (Receiver Operating Characteristic) curve, also called AUC. A receiver operating characteristic (ROC), or simply ROC curve, is a graphical plot which illustrates the performance of a binary classifier system as its discrimination threshold is varied. It is created by plotting the fraction of true positives out of the positives (TPR = true positive rate) vs. the fraction of false positives out of the negatives (FPR = false positive rate), at various threshold settings. TPR is also known as sensitivity, and FPR is one minus the specificity or true negative rate. Area Under the Curve (AUC) is a measure of a classifier/test performance across all possible values of the thresholds. The higher the AUC, the better the performance of the test.

Thus, preferably, the combination of at least 2 biological markers is chosen between the combinations of markers:

- PTX3, FOS, PLCB1 , POMT2, RPL7 and S100A13; and

- FOS, MERTK, POLR3K and POPDC2.

More particularly, the inventors have found that the highest AUC can be obtained either with the level of the mRNA transcripts corresponding to the combination of biological markers PTX3, FOS, PLCB1 , POMT2, RPL7 and S100A13, or with the level of the peptides corresponding to the combination of biological markers FOS, MERTK, POLR3K and POPDC2.

Thus, in an embodiment, the expression level of the combination of biological markers PTX3, FOS, PLCB1 , POMT2, RPL7 and S100A13 is determined by measuring the level of the corresponding mRNA transcripts. In another embodiment, the expression level of the combination of biological markers FOS, MERTK, POLR3K and POPDC2 is determined by measuring the level of the corresponding peptides.

Several techniques are available to the person skilled in the art to measure the level of mRNA transcripts in a sample. They include for instance PCR-based techniques such as quantitative polymerase chain reaction (Q-PCR), reverse- transcriptase polymerase chain reaction (RT-PCR), quantitative reverse- transcriptase PCR (QRT-PCR), rolling circle amplification (RCA) or digital PCR. These techniques are well known and easily available technologies for those skilled in the art and do not need a precise description.

Yet, preferably, in the context of the invention, mRNA transcripts are measured by quantitative polymerase chain reaction. In a preferred embodiment, the level of the mRNA transcripts corresponding to the combination of biological markers PTX3, FOS, PLCB1 , POMT2, RPL7 and S100A13 is measured by quantitative polymerase chain reaction.

Several techniques are available to the person skilled in the art to measure the level of a specific protein in a sample. They include for instance immunological techniques, among which Reverse Phase Protein Array, Western-blotting, ELISAs and Radio-lmmuno Assays, or various proteomics techniques such as mass spectrometry or chromatography.

Preferably, in the context of the invention, protein level is measured by immunological techniques, which comprise Reverse Phase Protein Array, Western- blotting, ELISAs and Radio-lmmuno Assays.

Immunological techniques are based on the use of molecules able to recognize an antigen with high specificity and high sensitivity. Most commonly antibodies specific for the antigen are used for this purpose. A large number of antibodies specific for the human proteins FOS, MERTK, POLR3K and POPDC2 are currently available on the market.

Reverse Phase Protein Array is a sensitive and quantitative technique allowing the detection of specific proteins from very small quantities of biological samples. This technique leads to minimal background signal and maximal signal/noise ratio. This technique also presents the advantage to be cost and material effective.

Thus, preferably, protein level is measured by Reverse Phase Protein Array.

In a preferred embodiment, the level of the peptides corresponding to the combination of biological markers FOS, MERTK, POLR3K and POPDC2 is measured by Reverse Phase Protein Array.

According to the invention, "determining the expression level of a combination of at least 2 biological markers" means determining the expression level of each of the biological markers comprised in said combination. In an embodiment, the method of the invention further comprises a step of comparing the expression level of the combination of the invention with a reference value. By "comparing the expression level of the combination of the invention with a reference value" it is herein meant comparing the expression level of each of the biological markers comprised in said combination with at least one reference value. In an embodiment the expression level of each of the biological markers comprised in said combination is compared to a distinct reference value. The reference value may for instance be a predefined value, or a value obtained in similar experimental conditions of analysis with a biological sample of reference. Preferably, the "reference value" being compared to a specific biological marker is the level of expression of said marker in cumulus cells from an oocyte yielding to a blastocyst after 5 or 6 days of in vitro culture (positive control) or from an oocyte arresting development at the embryo stage after 5 or 6 days of in vitro culture (negative control).

Preferably, the method of the invention comprises a further step of concluding that the oocyte or the embryo is competent if there is a positive difference between the level of expression of the combination of markers and the reference value.

