WO2011000805A1

WO2011000805A1 - Biomarkers of oocyte competency and method of use

Info

Publication number: WO2011000805A1
Application number: PCT/EP2010/059147
Authority: WO
Inventors: Nathalie Ledee; René FRYDMAN
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale)
Priority date: 2009-06-29
Filing date: 2010-06-28
Publication date: 2011-01-06

Abstract

The present invention relates to a method for determining the implantation potential of an oocyte from a female subject, comprising measuring, in the follicular fluid from which the oocyte is derived, the level of at least one of IL-7, IL- 17 and IL- 15, and optionally G-CSF, and determining the implantation potential of the oocyte from the level of follicular fluid IL-7 and/or IL- 17 and/or IL- 15 and optionally G-CSF.

Description

BIOMARKERS OF OOCYTE COMPETENCY AND METHOD OF USE

FIELD OF THE INVENTION

The invention relates to non invasive biomarkers of oocyte competency useful to predict implantation and birth in controlled natural cycle.

BACKGROUND OF THE INVENTION

Assisted reproductive technologies (ART) such as in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI) are currently used for treating infertility conditions. Ovarian stimulation has become a key component of said procedures. Its aim is to increase the number of oocytes in order to compensate for inefficiencies of the IVF procedure enabling the selection of one or more embryos for transfer. The most frequently used stimulation protocol combines high doses of follicle-stimulating hormone (FSH) with a long gonadotrophin-releasing hormone (GnRH) agonist pituitary suppression regimen. These medication regimens are complex, very expensive and require weeks of daily injections and intense ovarian response monitoring. Such regimens are associated with the risk of complications such as ovarian hyperstimulation syndrome. Other negative effects include emotional stress, high drop-out rates and abdominal discomfort. Moreover, uncertainties remain regarding long-term health risks (such as ovarian cancer) and an increased incidence of low birthweight and birth defects in the offspring conceived following IVF treatment (Verberg et al., 2009, Human Reproduction Update, 15(1): 13-29). Despite all these drawbacks, such policy is still applied because we are still facing in reproductive medicine with relatively low rates of implantation per transferred embryo. No criteria are available to define individual oocyte quality. Main rational for ovarian hyperstimulation is to select through subjective microscopic methods, such as embryo morphology, cleavage rate, appearance of pronuclei, fragmentation rate, blastomeres number and embryo symmetry, the best embryos in a significant cohort of generated embryos so as to increase their potentiality to implant. Such policy has as consequence to induce a huge wastage of oocytes. Only 5% of oocytes collected allow the birth of healthy baby (Patrizio et al. 2007 Reprod Biomed Online 14(1): 92-5). Accurate and extensive observation of the embryos during their preimplantation in vitro development even useful is insufficient to predict their potentiality for implantation (Guerif et al. 2007 Hum Reprod 22(7): 1973-81).

In consequence, new non invasive strategies are emerging to understand embryo and oocyte expressions and behaviors, and to improve embryo selection with the objective of successful single embryo transfer. Mass spectrometry, SELDI-TOF (Katz-Jaffe et al. 2006 Fertil Steril 85(1): 101-7; Katz-Jaffe et al. 2006 Fertil Steril 86(3): 678-85), near-infrared and Raman spectroscopy (SeIi et al. 2007 Fertil Steril 88(5): 1350-7; Scott et al. 2008 Fertil Steril 90(1): 77-83) and microarray of proteins (Dominguez et al. 2009 Reprod Sci 16(2): 188-90) are explored as non invasive techniques. Immunological expressions of soluble HLA-G or ICAM-I in embryo supernatants or follicular fluids are under investigation to determine whether they can be biomarkers of oocyte competency (Borgatti et al. 2008 PLoS ONE 3(12): e3970; Tabiasco et al. 2009 Reprod Biomed Online 18(3): 374-81). Therefore, there is a need for a non invasive method which can indicate the implantation potential of an oocyte before fertilization, so as to increase the chances of successful implantation.

The inventors previously suggested that the measurement of the level of follicular fluid granulocyte colony stimulating factor (G-CSF) can be used to determine the implantation potential of an oocyte in a context of ovarian hyperstimulation (Ledee et al 2008 Hum Reprod 23(9): 2001-9, WO2008/009705).

In the present invention, the inventors suggest that Interleukine-7 (IL-7), Interleukine-17 (IL-17) and combination of at least one of IL-7, IL-17 and IL-15 with G-CSF can be used as novel non invasive biomarkers of oocyte competence in a context of controlled natural cycle. In particular, the inventors demonstrated that G-CSF and IL-15, IL-7, IL-17 are novel, valuable and non invasive biomarkers of oocyte competency and can be used to predict not only implantation but also birth in controlled natural cycle.

SUMMARY OF THE INVENTION

One object of the invention is a method for determining the implantation potential of an oocyte from a female subject in a natural modified cycle, comprising: - measuring, in the follicular fluid from which the oocyte is derived, the level of at least one of IL-7 and IL- 17 and

- determining the implantation potential of the oocyte from the level of follicular fluid IL-7 and/or IL- 17.

In one embodiment of the invention, the oocyte having a high level of IL-7 and/or IL- 17 in the follicular fluid gives rise to an embryo with a high implantation potential.

In another embodiment of the invention, the oocyte having a low level of IL-7 in the follicular fluid gives rise to an embryo with a low implantation potential.

In another embodiment of the invention, the method as described here above further comprises

- measuring, in the follicular fluid from which the oocyte is derived, the level of G-CSF and optionally IL- 15, and

- determining the implantation potential of the oocyte from the level of follicular fluid G- CSF and at least one of IL-7, IL- 17 and IL-15.

In another embodiment, the invention also relates to a method for determining the implantation potential of an oocyte from a female subject in a natural modified cycle, comprising:

- measuring, in the follicular fluid from which the oocyte is derived, the level of G-CSF and the level of IL- 15 , and

- determining the implantation potential of the oocyte from the level of follicular fluid G- CSF and IL-15.

In another embodiment of the invention, the invention also relates to a method for determining the implantation potential of an oocyte from a female subject in a natural modified cycle, comprising

- measuring the embryo morphology score, said embryo being derived from said oocyte, and

- determining the level of follicular fluid IL-15.

According to the invention, the oocyte having a low level of IL-15 in the follicular fluid and a good embryo score regarding its morphology gives rise to an embryo with a high implantation potential. To the opposite, the oocyte having high level of IL-15 and a low score regarding the embryo morphology give rise to an embryo with a low implantation potential.

In one embodiment of the invention, the follicular fluid is obtained from a follicular aspirate.

In another embodiment of the invention, the levels of at least one of IL-7, IL- 17 and IL- 15 and the level of G-CSF are measured within 20 hours after collection of the follicular fluid.

In another embodiment of the invention, the levels of at least one of IL-7, IL- 17 and IL- 15 and the level of G-CSF are measured using an immunoassay.

In another embodiment of the invention, the levels of at least one of IL-7, IL- 17 and IL- 15 and the level of G-CSF are measured using an ELISA.

In another embodiment of the invention, the levels of at least one of IL-7, IL- 17 and IL- 15 and the level of G-CSF are measured using a luminex assay.

In another embodiment of the invention, the levels of at least one of IL-7, IL- 17 and IL- 15 and the level of G-CSF are measured by determining the levels of IL-7, IL- 17 and IL- 15 and G-CSF mRNA.