By positive difference, it is herein meant that the level of expression of the combination of markers is superior to the reference value. Preferably, the level of expression of the combination of markers is at least 10%, 15%, 20%, 30%, 40, 50% superior to the reference value

It is well known in the field that developmental competence of an embryo may be assessed by criteria relating to the morphology of the embryo in itself. Embryo morphology is determined by the number, size and shape of blastomeres, the proportion of fragments and the presence of multi-nucleated blastomeres. The use of morphologic parameters to select embryos with the best capacity to implant is well documented, and has been for instance reviewed in Gardner et al. (Placenta. ;24 Suppl B:S5-12; 2003), and in Sakkas et al. (Curr Opin Obstet Gynecol.; 17(3):283-8; 2005).

Briefly, selection of the most viable cleavage stage embryo is usually based on embryo morphology on day 2 or day 3. Embryo morphology is determined by the number, size and shape of blastomeres, the proportion of fragments and the presence of multi-nucleated blastomeres. It has been demonstrated that after 2 days of culture, the 4-cell stage is the optimal cleavage stage (Giorgetti et al., 1995; Ziebe et al., 1997). Embryos at this cleavage stage with little or no fragmentation and without multi-nucleated blastomeres are associated with a higher implantation rate compared to embryos at other cleavage stages with fragmentation or multi-nucleated blastomeres. The main parameters of embryo morphology utilized for this selection typically include the number of cells, regularity of cleavage and degree of fragmentation.

Thus, the method of the invention can advantageously be complemented by the addition of a step of assessing the status of the embryo of interest regarding those morphological criteria. It is to be understood that this additional step does not require the destruction of said oocyte or embryo, but only their observation. Preferably, said embryo is observed at day 2 following fertilization.

Thus, preferably, the method of the invention further comprises a step of assessing the embryo status regarding at least 2 criteria chosen in the list of morphological criteria of the embryo (MCE) consisting of: number of cells, regularity of cleavage and degree of fragmentation. According to the invention, the terms "number of cells" refer to the number of blastomeres of the embryo at day 2 post-insemination.

Hence, according to the invention, the status of the embryo regarding the criteria "number of cells" may be categorized as a unique number comprised between 2 to 8.

The number of cells can easily be assessed by direct observation of the embryo with a microscope, at between 44 h and 46h post-insemination.

According to the invention, the terms "regularity of cleavage" refer to the terms as defined in Ziebe et al., 1997. Typically, the regularity of cleavage is herein defined as generally so in the field, that is to say that an embryo is considered as regularly cleaved when all the cells of said embryo have a similar size. In the context of the invention, two cells of an embryo are considered to have a similar size when their respective volume are similar, that is to say when their respective volumes differ of less than 20%. In the context of the invention, when comparing the sizes of two cells, their respective volumes may be estimated by their respective diameter. Typically, as well known in the field, the regularity of cleavage is estimated on embryos comprising an even number of cells. Hence, according to the invention, the status of the embryo regarding the criteria "regularity of cleavage" may be categorized as regular or as not regular.

The regularity of cleavage can easily be assesses by direct observation of the embryo with a microscope, at between 44 h and 46h post-insemination.

According to the invention, the terms "degree of fragmentation" refer to a percentage of the total embryo volume occupied by anucleate cytoplasmic fragments. In the context of the invention, the term "fragment" should be understood according to the definition commonly accepted in the field, that is to say as an anuclear, membrane-bound extracellular cytoplasmic structure. Preferably, in the context of the invention, for Day-2 embryos a fragment is an anuclear, membrane-bound extracellular cytoplasmic structure which has a diameter inferior to 45 μιη, while for Day-3 embryos, a fragment is an anuclear, membrane-bound extracellular cytoplasmic structure which has a diameter inferior to 40 μιη. These definitions are consistent with the teachings of Johansson et al. (2003).

Hence, according to the invention, the status of the embryo regarding the criteria "degree of fragmentation" may be categorized into one of 3 categories chosen in the list consisting in fragmentation rate <20% of the volume of embryo [condition 1 ], between 20 and 50% of the volume of the embryo [condition 2], >50% of the volume of the embryo [condition 3].

The number of cells can easily be assessed by direct observation of the embryo with a microscope, at between 44 h and 46h post-insemination. In order to better define the most interesting set of markers and morphologic criteria to be used, the inventors have defined and used a greedy algorithm to analyze data collected from the two previously mentioned studies. As generally considered in the field, a greedy algorithm is an algorithm that follows the "problem solving heuristic" of making the locally optimal choice at each stage in order to find the optimal solution to said problem.

Interestingly, by using said greedy algorithm, the inventors have observed that the best AUCs are obtained when the following associations of combination of biological markers and of MCE are considered:

- the combination of biological markers PTX3, FOS, PLCB1 , POMT2, RPL7 and S100A13, together with the morphologic criteria of: number of cells and degree of fragmentation, and

- the combination of biological markers FOS, MERTK, POLR3K and POPDC2, together with the morphologic criteria of: number of cells, regularity of cleavage and degree of fragmentation. Therefore, in an embodiment, the method of the invention comprises:

- a step of determining the expression level of the combination of biological markers PTX3, FOS, PLCB1 , POMT2, RPL7 and S100A13 by measuring the expression level of the corresponding mRNA transcripts, and

- a step of assessing the status of the embryo regarding the morphologic criteria of: number of cells and degree of fragmentation. In another embodiment, the method of the invention comprises:

- a step of determining the expression level of the expression level of the combination of biological markers FOS, MERTK, POLR3K and POPDC2 is determined by measuring the expression of the corresponding peptides, and

- a step of assessing the status of the embryo status regarding the morphologic criteria of: number of cells, regularity of cleavage and degree of fragmentation.

Thus, in some embodiments, the method of the invention relies on both linear values (i.e. the expression levels according to the invention), and on nominal values (i.e. the status of the embryo regarding the above mentioned criteria).

In order to be able to combine those various criteria, which are of a very different nature, into a general scale, the inventors have established a specific mathematical model, herein called the Heterogeneous Value Difference Metric (HVDM). This mathematical model can be implemented in order to obtain a clear indicator, in the form of a number, which can for instance be used to help the person skilled in the art select the more appropriate oocyte or embryo to be implanted.

This mathematical model relies on a classical statistical type of algorithm called "the k-Nearest Neighbors algorithm (or k-NN for short)", which is a non- parametric method used for classification and widely used in pattern recognition.

In k-NN algorithms, the neighbors are taken from a set of objects for which the class (for k-NN classification) or the object property value (for k-NN regression) is known. The "nearest neighbors" may then be determined as the object(s) sharing the most similar relevant features with the tested oocyte. In the context of the invention, the competence for the given oocyte is given by the class associated to its nearest neighbor.

The inventors have therefore determined what are the said most relevant features, and their relative importance in determining the said nearest neighbor. To this end, a greedy dimension reduction has been performed to find the optimal combination of effective morphological fingerprints and biomarkers to correctly predict the class of each oocyte or embryo. The mathematical model of the invention therefore require a pre-set dataset comprising pre-analyzed oocytes, preferably fertilized oocytes, that is to say embryos (to be used as "neighbors"). Such dataset (and ensuing database) can easily be set up by any person skilled in the art, by simply collecting information regarding subjects who have already been implanted for instance. Such a dataset or database can be continuously implemented with new data, i.e. data obtained with new subjects.

In the context of the invention, the data regarding subjects who have already been implanted were classified according to their outcome when setting up a dataset in order to implement the method of the invention, the person skilled in the art may for instance want to use a similar classification.

For instance, the 4 following classes have been used which can be described as follows:

Class E: An oocyte arresting development at the embryo stage after 5 or 6 days. Class B1 : An oocyte yielding to a blastocyst after 5 or 6 days which has been implemented and does not give rise to pregnancy.

Class B2: An oocyte yielding to a blastocyst after 5 or 6 days which has not been yet implemented and/or for which pregnancy is unknown.

Class B3: An oocyte yielding to a blastocyst after 5 or 6 days which has been implemented and gives rise to a pregnancy.

Class B1 , B2, B3 are regrouped as positive examples (i.e. considered "competent"), and Class E as negative control (i.e. "not competent").

The mathematical model of the invention can be defined as follows:

Each given oocyte present in a Learning Set (L) can be represented as vector < X > = ( x₁,x₂,...,x_m) where x₁,x₂,...,x_m design the three morphological criteria (number of cells, regularity of cleavage and degree of fragmentation) and m-3 biological markers (gene or proteins).

For each oocyte present in L, an identification of its nearest neighbor is then performed. Let < X > = (xi,x₂,.-,Xm) and < Y > = (yi,y₂,...,y_m), two oocytes, the HVDM distance between them is given by

Where for each attribute a, morphological (nominal) criterion or biological markers (genes or protein)

if iV y unknown

d_a x, y)= ndm x, y) if a nominal

ndiff _a{x, y_j if a linear

With

Where a_' ^x : design number of instances (oocytes) present in learning set that have value x for the morphological attribute a, a,x,c _: design number of instances (oocytes) in L that have a value x for the morphological attribute a according a given class (B1 , B2, B3 or E) c . : Number of output of the classes presented above: B1, B2, B3, and E.

And for linear attribute A ndiff_a {x, y)

Where design standard deviation for attribute a.