Another object of the invention is a kit consisting of at least one reagent suitable for detection of at least one of IL-7, IL- 17 and IL- 15 level and at least one reagent suitable for detection of G-CSF level.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the unexpected finding by the inventors that there is a strong correlation between the level of G-CSF, IL-7, IL- 17 and IL- 15 present in the follicular fluid of the follicle from which an oocyte is derived and a high or low implantation potential of an embryo obtained by fertilisation of said oocyte in natural modified cycle. The method disclosed in the present invention thus provides the possibility to determine with accuracy the implantation potential of the oocyte collected in natural modified cycle, without interference to the embryo or oocyte. The invention thus relates to methods and kits which can be used to predict the outcome of assisted fertilisation in a female subject. The methods and kits of the invention can also be used in a method of fertilisation treatment, to improve implantation.

As used herein, the term "female subject" refers to any mammalian female including for example a human female, a mare or a cow. Preferably, the female subject is a human female.

As used herein, the term "assisted fertilisation" refers to ex vivo fertilisation methods where the oocytes are fertilised outside the female body, such as IVF or ICSI.

As used herein, the term "IL-7" has its general meaning in the art. IL-7 is a gamma chain cytokine sharing with IL-15, 11-9, IL-4 and IL-2 a common receptor interleukin-2 gamma chain and was mainly describes in the past decade for its critical role on lymphopoiesis in the thymus and peripheral T cell homeostasis.

As used herein, the term "IL- 17" has its general meaning in the art. IL- 17 is a family of structurally related cytokines. Representative examples of IL- 17 cytokines include, but are not limited to, IL-17/IL17A, IL-17B, IL-17C, IL-17D, and IL-17F.

As used herein, the term "IL-15" has its general meaning in the art. As described above,

IL-15 shares with IL-7 a common receptor interleukin-2 gamma chain. This cytokine induces cell proliferation of natural killer cells.

As used herein, the term "G-CSF" has its general meaning in the art. G-CSF has been previously shown to be secreted by granulosa cells at the time of ovulation, followed in the luteal phase by a secretion within the endometrium itself and then the placenta during gestation.

One object of the invention is the use of IL-7, IL- 17, IL-15 and/or G-CSF as biomarkers of the implantation potential of an oocyte in the context of controlled natural cycle. Another object of the invention is the use of IL-7, IL-17, IL-15 and/or G-CSF as biomarkers of birth rate in the context of controlled natural cycle.

As used herein, the term "controlled natural cycle" or "natural modified cycle" refers to ovarian cycle in which previous ovarian desensibilisation has not been applied (GnRH agonist), in which FSH and LH administration do not begin with the beginning of the cycle and in which the physiological dominance of one oocyte over its own cohort is respected. One object of the invention is an in vitro method for determining the implantation potential of an oocyte from a female subject, comprising measuring, in the follicular fluid from which the oocyte is derived, the level of at least one of IL-7 and IL- 17, and determining the implantation potential of the oocyte from the level of follicular fluid IL-7 and/or IL- 17. As used herein, the term "implantation potential" refers to the capacity of the oocyte to implant into the uterus. A low implantation potential is a probability of implantation of 10%, 9%, 8% or less. A high implantation potential is a probability of implantation of more than 30%, preferably more than 35%. In one embodiment of the invention, said method as described here above is also predictive of subsequent pregnancy and birth.

A low pregnancy rate is a probability of less than 5%

A high pregnancy rate is a probability of more than 30%

A low birth rate is a probability of less than 5%

A high birth rate is a probability of more than 30%

The inventors demonstrated that IL-7, IL- 17 are specific biomarkers of the implantation potential of an oocyte in the context of a controlled natural cycle, as they are not detectable in follicular fluid in the context of ovarian hyperstimulation.

According to the invention, an oocyte from a follicle having a high level of IL-7 and/or IL- 17 in the follicular fluid gives rise to an embryo with the greatest implantation potential. According to the invention, an oocyte from a follicle having a low level of IL-7 in the follicular fluid gives rise to an embryo with the lowest implantation potential and eventually is predictive of no subsequent pregnancy and birth.

According to the invention, an oocyte from a follicle having a detectable level of IL- 17 gives rise to an embryo with a high implantation potential.

In another embodiment of the invention, said method further comprises measuring, in the follicular fluid from which the oocyte is derived, the level of G-CSF and determining the implantation potential of the oocyte from the level of follicular fluid G-CSF and at least one of IL-7, IL- 17 and IL-15. According to the invention, an oocyte from a follicle having a high level of G-CSF in the follicular fluid gives rise to an embryo with the greatest implantation potential.

According to the invention, an oocyte from a follicle having a low level of G-CSF and a low level of IL-7 in the follicular fluid gives rise to an embryo with the lowest implantation potential and eventually is predictive of no subsequent pregnancy and birth.

According to the invention, an oocyte from a follicle having a high level of G-CSF and a high level of IL-7 in the follicular fluid gives rise to an embryo with the greatest implantation potential and eventually is predictive of very high chance of pregnancy and birth.

According to the invention, an oocyte from a follicle having a high level of G-CSF and a low level of IL- 15 in the follicular fluid gives rise to an embryo with the greatest implantation potential and eventually is predictive of very high chance of pregnancy and birth.

The level of follicular fluid IL-7, IL- 17, IL- 15 or G-CSF (FF IL-7, FF IL- 17, FF IL- 15 and FF G-CSF) is preferably measured within the day of oocyte collection. Generally, follicular aspiration is guided using transvaginal sonography after local or general anaesthesia. The follicular fluid corresponding to the dominant ovarian follicle visualised through vaginal sonography is aspirated individually. The capture of each oocyte does not require any other manipulation because the follicular fluid, which surrounds the oocyte, is aspirated along with the oocyte. Inspection of the follicular fluid under microscope allows immediate identification of the presence of the oocyte. When collecting the follicular fluid and the oocyte, the follicular fluid is separated and the level of IL-7, IL- 17, IL- 15 and G- CSF can be measured.

In one embodiment of the invention, the level of FF IL-7, FF IL- 17, FF IL- 15 and FF G- CSF is measured 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 hours after oocyte and follicular fluid collection, or within a time between any two of the aforementioned values. Preferably the level of FF IL-7, FF IL- 17, FF IL- 15 and FF G-CSF is measured within 1 to 6 hours after oocyte collection, before fertilisation of the oocyte. The level of FF IL-7, FF IL- 17, FF IL- 15 and FF G-CSF associated with an oocyte can be measured using any suitable quantitative assay known by the person skilled in the art. The measuring may be performed, for example, using a method selected from biochemical assay (e.g., solid or liquid phase immunoassay), surface plasmon resonance, fluorescence resonance energy transfer, fluorescence quenching, and fluorescence polarisation. Such techniques are well known in the art and are briefly described herein below.

Biochemical assays generally rely on the immobilisation of an analyte component, for example, to a membrane or other solid support, and exposure to a ligand. After washing away excess ligand, bound ligand is detected by immunoassay, or by using labelled ligand (e.g., radio-labelled ligand, fluorescently labelled ligand, particulate labelled ligand etc.). Methods to determine and obtain ligands which bind with high affinity to a specific analyte are also available in the art; see for example WO89/09088 entitled "Paralog Affinity Chromatography". In an example of an immunoassay, antibodies against IL-7, IL- 17, IL- 15 and/or G-CSF may be immobilised onto magnetic beads and exposed to a sample of follicular fluid. Bound IL-7, IL-17, IL-15 and/or G- CSF can be detected using primary and secondary antibody immunoassays. Typically, an immunoassay is calibrated using a set of standards. Solid phase immunoassays are described for example in US 4,376,110. Variations of the immunoassays within the scope of the invention include any competitive or immunometric assay format using anti-IL-7, anti-IL-17, anti-IL-15 and/or anti-G-CSF antibodies, for instance RIA (radio-immunoassay), ELISA (enzyme-linked immunosorbent assay), ELISPOT (enzyme-linked immunosorbent spot) or Luminex (bead-based multiplex sandwich immunoassay).