Accuracy of the given algorithm is given by m i, c =Bl E

Acc

N total corresponding to number of oocytes for which Class B (B1 /B2/B3) or Class

E has been correctly predicted in regard to the whole set of oocytes. Each study presented below has been cross-validated in Leave-One-Out Manner. Each model has been learnt on N-1 oocytes labeled B1 / B2/B3 or E and prediction made on the oocyte excluding from the learning set. This Stage has been repeated N times corresponding to the N oocytes of the learning set.

Thus in an embodiment, the method of the invention further using the Heterogeneous Value Difference Metric (HVDM) as herein defined.

Preferably, the method according to the present invention may be useful in assisted reproduction techniques, i.e. wherein oocytes and/or embryos are selected in view of future in vitro fertilization, and further, implantation in a female uterus. For example, said assisted reproduction techniques comprise in vitro fertilization, intracytoplasmic sperm injection, embryo transfer and long- term storage. The method according to the present invention may also be useful oocyte long-term storage for later use. For example, long-term storage can be done by freezing.

Preferably, said method is being performed before implantation, after fertilization, to assist in maximizing the generation of chromosomally normal embryos or to assist in minimizing the generation of chromosomally abnormal embryos. According to another aspect, the present invention is relative to a method of assisted reproduction, comprising a step of evaluating developmental competence of the oocyte or embryo as described above. Said method of assisted reproduction can further include at least one of the steps comprised in the group consisting of: in vitro fertilization, intracytoplasmic sperm injection, embryo transfer and long-term storage.

According to another aspect, the present invention is relative to a method of oocyte or embryo conservation, comprising a step of evaluating developmental competence of said oocyte or embryo as described above. Said method of conservation can include a subsequent step of freezing the selected oocyte or embryo. According to another aspect, the invention is also relative to method for selecting embryo to be transferred to a subject, comprising the comprising a step of evaluating developmental competence of the oocyte giving rise to said embryo or of said embryo as defined above. By the "oocyte giving rise to said embryo", one should understand an oocyte which by fertilization becomes said embryo to be selected.

The invention further pertains to an in vitro method for assessing the efficiency of a treatment of a patient comprising a step of determining the expression level of a combination of at least 2 biological markers in a biological sample, wherein said biological markers are chosen in the list consisting in PTX3, FOS, PLCB1 , POMT2, RPL7, S100A1 3, MERTK, POLR3K and POPDC2. Preferably, said combination of at least 2 biological markers is chosen between the combinations of markers:

- PTX3, FOS, PLCB1 , POMT2, RPL7 and S100A1 3; or

- FOS, MERTK, POLR3K and POPDC2.

Preferably, said treatment is a hormonal treatment. For instance, said treatment may be chosen in the group consisting of: a follicle-stimulating hormone (FSH) treatment, luteinizing hormone (LH) treatment, Gonadotropin-releasing hormone (GnRH) treatment, Gonadotropin-releasing hormone analogs treatment or combination thereof. Alternatively, said treatment may be a contraceptive treatment. The invention further pertains to an in vitro method for identifying compounds capable of modulating developmental competence of an oocyte, comprising the steps of:

a) contacting at least one cumulus cell with a test compound; and b) determining the expression level of a combination of at least 2 biological markers in the cumulus cell of step a), wherein said biological markers are chosen in the list consisting in PTX3, FOS, PLCB1 , POMT2, RPL7, S100A13, MERTK, POLR3K and POPDC2

Preferably, said combination of at least 2 biological markers is chosen between the combinations of markers:

- PTX3, FOS, PLCB1 , POMT2, RPL7 and S100A13; or

- FOS, MERTK, POLR3K and POPDC2.

In a particular embodiment, said method can include a further step c) of comparing the expression level of said combination of at least 2 biological markers of step b) with a reference value. In an embodiment the reference value is the expression level of said combination of markers obtained with cumulus cells not contacted with the test compound. In another embodiment the reference value is the expression level of said combination of markers obtained with cumulus cells contacted with a reference compound.

The invention further pertains to a kit for evaluating the developmental competence of an oocyte or an embryo comprising means for determining the expression level of a combination of at least 2 biological markers in a biological sample, wherein said biological markers are chosen in the list consisting in PTX3, FOS, PLCB1 , POMT2, RPL7, S100A13, MERTK, POLR3K and POPDC2.

Preferably, the combination of at least 2 biological markers is chosen between the combinations of markers: - PTX3, FOS, PLCB1 , P0MT2, RPL7 and S100A13; and

- FOS, MERTK, POLR3K and POPDC2.

By "means for determining the expression level of a combination of at least 2 biological markers", it is herein referred to molecular tool adapted for the determination of the expression level of biological markers. Such molecular tools comprise for instance amplification primers and antibodies, such as monoclonal or polyclonal antibodies.

Such as used here, amplification primers are defined as being a pair of nucleic acid molecules that can respectively pair with the 3' and 5' regions of a gene in a specific manner (positive and negative strands or vice versa) and encompassing a short region of said gene. Generally, amplification primers have a length of 10 to 30 nucleotides and allow amplifying a region of a length comprised between 50 and 200 nucleotides.

The kit may also contain additional elements, such as for example elements needed to practice the method of the invention, or appropriate buffers to be used with the means for determining the expression level of the invention measured in step b) with the expression level of said at least cumulus cells

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : The maximum AUC obtained when mRNA level of biological markers is measured is of 0.75 and is obtained with the biological markers PTX3, FOS, PLCB1 , POMT2, RPL7 and S100A13, and the following morphologic criteria number of cells, and degree of fragmentation.

Figure 2: The maximum AUC obtained when protein level of biological markers is measured is of 0.87 and is obtained with the biological markers FOS, MERTK, POLR3K and POPDC2, and the following morphologic criteria number of cells, regularity of cleavage and degree of fragmentation.

The examples that follow are merely exemplary of the scope of this invention and content of this disclosure. One skilled in the art can devise and construct numerous modifications to the examples listed below without departing from the scope of this invention. EXAMPLES

Materials and Methods Patient selection and IVF treatment

Seventy two patients were included in this study, all undergoing intracytoplasmic sperm injection (ICSI) procedure for male infertility. A group of twenty nine patients was selected for the qPCR analysis on the basis that at least one embryo had reached the blastocyst stage while at least one embryo arrested its development after 6 days of culture. The ovarian stimulation protocol, the ICSI and the embryo culture procedures have been described by Guerif F. et al., 2003.

Cumulus cell recovery and assessment of oocyte and embryo quality

Individual CCs were collected as described in Feuerstein P. et al., 2012.

Assessment of oocyte and embryo quality was described in Feuerstein P. et al., 2012. CCs from an oocyte yielding a blastocyst after 5/6 days of in vitro culture were denominated CCB+ and CCs from an oocyte arresting development at the embryo stage after 5/6 days of in vitro culture were denominated CCB-.

Identification of most differentially expressed genes according to oocyte developmental competence. Microfluidic-based qPCR assay

The following procedures were used in order to comply as far as possible with the Minimum Information for Publication of Quantitative PCR experiments MIQE guidelines (Bustin SA. et al., (2009) The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments, Clinical Chemistry 55: 611 -622).

RNA extraction and cDNA synthesis

Total RNA extraction and removal of genomic DNA were performed using the RNeasy® Micro Kit (Qiagen, Courtaboeuf, France) according to the manufacturer's recommendations. The quality and integrity of RNA samples were assessed using the 2100 Bioanalyser and RNA 6000 Nano LabChip kit series II (Agilent Technologies). Total RNA was quantified using a Nanodrop® ND-1000 spectrophotometer (Nyxor Biotech, Paris, France). Only RNA samples that displayed a RIN (RNA integrity number) greater than or equal to 7 were reverse transcribed to cDNA. The mean quantity of RNA per cumulus was 99 ng (range 21 - 205 ng). Total RNA from each sample was reverse transcribed into cDNA using the iScriptTM cDNA Synthesis kit (Bio-Rad Laboratories, Marnes-la-Coquette, France) with a blend of oligo(dT) and random hexamer primers to provide complete RNA sequence representation. Gene selection, primer design and qPCR design

Among 96 genes selected, 95 were the most differentially expressed transcripts of the microarray study (GSE37277) and RPL19 was selected as reference gene. All primers were designed by DELTAgene Assays with wet validation (Fluidigm Europe B.V., Amsterdam, Netherlands). The level of gene expression was assessed over cumulus cells from 102 mature oocytes, including 54 CCB+ and 48 CCB-.