In one embodiment of the invention, the levels of FF IL-7, FF IL-17, FF IL-15 and FF G- CSF are measured by using Luminex technology. Luminex is a highly sensitive method for measuring simultaneously the levels of specific components in a system. It makes use of solid phase, colour (dye) coded microspheres that are small enough to behave almost as a solution in a liquid. Each microsphere is coated with an antibody, or other ligand-binding reagent specific for the detected components (e.g. FF IL-7, FF IL-17, FF IL-15 and FF C- GSF). The components of the sample are captured and detected on the microspheres. Within an analyzer, lasers excite the internal dyes that identify each microsphere particle, and also any reporter dye captured during the assay. Many readings are made on each bead set, to validate the results. In this way, a sensitive multiplex assay is made that is both rapid and precise.

Examples of kits allowing the measuring of the levels of FF IL-7, FF IL- 17, FF IL- 15 and FF-G-CSF include, but are not limited to, kits manufactured by Biorad® or R&D®. such as the Bio-Plex Pro Human Cytokine IL-7, IL- 17, IL-15 or G-CSF sets (171-B5007M, 171-G5014M, 171-B5013M and 171-B5017M Biorad®) and Human G-CSF or IL- 17 Fluorokine MAP (LUH214 and LUH 317 R&D®) .

For the purposes of this invention, the term "antibody", unless specified to the contrary, includes monoclonal antibodies, polyclonal antibodies, and fragments of whole antibodies which retain their binding activity for a target antigen. Such fragments include Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies.

Antibodies against FF IL-7, FF IL- 17, FF IL-15 or FF G-CSF may be monoclonal or polyclonal antibodies. Monoclonal antibodies may be prepared by conventional hybridoma technology using the proteins or peptide fragments thereof, as an immunogen. Polyclonal antibodies may also be prepared by conventional means which comprise inoculating a host animal, for example a rat or a rabbit, with a peptide of the invention and recovering immune serum.

Alternatively, levels of FF IL-7, FF IL- 17, FF IL-15 and FF G-CSF may be estimated by analysing the levels of FF IL-7, FF IL- 17, FF IL-15 and FF G-CSF mRNA in the granulosa cells. Granulosa cells around the corona radiata may be stored at the stage of the decoronisation of each oocytes and be stored in RNA stabiliser (e.g. at 8O⁰C) until assay. Probes for the IL-7, IL- 17, IL-15 and G-CSF gene may be designed, for example for use in a nucleic acid (PCR) amplification assay and/or hybridisation. Methods and conditions for performing a PCR and hybridisation reactions are known in the art, and can be found, for example, in Molecular Cloning: A Laboratory Manual (Third Edition) (Joseph Sambrook, Peter MacCallum, David Russell, Cold Spring Habor Laboratory Press) or could be performed by a quantigene plex assay, which is designed to quantitate multiple target- specific RNA molecules (Panomics).

A surface plasmon resonance assay may, alternatively, be used as a quantitative method to measure the level of IL-7, IL-17, IL-15 and G-CSF in a follicular fluid sample. A chip- bound anti-IL-7, anti-IL-17, anti-IL-15 and anti-G-CSF antibody is challenged with a follicular fluid and the surface plasmon resonance measured. Binding reactions are performed using standard concentrations to arrive at the levels of IL-7, IL- 17, IL- 15 and G- CSF in the follicular fluid.

FRET (fluorescence resonance energy transfer) may also be used to measure the level of IL-7, IL- 17, IL- 15 and G-CSF in a follicular fluid sample. IL-7, IL- 17, IL- 15 and G-CSF and antibodies anti-IL-7, anti-IL-17, anti-IL-15 and anti-G-CSF are labelled with a complementary pair of donor and acceptor fluorophores. While bound closely together by the ligand/antibody interaction, the fluorescence emitted upon excitation of the donor fluorophore will have a different wavelength than that emitted in response to that excitation wavelength when the ligand/antibody complexes are not bound, providing for quantification of bound versus unbound molecules by measurement of emission intensity at each wavelength. Binding reactions can be compared with a set of standards to arrive at the level of IL-7, IL- 17, IL- 15 and G-CSF in the follicular fluid.

BRET (bioluminescence resonance energy transfer) may also be used to measure the level of IL-7, IL-17, IL-15 and G-CSF in a follicular fluid sample. Light is emitted by an acceptor when in close proximity to the donor, i.e., when a ligand/antibody interaction complex is formed. By comparing the interaction with a set of standards, the level of IL-7, IL-17, IL-15 and G-CSF in the follicular fluid is determined.

Fluorescence quenching fluorescence similarly provides a measurement of IL-7, IL-17, IL- 15 and G-CSF levels. Generally, a decrease in fluorescence of the labelled antibody is indicative that the ligand bearing the quencher has bound. Of course, a similar effect would arise when a ligand is fluorescently labelled and the antibody bears the quencher. By comparing the interaction with a set of standards, the level of IL-7, IL-17, IL-15 and G- CSF in a follicular fluid sample can be measured.

Fluorescence polarisation measurement can also determine the level of IL-7, IL-17, IL-15 and G-CSF in a follicular fluid sample. Ligand/antibody complexes would have higher polarisation values than uncomplexed, labelled antibody. This form the basis for determining the levels of IL-7, IL-17, IL-15 and G-CSF in a follicular fluid sample, which measurements are typically performed concurrent with a set of standard IL-7, IL-17, IL-15 and G-CSF concentrations.

Others methods than can be used for quantitatively assaying IL-7, IL-17, IL-15 and G-CSF in the FF include mass spectrometry (MS), high performance liquid chromatography (HPLC), HPLC/MS, HPLC/MS/MS, capillary electrophoresis and rod or slab gel electrophoresis associated with image analysis. Such techniques are well known in the art as described, for example, in Modern HPLC for Practicing Scientists (Dong, M, Wiley- lnterscience, June 2006), Tandem Mass Spectrometry (McLafferty F. W. John Wiley & Sons Inc, November, 1983), Mass Spectrometry for Biotechnology (Siuzdak, G., Academic Press, February 1996), Clinical Applications of Capillary Electrophoresis (Methods in Molecular Medicine, Palfrey S. M., Humana Press, June 1999), Handbook of Capillary Electrophoresis, Second Edition, (Landers J. P. CRC; December 1996), High- Resolution Electrophoresis and Immunofixation: Techniques and Interpretation (Keren, D. F. Hodder Arnold, January, 1994).

Once the levels of FF IL-7, FF IL- 17, FF IL- 15 and/or FF G-CSF have been measured in the dominant follicle from a single subject, the results may be used to establish the relative implantation potential of the embryo obtained by fertilization of said oocyte. The level of FF IL-7, FF IL- 17, FF IL- 15 and/or FF G-CSF may be used to determine over few natural modified cycles, whether all, some or none of the oocytes in natural modified cycle will after fertilisation establish implantation in a female subject undergoing assisted fertilisation treatment. In addition, the level of FF IL-7, FF IL- 17, FF IL- 15 and/or FF G-CSF may be used to determine whether all, some or none of the embryos will implant in a female subject undergoing assisted fertilisation.