Microfluidic-based qPCR assay qPCRs were performed on Biomark HD (Fluidigm Europe B.V.), in 2 microfluidic multiplex 96.96 dynamic array chip according to the Genotoul Protocol. Briefly, a 14-cycle pre-amplification reaction was performed for each sample in 5 μΐ by pooling 1.25 μΐ of all primer pairs (each primer at a concentration of 20 nM), 1.3 μΐ of cDNA and 2.5 μΐ of TaqMan® PreAmp Master Mix Kit (Applied Biosystems, 4391128). Pre-amplification was performed with the following thermal cycling conditions: initial activation at 95°C for 10 minutes, followed by 14 cycles of 15 s at 95° C and 4 min at 60° C. All samples of pre-amplified cDNA was diluted by 5- fold in TE-buffer 1X (Dutscher, 091575). Sample Mix contained 440 μΐ of 2X TaqMan Gene Expression Master Mix (Applied Biosystems, 4369510), 44 μΐ of 20X DNA Binding Dye Sample Loading Reagent (Fluidigm, 100-0388), 44 μΐ of 20X EvaGreen (Interchim, Bl 1790) and 132 μΐ of TE buffer 1X. For each sample 6 il of Sample Pre-Mix was added to 2 μΐ of pre-amplified cDNA 1 /5 diluted and 5 μΐ was loaded in Sample Inlets on the chip. For each individual assay, 4 μΐ of 2X Assay Loading Reagent (Fluidigm, 85000736), 2 μΐ of TE Buffer 1X and 2 μΐ of primer (20 nm) were pooled and 5 μΐ were loaded into one of the Assay Inlets on the chip. The Biomark's cycling program (10min Hot Start, 35 eye) with a final melting was used. Pooled human cumulus cells were used to establish the standard curve by 4- fold serial dilutions in TE buffer 1X and repeated for each array chip. A no template control (NTC) and a positive control were included in all assays.

Data analysis

For each gene, the threshold of Cq was defined for the 2 array chips on the first point of the standard curve. All gene analysis with a variation between array chip of the positive control or the first point of the standard curve greater than 0.5 were discarded (see supplemental data). The presence of multiplex on the melting curve was another criterion to discard the data point. Among 96 genes, 74 fulfilled all the criteria. RPL19 and RPL13 were selected by the GeNorm algorithm (Vandesompele J. et al., (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes, Genome Biology 3: RESEARCH0034) as the most stables genes. Thus, the data were normalized to the mean of RPL19 and RPL13 relative concentrations.

Identification of most differentially expressed proteins according to oocyte developmental competence.

Materials and Methods Patient selection and IVF treatment

Seventy two patients were included in this study, all undergoing intracytoplasmic sperm injection (ICSI) procedure for male infertility. Forty three patients were included in RPPA analysis. The ovarian stimulation protocol, the ICSI and the embryo culture procedures have been described by Guerif F, et al., 2003. Cumulus cell recovery and assessment of oocyte and embryo quality

Individual cumulus cells (CCs) were washed in cold phosphate buffer saline (80 lU/ml, SynVitro Hyadase, Medicult, Jyllinge, Denmark). Cumulus were dissociated as described in Feurestein et al., equally divided in 2 tubes and then centrifuged at 300 g for 5 minutes. The supernatants were removed and for one tube the pellet was stored at -80° C until potential RPPA assay and for the other tube the pellet was re-suspended in 50 μΐ of RLT buffer of the RNeasy® Micro Kit (Qiagen, Courtaboeuf, France) before storage at -80° C.

Assessment of oocyte and embryo quality

Assessment of oocyte and embryo quality was described in Feuerstein et al. , 2012. CCs from an oocyte yielding a blastocyst after 5/6 days of in vitro culture were denominated CCB+ and CCs from an oocyte arresting development at the embryo stage after 5/6 days of in vitro culture were denominated CCB-.

Reverse Phase Protein Array (RPPA)

Cumulus cells lysis The lysis buffer (MPER, Thermo Scientific) was supplemented with protease inhibitor (miniComplete, Roche) and phosphatide inhibitor (complete ultra tablet, Roche). CCs were suspended in 40 μΐ of lysis buffer and lysed for 20 min at 4° C on a rotating wheel. After centrifugation, approximately 40 μΐ of protein extract was obtained. Reverse Phase Protein Array design

Cells from 92 individual cumulus from mature oocytes were analysed by RPPA (representing a mean of 14500 cells per cumulus), including 52 CCB+ and 40 CCB-. A two-fold dilution corresponding to a range of equivalent of 0.5 individual CC to 0.25 individual CC were also spotted in two replicates as described in example 2. Reverse Phase Protein Array