In our studies, we have measured levels of FF IL-7, FF IL-17, FF IL-15 and/or FF G-CSF using immunoassays, in particular using Luminex technology from Biorad.

We have found that those embryos derived from oocytes having a concentration of FF G- CSF equal to or less than 9 pg/ml defined the group with the higher predictive value of failure of implantation, pregnancy and birth. In contrast, embryos derived from oocytes having a concentration of FF G-CSF above 12 pg/ml defined the group with the higher predictive value of success of implantation, pregnancy and birth.

We have found that those embryos derived from oocytes having a concentration of FF IL- 17 that can not be detected defined the group with the higher predictive value of failure of implantation, pregnancy and birth. In contrast, embryos derived from oocytes having a concentration of FF IL-17 that can be detected defined the group with the higher predictive value of success of implantation, pregnancy and birth. We have found that those embryos derived from oocytes having a concentration of FF IL-7 equal to or less than 11 pg/ml defined the group with the higher predictive value of failure of implantation, pregnancy and birth. In contrast, embryos derived from oocytes having a concentration of FF IL-7 above 30 pg/ml defined the group with the higher predictive value of success of implantation, pregnancy and birth.

We have found that those embryos derived from oocytes having a high level of G-CSF (above 12 pg/ml) and a low level of IL-15 (less than 7 pg/ml) in the follicular fluid define the group with the higher predictive value of success of implantation, pregnancy and birth. Thus, those embryos derived from oocytes having a concentration of FF IL-15 above 7 pg/ml and a concentration of G-CSF less than 12 pg/ml defined the group with the higher predictive value of failure of implantation, pregnancy and birth. In contrast, embryos derived from oocytes having a concentration of FF IL-15 equal to or less than 7 pg/ml and a concentration of FF G-CSF above 12 pg/ml defined the group with the higher predictive value of success of implantation, pregnancy and birth.

The persons skilled in the art will appreciate that although in our research we have determined the "threshold" levels of FF IL-7, FF IL- 17, FF IL-15 and FF G-CSF below which embryos are not implanted (and above which patients have significantly improved probability of implantation), the value is a statistical measure and other measurements and thresholds can be used. In practicing the invention, it is most important to achieve consistency of assay, and so each individual practitioner (or assisted fertilisation team) will be capable of establishing their own particular assay method and determining their own threshold level. This could be established by first conducting a historical study on samples from previous patients.

Thus, the level of FF IL-7, FF IL-17, FF IL-15 and FF G-CSF mentioned above represents the measure we have used in our studies as a suitable limit. However, if levels of FF IL-7, FF IL-17, FF IL-15 and FF G-CSF were to be measured in any of the other ways mentioned above, it would be desirable to conduct, using routine procedures, a control using our method of assay in order to determine the relationship between our results and the results of other methods, in order to make direct comparisons. According to one embodiment of the invention, an embryo derived from an oocyte in a context of controlled natural cycle where a level of FF IL- 17 in its follicle is not detected, is predicted to have a low implantation potential, pregnancy rate and birth rate.

According to another embodiment of the invention, an embryo derived from an oocyte in a context of controlled natural cycle where a level of FF IL- 17 in its follicle is detected is predicted to have a high implantation potential, pregnancy rate and birth rate.

According to one embodiment of the invention, an embryo derived from an oocyte in a context of controlled natural cycle where a level of FF IL-7 in its follicle is equal to or less than 11.3 pg/ml, 11.2 pg/ml, 11 pg/ml, 10.8 pg/ml, 10.6 pg/ml, 10.4 pg/ml, 10.2 pg/ml, 10 pg/ml, 9.8 pg/ml, 9.6 pg/ml, 9.4 pg/ml, 9.2 pg/ml, 9 pg/ml, 8.8 pg/ml, 8.6 pg/ml, 8.4 pg/ml, 8.2 pg/ml, 8 pg/ml, or a level between any two of the aforementioned values, is predicted to have a low implantation potential, pregnancy rate and birth rate.

According to another embodiment of the invention, an embryo derived from an oocyte in a context of controlled natural cycle where a level of FF IL-7 in its follicle is equal to or more than 32 pg/ml is predicted to have a high implantation potential, pregnancy rate and birth rate.

According to another embodiment of the invention, an embryo derived from an oocyte in a context of controlled natural cycle where a level of FF IL-7 in its follicle is less than 32 pg/ml but more than 11.3 pg/ml is predicted to have a medium implantation potential, which means that it is likely to be implanted.

"Likely to be implanted" means a higher chance of success than no certainty of implantation; a likely potential of implantation means a probability of implantation of 15 % to 25 %.

According to one embodiment of the invention, an embryo derived from an oocyte in a context of controlled natural cycle where a level of FF IL- 15 in its follicle is equal to or less than 7 pg/ml, 6.9 pg/ml, 6.8 pg/ml, 6.7 pg/ml or a level between any two of the aforementioned values, is predicted to have a high implantation potential, pregnancy rate and birth rate if the embryo morphology score is higher than 14 and/or the level of FF G- CSF in its follicle is equal to or more than 12 pg/ml.

According to another embodiment of the invention, an embryo derived from an oocyte in a context of controlled natural cycle where a level of FF IL- 15 in its follicle is greater than 7 pg/ml is predicted to have a low implantation potential, pregnancy rate and birth rate if the embryo morphology score is less than 14 and/or the level of FF G-CSF in its follicle is less than 12 pg/ml.

Embryos morphology scores are defined the second day after fertilization (d2) as number of blastomeres x (5-embryo grade), wherein the embryo grade is defined as follows:

Grade 1: 10% or less of fragmentation,

Grade 2: 10-30% of fragmentation,

Grade 3: 30-50% of fragmentation,

Grade 4: over 50% of fragmentation, embryo was downgraded when blastomeres were unequal in size. Optimal category is defined as those with 4-5 cells and 10% or less of fragmentation and equal cells.

According to one embodiment of the invention, an embryo derived from an oocyte in a context of controlled natural cycle where a level of FF G-CSF in its follicle is equal to or less than 8.8 pg/ml, 8.6 pg/ml, 8.4 pg/ml, 8.2 pg/ml, 8 pg/ml, 7.8 pg/ml, 7.6 pg/ml, 7.4 pg/ml, 7.2 pg/ml, 7 pg/ml, 6.8 pg/ml, 6.6 pg/ml, 6.4 pg/ml, 6.2 pg/ml, 6 pg/ml, 5.8 pg/ml, 5.6 pg/ml, 5.4 pg/ml, 5.2 pg/ml, 5 pg/ml, or a level between any two of the aforementioned values, is predicted to have a low implantation potential, pregnancy rate and birth rate. According to another embodiment of the invention, an embryo derived from an oocyte in a context of controlled natural cycle where a level of FF G-CSF in its follicle is equal to or more than 12 pg/ml is predicted to have a high implantation potential, pregnancy rate and birth rate.

According to another embodiment of the invention, an embryo derived from an oocyte in a context of controlled natural cycle where a level of FF G-CSF in its follicle is less than 12 pg/ml but more than 9 pg/ml is predicted to have a medium implantation potentiel, which means that it is likely to be implanted.