RPPA were adapted from Dupuy L. et al. , 2009. Briefly, desiccated nitrocellulose- coated slides (Fast Slides, Whatman, Maidstone, UK) were printed with samples, using a 32-pin manual arrayer (G lass Slide Microarrayer, VP478, V&P Scientific, San Diego, CA, USA) and desiccated again overnight. According to the manufacturer's indications, 3-1 3 nl_ of sample were spotted on Fast-slide per array pin touch. The immunodetection procedure was adapted from Chan SM et al, 2004. All antibodies were pre-cleared in FBS for 1 h at 37° C prior to use. After rehydration with PBS- Tween 20 0.1 % (PBST), slides were blocked overnight at 4° C with 3% casein in PBST. Slides were probed with appropriate antibodies 3h at room temperature (RT) in PBST with 20% FBS (PBST-FBS). Slides then were washed three times for 5min with three rinses with PBST between washes and then probed for 45min at RT with Peroxidase-conjugated F(ab')2 Fragment Donkey anti-rabbit IgG (1 :2000) (Jackson ImmunoResearch Laboratories ref: 711 -036-152) or Peroxidase- conjugated F(ab')2 Fragment Goat anti-Mouse IgG (1 :2000) (Jackson ImmunoResearch Laboratories ref: 115-036-072) diluted in PBST-FBS. Slides were washed as previously described for signal amplification and slides were subsequently incubated for 10min at RT with BioRad Amplification Reagent (Amplified Opt CN Substrate Kit, BioRad, Hercules, CA, USA). Then, slides were rinsed six times, washed once for 5min in PBST with 20% DMSO (PBST-DMSO) and washed twice for 5min and rinsed in PBST as above. Slides were then probed for 1 h at RT with streptavidin AlexaFluors 680 conjugate (0.2 mg/mL) (AF680- streptavidin) (Invitrogen LifeTechnologies) in PBST-FBS. Finally, slides were air- dried by centrifugation at 2500 rpm for 15min and scanned at 700 nm with the Odyssey IR imaging system (LI-COR Biosciences, Lincoln, NE, USA) at a 42 μιη resolution.

Data analysis

Scanned images of arrays were analysed with GenePix Pro6.0 software (Molecular Devices, Sunnyvale, CA, USA). The mean value of intensity was determined for each spot with subtraction of the local background intensity. The mean value of signals was normalized according to the mean value of signals of the reference protein (i.e. VCL). The outliers corresponding to the maximum and the minimum values in each group were deleted for the statistical analysis of RPPA results.

Results Conclusions

Among 95 genes differentially expressed according the oocyte developmental competence by microarray, the Inventors showed that the genes CUL4B, LGALS9, POLR3K, NFYB, TRSPAP1 and MERTK were differentially expressed with a statistical difference. Interestingly, they also demonstrated that the proteins RGS2, POLR3K and MERTK were differentially expressed according the oocyte developmental competence. Their strategy was based on two stages: 1 ) identify new potential biomarkers of oocyte developmental competence from results of a transcriptomic approach on individual CCs by high throughput qPCR; 2) evaluated the level of expression of the proteins coded by some of these genes in regard to oocyte quality. Most of the recent studies focused on the somatic cells surrounding the oocyte which identified through microarray approach potential biomarkers of oocytes quality validated their results by qPCR (Assou S. et al.; Human cumulus cells as biomarkers for embryo and pregnancy outcomes, Molecular Human Reproduction 2010, 16:531 -538). However, these two approaches have different output and while microarray can highlight a large number of differentially expressed genes, the validation by qPCR have to focus on a part of them. The Inventors used BiomarkHD system to extend the potential biomarkers list by assessing 95 genes of the 308 differentially expressed genes according oocyte developmental competence identified by microarray in a previous study (Feuerstein et al, 2012). Moreover, they used independent set of samples to ensure that the expression profile was really correlated with the situations being compared and was not sample dependent (van Montfoort AP et al. (2008) Differential gene expression in cumulus cells as a prognostic indicator of embryo viability: a microarray analysis. Molecular Human Reproduction 14: 157-168). This approach led to identified "only" 10 new potentials biomarkers over the 94 selected genes. This apparent 'loss' of differentially expressed genes may partly be attributed to technical differences between microarray and qPCR approaches (Morey J, et al. (2006) Microarray validation: factors influencing correlation between oligonucleotide microarrays and real-time PCR. Biological Procedures Online 8: 175-193) and partly be the result of inter-patient variation due to the utilization of independent set of samples between the two approaches.

Transcriptomic analyses do not warrant information about the ultimate effectors in cells which remain based on protein -protein interactions or cellular signalling pathways. However, few studies addressed on the CCs proteome according to the oocyte quality mainly because of a lack of sensitivity of the techniques. Recently, 2D electrophoresis was used to analysed the proteome of pooled CCs according ovarian protocol stimulation [Hamamah] and MALDI-TOF MS was used to study the lipid profile of cumulus cells and potentially be used as a supporting tool to predict pregnancy (Montani D, et al. (2012) The follicular microenviroment as a predictor of pregnancy: MALDI-TOF MS lipid profile in cumulus cells. Journal of Assisted Reproduction and Genetics 29: 1289-1297). Thereby, RPPA was used to detect and analyse potential biomarkers related to oocyte developmental competence through a non-invasive procedure involving individual cumulus cells in human.

Although mRNA abundance does not always correlate with the level of the corresponding proteins (Shankavaram UT, et al. (2007) Transcript and protein expression profiles of the NCI -60 cancer cell panel: an integromic microarray study. Molecular Cancer Therapeutics 6: 820-832.), FOS was selected in both proteins to be targeted among the potential biomarkers which were identified at mRNA level.

In conclusion, the Inventors have surprisingly identified new combinations of biomarkers which are relevant at the DNA and protein levels for determining the oocyte developmental competence.

Claims

An in vitro method for evaluating developmental competence of an oocyte or an embryo from a subject, comprising the step of determining the expression level of a combination of at least 2 biological markers in a biological sample, wherein said biological markers are chosen in the list consisting in PTX3, FOS, PLCB1 , POMT2, RPL7, S100A13, MERTK, POLR3K and POPDC2.

The method according to claim 1 , wherein said combination of at least 2 biological markers is chosen between the combinations of markers:

- PTX3, FOS, PLCB1 , POMT2, RPL7 and S100A13; and

- FOS, MERTK, POLR3K and POPDC2.

The method according to claim 2, wherein the expression level of the combination of biological markers PTX3, FOS, PLCB1 , POMT2, RPL7 and S100A13 is determined by measuring the expression level of the corresponding mRNA transcripts.

The method according to claim 3, wherein the expression level of the mRNA transcripts corresponding to the combination of biological markers PTX3, FOS, PLCB1 , POMT2, RPL7 and S100A13 is measured by quantitative polymerase chain reaction.

The method according to claim 2, wherein the expression level of the combination of biological markers FOS, MERTK, POLR3K and POPDC2 is determined by measuring the expression of the corresponding peptides.

6. The method according to claim 5, wherein the expression level of the peptides corresponding to the combination of biological markers FOS, MERTK, POLR3K and POPDC2 is measured by Reverse Phase Protein Array.

7. The method according to any of claims 1 to 6, wherein the biological sample comprises or consists in cumulus cells from said subject.

The method according to any of claims 1 to 7, further comprising a step of assessing the status of the oocyte or embryo status regarding at least 2 criteria chosen in the list of morphologic criteria consisting of: number of cells, regularity of cleavage and degree of fragmentation.

9. The method according to claim 8, comprising the steps of: determining the expression level of the combination of biological markers PTX3, FOS, PLCB1 , POMT2, RPL7 and S100A1 3 by measuring the expression level of the corresponding mRNA transcripts, and

assessing the status of the oocyte or embryo status regarding the morphologic criteria of number of cells and degree of fragmentation.

10. The method according to claim 8, comprising the steps of:

- determining the expression level of the expression level of the combination of biological markers FOS, MERTK, POLR3K and POPDC2 is determined by measuring the expression of the corresponding peptides, and

- assessing the status of the oocyte or embryo status regarding the morphologic criteria of number of cells and degree of fragmentation.

1 1 . A method for evaluating the efficiency of a fertility treatment of a patient comprising a step of determining the expression level of a combination of at least 2 biological markers in a biological sample, wherein said biological markers are chosen in the list consisting in PTX3, FOS, PLCB1 , POMT2, RPL7, S100A1 3, MERTK, POLR3K and POPDC2.

12. An in vitro method for identifying compounds capable of modulating developmental competence of an oocyte, comprising the steps of:

a) contacting at least one cumulus cell with a test compound; and b) determining the expression level of a combination of at least 2 biological markers in the cumulus cell of step a), wherein said biological markers are chosen in the list consisting in PTX3, FOS, PLCB1 , POMT2, RPL7, S100A1 3, MERTK, POLR3K and POPDC2

1 3. The in vitro method of claim 12, wherein said combination of at least 2 biological markers is chosen between the combinations of markers:

- PTX3, FOS, PLCB1 , POMT2, RPL7 and S100A1 3; and

- FOS, MERTK, POLR3K and POPDC2.

1 . Kit for evaluating the developmental competence of a oocyte or an embryo comprising means for determining the expression level of a combination of at least 2 biological markers in a biological sample, wherein said biological markers are chosen in the list consisting in PTX3, FOS, PLCB1 , POMT2, RPL7, S100A1 3, MERTK, POLR3K and POPDC2.

1 5. The kit of claim 14, wherein said combination of at least 2 biological markers is chosen between the combinations of markers:

- PTX3, FOS, PLCB1 , POMT2, RPL7 and S100A1 3; and

- FOS, MERTK, POLR3K and POPDC2.