Consequently, an embryo derived from an oocyte in a context of controlled natural cycle where a level of FF G-CSF in its follicle is high or medium (over 8.8 pg/ml) and a level of FF IL-7 is high (over 32.41 pg/ml) in its follicle is high is to be implanted.

An embryo derived from an oocyte in a context of controlled natural cycle where a level of FF G-CSF in its follicle is high or medium (over 8.8 pg/ml) and a level of FF IL-7 in its follicle is medium (from 11.3 pg/ml to 32.4 pg/ml) is likely to be implanted. An embryo derived from an oocyte in a context of controlled natural cycle where a level of FF G-CSF in its follicle is high or medium (over 12 pg/ml) and a level of FF IL- 15 in its follicle is low (less than 7 pg/ml) is likely to be implanted. If levels of FF IL-7, FF IL- 17, FF IL- 15 and/or FF G-CSF in such patients are significantly below the level associated with likely or certain implantation in the collected oocyte, then there would be a saving in time, money and stress to the subject not to undertake implantation. In such cases, it will be possible for the practitioner (or assisted fertilisation clinic) to decide whether or not to even attempt a first implantation. On the other hand, if the oocyte indicates a high or complete certainty of implantation, this oocyte alone may be fertilized and the embryo so obtain implanted, so saving money and resources by fertilizing only the oocyte likely to become established as implanting embryo.

The present invention significantly increases the implantation rate while decreasing the number of embryos replaced. It also allows a specialist to become more efficient in preventing multiple pregnancies and all the related foetal and maternal morbidity especially the syndrome of ovarian hyper stimulation. The embryo with a known potential for implantation can thus be implanted, therefore allowing a policy of single embryo transfer while not decreasing the overall pregnancy rate.

In one embodiment of the invention, the embryo obtained by the methods as described here above may be further subjected to microscopic methods, such as embryo morphology, cleavage rate, appearance of pronuclei, fragmentation rate, blastomeres number and embryo symmetry, to check its quality. The embodiments described above in regard of the method apply to corresponding embodiments of the method of assisted fertilisation. The threshold values are indicated elsewhere herein. The skilled person will understand that intervening steps may be present such as freezing after oocyte collection. By use of the present invention, it will be possible for assisted fertilisation clinics to allocate resources more efficiently, so that patients with low levels of FF IL-7 and/or not detectable level of FF IL-17 and/or high level of FF IL-15 and/or low level of FF G-CSF in the follicle of a recovered oocyte who are unlikely to become pregnant by assisted fertilisation treatment are not treated. In one embodiment of the invention, the method as described here above may be carried out for preserving oocytes of a female subject in need thereof.

An example of female subject in need to preserve her oocytes is a female subject who is going to be subjected to chemotherapy, radiotherapy or to undergo bilateral oophorectomy, loss of ovarian function. According to this embodiment, the oocyte is tested for its implantation potential according to the method of the invention, and depending on the implantation potential, the oocyte is vitrified to be preserved (Domingo et al, 2009, Clin

Transl Oncol. 11(3): 154-9). This procedure may be repeated at each cycle, to create an oocyte bank.

The invention also pertains to kits for use in performing the methods of the invention. Said kits include at least one reagent useful for the detection of FF IL-7 and/or FF IL- 17 and/or FF IL- 15 and/or FF G-CSF. Suitable reagents include antibodies, or other suitable ligand-binding reagents, against FF IL-7 and/or FF IL-17 and/or FF IL-15 and/or FF G- CSF, said antibodies or reagents being optionally linked to a label. Typical labels are those commonly used in immunoassay procedures, for example horse radish peroxidase. The kit may also contain standards, for examples predetermined amounts of FF IL-7 and/or FF IL- 17 and/or FF IL-15 and/or FF G-CSF (e.g. protein or RNA), that may be labelled with a detectable label. The kit may also contain disposable aspirator tips for use in extracting the oocytes and follicular fluid.

The kit may be used for the measurement of the levels of FF IL-7 and/or FF IL-17 and/or FF IL-15 and/or FF G-CSF for use in a method of diagnosis, prognosis, and/or assisted fertilisation treatment of a female subject. The invention further provides the use of a reagent for the detection of FF IL-7 and/or FF IL-17 and/or FF IL-15 and/or FF G-CSF for the prognosis of the likelihood of establishing pregnancy by assisted fertilisation in a female subject.

The abovementioned antibodies, fragments and variants thereof, and other suitable ligand- binding reagents, which may optionally be labelled with a detectable label, may be used for the manufacture of a diagnostic kit for use in the treatment or diagnosis of suitability for assisted fertilisation. Levels of FF IL-7 and/or FF IL- 17 and/or FF IL- 15 and/or FF G-CSF may also be assayed via analysis of the levels of mRNA present in samples obtained. In order to achieve this, FF IL-7 and/or FF IL- 17 and/or FF IL- 15 and/or FF G-CSF or fragments thereof may be used as a probe to determine levels of IL-7, IL- 17, IL- 15 and G-CSF in the follicular fluid. Alternatively, levels of FF IL-7 and/or FF IL- 17 and/or FF IL- 15 and/or FF G-CSF may be estimated by analysing the levels of mRNA expressed in the follicular fluid or in the granulosa cells. Granulosa cells around the corona radiate may be stored at the stage of the decoronisation of each oocytes and stored in RNA stabiliser (e.g. at 8O⁰C) until assay. Such probes may also be formulated into kits in a manner analogous to those described for antibodies, and may contain control nucleic acids. Probes for the FF IL-7 and/or FF IL- 17 and/or FF IL- 15 and/or FF G-CSF gene may be designed for use as probes, for example for use in a nucleic acid amplification assay.

The invention will further be illustrated in view of the figures and examples.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Area under ROC curve for G-CSF (a), IL- 17 (b) and IL-7 (c) as predictors of birth by oocytes puncture.

Figure 2: Correlation between G-CSF, IL-7 and IL- 17.

Figure 3: Comparison of ROC curves evaluating FF G-CSF and combination of FF G-CSF and FF IL- 15 as predictors of birth.

AUCroc curve were 0.81 [0.70-0.89], p<0.0001 for FF G-CSF and 0.85 [0.74-0.92], p<0.0001 for combination of FF G-CSF and FF-IL-15. Predictibility of birth per retrieval was evaluated on 83 samples in multivariated models including age, range of attempt,

FSH, antral follicle count, estradiol level and endometrial thickness.

Figure 4: Correlation of FF-GSF over two cycles.

EXAMPLE 1

Materials and Methods

Patients All the patients from whom a follicular fluid was selected were enrolled in modified natural IVF/ICSI protocol because they previously failed to get pregnant despite at least 2 attempts in conventional IVF/ICSI cycles below 40 years old. Only the first attempt from the time of inclusion in the protocol of IVF/ICSI modified natural cycle was recorded. 83 follicular fluids were blindly analyzed. The outcome following the IVF/ICSI procedure was pregnancy for 25 patients (19 deliveries, 6 spontaneous abortions), no pregnancy despite the embryo transfer for 29 patients. For 19 patients, the oocyte was collected but did not successfully fertilize and in 10 cases the oocyte was not retrieved. Patient's characteristics in the observed four groups are detailed in table 1. No difference was observed regarding their age, number of previous conventional attempts, ovarian reserve, estradiol level and endometrial thickness the day of triggering or embryo morphology scoring at the day of transfer. 34 oocytes were fertilized by a standard in vitro fertilization and 39 by intracytoplasmic sperm injection. Infertility was due to sperm abnormalities (39%), tubal abnormalities (30%), or endometriosis (12%) or was unexplained (19%). An informed consent was obtained from all women, and this investigation received the approval of the internal institutional review board.

Table 1: characteristics of patients included

Modified natural IVF/ICSI protocol

On d3, women underwent blood sampling and the number and sizes of early antral follicles were assessed by ultrasound. From cycle d 8 onward, selection of the dominant follicle was monitored by ultrasound. When the mean diameter of the dominant follicle exceeded 13 mm, to prevent the risk of premature LH peak and to control additional follicular maturation, women were administered sc 0.5 mg of a GnRH antagonist (cetrorelix acetate, Cetrotide 0.25 mg; Serono Pharmaceuticals, Boulogne, France) and 150 IU human menopausal gonadotropin (hMG) (Menopur; Ferring Pharmaceuticals, Gentilly, France) daily until the day of human chorionic gonadotropin (hCG) (Gonadotrophine Chorionique Endo; Organon Pharmaceuticals, Saint-Denis, France) administration. The choice of starting hMG treatment once follicle dominance had been achieved aimed at preventing the rescue of additional subordinated follicles. Eventually, women received a 5000-IU hCG injection im when the dominant follicle diameter exceeded 16 mm. The single oocyte was picked up approximately 34 h after hCG administration [day of oocyte pickup (dOPU)], and ET was performed 2 d after oocyte pickup. Embryos morphology were defined on d 2 in relation to (a) their fragmentation (Grade 1: 10% or less, Grade 2: 10-30%, grade 3: 30- 50%, Grade 4: over 50%, embryo was downgraded when blastomeres were unequal in size), (b) their embryo score defined as number of blastomeres x (5-embryo grade). Optimal category was defined as those with 4-5 cells on d 2 and 10% or less of fragmentation and equal cells. Each patient had only one oocyte retrieved and only one embryo obtained and transferred. Luteal phase was supported with micronized progesterone (Estima Ge; Effik Pharmaceuticals, Bievres, France; 600 mg/d) administered daily by vaginal route starting on the evening of ET. FF collection

Under transvaginal ultrasound guidance, the FF from the dominant follicle was gently and thoroughly aspirated using a 10-ml syringe and then maintained at steady temperature conditions (37⁰C) until the oocyte was found and isolated. Meanwhile, the aspiration needle was kept steady inside the follicle and, in case of negative oocyte recovery, sequential follicular flushings were performed using 10-ml syringes filled with 3 ml of a balanced salt solution (Tyrode's salt solution; Eurobio Pharmaceuticals, Courtaboeuf,

France) (Mendez Lozano et al. 2008). All flushing volumes were discarded. Oocyte pickup failure was defined by a negative oocyte recovery after three consecutive follicular flushings.

Follicular fluid samples

FF samples corresponding to the dominant follicle were collected. The FF was centrifuged at 3000 x g for 15 min at 4⁰C to eliminate cellular elements and subsequently frozen at - 8O⁰C for subsequent analysis. Time elapsed between follicular aspiration and FF cryopreservation did not exceed 30 min.

For the multiplex analysis, each sample was blindly identified by the anonymous number of the patient, the anonymous number of the stimulation and the anonymous number of the oocyte within the cohort.

Multiplex sandwich assays

Multiplexed microsphere-based immunoassays were tested to simultaneously measure multiple analytes in individual follicular fluids by flow cytometric resolution of spectrally distinct microspheres coupled with capture molecules and reporter fluorochromes bound to detection antibodies.

The bead-based multiplex sandwich immunoassay purchased from Biorad, read with a Luminex system (Luminex Map Technology) was used to measure the concentrations of the following cytokines and chemokines:IL-lα, IL-IRa, IL-2, IL-4JL-5, IL-6, IL-8, IL-9, IL-10, IL- 12, IL- 13, IL- 15, IL-17,IFN-γ ,TNF-α, G-CSF, GM-CSF, VEGF, PDGF, FGF, IP-10, MCP-I, CCL5, eotaxin, MIP-Ia and MIP-1-β (Bio-Rad Laboratories, Hercules, CA, USA).146 samples were evaluated. In brief, 50 μl of each individual FF sample was added to 50 μl of antibody-conjugated beads directed against the cytokines listed above in a 96-well filter plate. After a 30- minutes incubation, the plate was washed and 25 μl of biotinylated anti-cytokine antibody solution was added to each well before another 30-min incubation. The plate was then washed and 50 μl of streptavidin-conjugated PE was added to each well. After a final wash, each well was resuspended with 125 μl of assay buffer (from Bio-Rad) and analysed by the Luminex array system.

The limit of detection for each analyte was defined as the mean ± standard deviation of the blanks in which the analyte was absent (Table X). The cytokine concentrations were calculated and a standard curve was derived from the results obtained with graded concentrations of a cytokine standard included in the assay.

The intratest variation was assessed by measuring the same FF samples 5 times.

Table 2: Mean concentration, detection limit and predictability for subsequent birth in univariate and multivariate regression models

The multivariate logistic regression models included age, range of attempt, basal FSH on day 3, Antral follicle count on day 3, Endometrial thickness, estradiol level the day of triggering , and embryo score if specified (last column).

AUCroc: Area under the receiving operating curve.

Emb.S: Embryo score= [number of blastomere x (5-grade)] on Day 2. Embryo score was included in the final predictive model for G-CSF, IL-15, IL- 17 and IP-IO. IL-lβ, IL- 5, IL-IO, IL-13, TNF -alpha, PDGF and FGF were excluded because of their low detection rates (over 50% undetectable).

0.6

IL-7 23.8 + 1 .01+0.95 0.76 0.74 (p=0.002) 0.74 (p=0.005)

1.7 (p=0.0001)

IL-8 195 + 15 1 .69+0.40 0.49 (p=0.88) -

IL-9 31 + 2.5 1 .62+0.12 0.61 (p=0.13) -

IL- 12 2.8 + 0.3 1 .56+0.64 0.48 (p=0.88)

IL- 15 6.58+0.6 1 .37+0.48 0.60 (p=0.07) 0.65 (p=0.03) 0.87 (P=O OOOi)

Z

IL- 17 9.53 + 0 .8+0.84 0.76 0.75 0.85(p<o oooi)

1.6 (p=0.001) (p=0 .0002)

Eotaxin 204 + 22 4 .80+3.84 0.68 (p=0.004) 0.68 (p=0.08) Excluded from model

MIP-α 3.8 + 0.3 1 .53+0.53 0.51 (p=0.61) -

MIP-β 75 + 4.5 1 .97+0.21 0.56 (p=0.38)

RANTES 64 + 16 1 .68+0.22 0.61 (p=0.13)

VEGF 2107+14 1 .90+0.25 0.65 (p=0.03) 0.67 (p=0.01) Excluded from model

7 /

MCP-I 3.8 + 0.3 1 .48+0.22 0.53 (p=0.63)

IP-IO 192 9 .09+5.11 0.68 (p=0.01) 0.73 (p=0.004) 0.82(p=0002)

+18.5

IFN-γ 51.6 + 1 .83+0.32 0.53 (p=0.68)

6.3

G-CSF +IL- 0.85 0.85 (p<0.0001)

15 (p<0 .0001)

Statistical analysis

Multivariate logistic regression analysis was performed to identify factors predictive of birth. We log-transformed non-normally distributed data prior to analysis. As covariate factors, some known factors able to influence the outcome "birth" were included as: age, antral follicle count on d 3 of the cycle (AFC), FSH levels on d 3, range of the attempt, endometrial thickness and oestradiol level the day of hCG injection with and without the embryo score (Table 1). To take in account that no transfer results from natural monitored IVF/ICSI cycles in 35% of the cases, the only outcome tested was birth rate per retrieval (n=83) for all multivariate analysis excluding embryo score (n=54). A P- value > 0.1 was used as a criterion for exclusion according to the literature on multivariate prognostic modelling. We apply the Area under the Receiver Operating Characteristic (ROC) (AuROC) curve methodology to determine the efficiency of potential biomarkers as predictors of implantation and birth. In ROC curve, the true positive rate (sensitivity) is plotted as a function of the false positive rate (100-specificity) for different cut-off points (Zweig and Campbell 1993). The calculation of the area under the ROC (AUC-ROC) curve measured the accuracy, i.e. the ability of a factor to discriminate between two distinct outcomes. We then evaluate ability of each factor in regard to the following outcome: birth/no birth -pregnancy/no pregnancy- fecundation/no fecundation- oocytes collection/no oocyte collected. Proposed thresholds to help to interpret the AUC: 0.9-1: perfect separation; 0.8-0.9: excellent discrimination; 0.7-0.8: acceptable discrimination; 0.6-0.7: poor discrimination; 0.5-0.6: no discrimination. Analytes with > 50% of values below the limit of sensitivity (IL-5, IL-lbeta, IL-IO, IL-13, TNF-α, FGF and PDGF) were excluded, owing to a certain level of uncertainty. The ROC analysis allowed also to define two essential thresholds: first the cut-off value predicting the failure with the highest negative predicting value, then the cut-off value to predict the success with the highest positive predicting value in regard to the outcome "birth or no birth/punction". Ranged levels of key cytokines (low-medium and high levels) are subsequently compared in regard their outcome by anova test.

Pregnancy was defined by a pregnancy test (bHCG) over 1000 IU and a miscarriage as a loss of a histologically or ultrasonographic recognized pregnancy in the first trimester. A P-value below 0.05 is considered as statistically significant.

Results

IVF /ICSI results and pregnancy/delivery rates in the cohort

The outcome following the first IVF/ICSI procedure was pregnancy for 25 patients (19 deliveries, 6 spontaneous abortions), and no pregnancy for 29. For 19 patients, an oocyte was collected but not successfully fertilized (12 ICSI-I l matures oocytes, 7 FIV) and in 10 cases no oocyte was retrieved. Infertility was due to sperm abnormalities (39%), tubal abnormalities (30%), or endometriosis (12%) or was unexplained (19%). Table 1 details the characteristics of the patients in these four groups. Standard IVF was used for 44 oocytes, and intracytoplasmic sperm injection for 39. Embryo score was significantly higher in the pregnant group (p=0.002). The AUC_roc of embryo score to predict subsequent birth was set at 0.71 (p<0.0006) in univariate analysis and reach 0.76 (p=0.0002) in multivariate analysis including known variables and will be used as reference for subsequent analysis (table T). Distribution of age, FSH, logarithm of range of the attempt and AFC were normal in the cohort.

Cytokine s/chemokines/ growth factors content and prediction of the outcome of the attempt In regression models including covariates as -age, range, AFC and FSH concentration on Day-3, endometrial thickness and estradiol level the day of triggering- FF G-CSF, IL-7, IL- 15, IL- 17, Eotaxin, VEGF and IP-IO were significantly predictive (Table T).

AU_rocC for FF-GCSF as a biomarker of birth/punction and birth/transfer were respectively both at 0.78 and 0.80 (p=0.0001) (Figure Ia). AU_rocC for FF-GCSF as a biomarker of pregnancy/punction and transfer were respectively at 0.72 and 0.73 (p=0.0008).

AU_rocC for FF-IL- 17 as a biomarker of birth/punction and birth/transfer were respectively both at 0.77 and 0.78 (p=0.0001) (Figure Ib). AU_rocC for FF-IL-17 as a biomarker of pregnancy/punction and transfer were respectively at 0.68 and 0.73 (p=0.001).

AU_rocC for FF-IL-7 as a biomarker of birth/punction and birth/transfer were respectively both at 0.75 (p=0.0002) and 0.75 (p=0.0004) (Figure Ic). AU_rocC for FF-IL-7 as a biomarker of pregnancy/punction and transfer were respectively at 0.69 (p=0.004) and 0.72 (P=OOOl).

Inclusion of embryo score in the model increased the AUCroc for FF G-CSF from 0.81 to 0.86 (p <0.0001) to predict birth. Introduction of embryo score also increased the power of predictability of IL- 15 , IL- 17 and IP- 10 while VEGF and Eotaxin were excluded (Table T) .

For FF G-CSF, concentration lower than 8.74 pg/ml defined the group with the higher negative predicting value to predict the failure and the group over 12.08 pg/ml the group with the higher positive predicting value to predict the success (sensibility at 89.5% for a specificity at 54.7%). FF G-CSF was lower than 12.08 pg/ml in 45% (low group: 37 samples - 21 transferred), and at or above it in 55% (high group: 46 samples - 33 transferred) and defined low-medium and high G-CSF ranged groups.

Birth rates/punction and transfer were respectively 0%-14%-37% and 0%-28%-51% in low-medium-and high G-CSF ranged groups (p=0.002 and 0.001). Pregnancy rates/punction and transfer were respectively 12.5%-28%-40% and 14%-43%-54% in low- medium and high G-CSF ranged groups (p=0.03 and 0.06). For IL- 17, since the highest negative predicting value was absence of detection and the highest positive predictive value a positive detection, we only took in account the detection of IL- 17 in samples. IL- 17 was detected in 41 samples and not detected in 42 samples. Birth rates/punction and transfer were respectively 4.8% versus 40% and 8 % versus 58 % in IL-17 not detected-detected ranged groups (p<0.001). Pregnancy rates/punction and transfer were respectively 17% versus 43% and 20 versus 62% in the two groups in which IL-17 was not detected versus detected (p=0.01 and 0.001).

For FF IL-7, concentration lower than 11.31 pg/ml defined the group with the higher negative predicting value of failure and the group over 32.41 pg/ml the group with the higher positive predicting value (sensibility 63% and specificity at 89%) to predict the birth. FF IL-7 was lower than 11.31 pg/ml in 17 samples (7 transferred), from 11.31 to 32.4 pg/ml in 43 samples (30 transferred), and over 32.4 pg/ml in 23 samples (17 transferred) and defined low-medium and high IL-7 ranged groups. Birth rates/punction and transfer were respectively 0%-16%-52% and 0%-23%-70% in low-medium-and high G-CSF ranged groups (p<0.001). Pregnancy rates/punction and transfer were respectively 5%-25%-56% and 0%-33%-76% in low-medium and high G-CSF ranged groups (p=0.001 and <0.001).

Results are summarized in table 3.

Table 3

Birth and Pregnancy rates according to G-CSF, IL-17 and IL-7 concentration (pg/ml)

N° N° of Pregnancy Pregnancy Birth Birth P value of transfer rate/ Rate Rate rate for punct Punctio, /transfer / punction /transfer Birth

(%) (%) (%) (%) /punct°

G-CSF Low 24 14 12.5 14 0 0

(Pg/m) 0.002

Medium 14 7 28 43 14 28 high 45 33 40 54 37 51

IL-17 Not 42 25 17 20 4.8 8

(pe/m) detectable 0.002

41 29 43 62 40 58

detectable

IL-7 Low 17 7 5 0 0 0

(Pg/m) <0.001

Medium 43 30 25 33 16 23 high 23 17 56 76 52 70

As quantification of G-CSF in regard to the potentiality to predict birth had the higher sensibility and IL-7 the higher sensibility, we combined the both in regard to the outcome. Results are summarized in Table 4. The combination of IL-7 and G-CSF allow to identify a group of patients with no chance of birth (low G-CSF with IL-17 low or medium) and a group of patients with very high chance of birth (G-CSF high or medium with IL-7 high) in natural modified cycle.

Table 4

a p=0.02 between IL-17/G-CSF high-high and IL-7/G-CSF low/low

b p=0.008 between IL-17/G-CSF high-high versus IL-7/G-CSF low/low and medium/high (b')

c p=0.001 between IL-17/G-CSF high-high versus IL-7/G-CSF low/low and medium/low and medium/medium

d p=0.001 between IL-17/G-CSF high-high versus IL-7/G-CSF low/low and medium/low, medium/medium and medium/high

G-CSF, IL-7 and IL- 17 were significantly correlated each others as represented in figure 2. Correlation coefficients between G-CSF and IL-17 were 0.68 (p<0.0001), between G-CSF and IL-7: 0.62 (p <0.0001), between IL-17 and IL-7 : 0.77 (p>0.0001).

Follicular cytokines/ 'chemokines/ 'growth factors content and prediction of a positive collection and fertilization of the oocyte.

Oocytes failed to be retrieved for 10 patients and failed to get fertilized in 19 cases (12 ICSI and 7 IVF).

IL-4, IL-IO, IL-12 and GM-CSF were significantly higher than observed in the ones where pick-up allowed retrieving the oocyte. All were significantly predictive of the failure to pick-up the oocyte. On the opposite, when the oocyte failed to be fertilized, concentration of IL-6, IL-9, IL-15, IL-2, IFN-G and G-CSF were significantly lower than observed in the one which successfully fertilized. Both high concentrations of GM-CSF and IL- 15 were predictive of the failure to collect the oocyte with AUCroc curve respectively at 0.85 (p=0.0068) and 0.88 (p=0.0001) if evaluated with covariates. GM-CSF and IL- 15 were correlated. Integrated with all the other analytes in a prediction model, only IL- 15 and oestradiol the day of triggering were retained in the model with an AUCroc curve at 0.89 (p=0.0001) to predict failure of oocyte retrieval. IL- 15 threshold above 7 pg/ml had a sensibility of 90% and a specificity of 77.8% to predict oocyte failure retrieval. 31% (26/83) of patients had a value FF IL-15 above 7 pg/ml. Using the same methodology, we failed to construct a model able to predict failure of fertilisation.

Table 2 summarizes sensibility/specificity of significant analytes either to predict failure of pick-up or failure of fertilization and the corresponding Area under ROC curve.

Cytokines/ 'chemokines/ 'growth factors content and embryo quality in modified natural cycle Using the ROC curve methodology, none of the analyte was able to discriminate between TOP and no TOP embryos and reach a significant specificity or sensitivity.

Optimal Model

In order to optimize the model of prediction of birth, all the analytes found to be significant in the multivariate regression model (G-CSF, IL-7, IL-15, IL- 17, Eotaxin, VEGF, IP-IO) and covariates excluding and including embryo score were evaluated. In both cases, only FF G-CSF and IL-15 were retained in the model to predict birth with an AUCroc at 0.85 (p<0.0001) (figure 1). Birth rates per retrieval were 48.88% (16/33) if two good-prognosis criteria were present (FF G-CSF>12 pg/ml and IL-15<7pg/ml), 8% (3/36) and 0% (0/14) if respectively only one or none were present (Table 5).

Table 5: Predictive value of combinations of follicular G-CSF and IL-15 on births rates

Cut-off values were defined from the area under ROC curves. The P values between FF with FF G-CSF over >12 pg/ml and FF IL-15 below < 7 pg/ml if compared with the three others combinations were: 0.001 (***)or birth/OR and birth /ET, 0.005 for pregnancy/OR and 0.02 (*) for pregnancy/ET using ANOVA test.

G-CSF and IL-15 Pregnancy Pregnancy Birth/initiated

Birth/ET /initiated cycle /ET cycle

Exploration of follicular fluids over two natural modified IVF/ICSI cycles.

15 patients were explored through a subsequent modified natural IVF/ICSI cycles. The issue at the second cycles resulted in 6 births, 1 abortion, 5 no embryo transfer and 4 no pregnancy. FF G-CSF was the only cytokine significantly correlated over two cycles (r=0.71, p=0.008) as represented in figure 4 while FF IL-15 showed variations from a cycle to another. All the six women which delivered had the optimal combination (FF G-CSF over 12 pg/ml with IL-15 below 7 pg/ml) over the second cycle and 5/6 over the first observational cycle (one case of elevated FF IL-15).

Claims

1. A method for determining the implantation potential of an oocyte from a female subject in a natural modified cycle, comprising:

- measuring, in the follicular fluid from which the oocyte is derived, the level of at least one of IL-7 and IL- 17, and

2. The method according to claim 1, wherein the oocyte having a high level of IL-7 and/or IL- 17 in the follicular fluid gives rise to an embryo with a great implantation potential.

3. The method according to anyone of claim 1 or 2, wherein the oocyte having a low level of IL-7 in the follicular fluid gives rise to an embryo with a low implantation potential.

4. The method according to anyone of claim 1 to 3, further comprising:

- measuring, in the follicular fluid from which the oocyte is derived, the level of G-CSF and optionally the level of IL- 15, and

- determining the implantation potential of the oocyte from the level of follicular fluid G- CSF and at least one of IL-7, IL- 17 and IL- 15.

5. The method according to claim 4, wherein the oocyte having a high level of G-CSF and a low level of IL- 15 in the follicular fluid gives rise to an embryo with a great implantation potential.

6. The method according to anyone of claim 1 to 5, wherein the follicular fluid is obtained from a follicular aspirate.

7. The method according to anyone of claim 1 to 6, wherein the levels of at least one of IL- 7, IL- 17 and IL- 15 and the level of G-CSF are measured within 20 hours after collection of the follicular fluid.

8. The method according to anyone of claim 1 to 7, wherein the levels of at least one of IL- 7, IL- 17 and IL- 15 and the level of G-CSF are measured using an immunoassay.

9. The method according to anyone of claim 1 to 8, wherein the levels of at least one of IL- 7, IL- 17 and IL- 15 and the level of G-CSF are measured using an ELISA.

10. The method according to anyone of claim 1 to 8, wherein the levels of at least one of IL-7, IL-17 and IL-15 and the level of G-CSF are measured using a luminex assay.

11. The method according to anyone of claim 1 to 7, wherein the levels of at least one of IL-7, IL-17 and IL-15 and the level of G-CSF are measured by determining the levels of IL-7, IL-17, IL-15 and G-CSF mRNA.

12. A kit consisting of at least one reagent suitable for detection of at least one of IL-7, IL- 17 and IL-15 level and at least one reagent suitable for detection of G-CSF level.

13. A method for determining the implantation potential of an oocyte from a female subject in a natural modified cycle, comprising:

14. A method for determining the implantation potential of an oocyte from a female subject in a natural modified cycle, comprising:

- determining the level of follicular fluid IL-15.