USE OF A CYCLIC TRIPEPTIDE TO IMPROVE CELLULAR ENERGY METABOLISM

Abstract

The present invention relates to the use of a cyclic peptide comprising the tripeptide reproducing a binding site of fertilin beta to the oocyte integrin, for improving cellular energy metabolism. More particularly, the invention concerns the use of a cyclic peptide comprising the tripeptide FEEc for stimulating the energy metabolism of gametes or embryonic cells in the context of medically assisted procreation (MAP) protocols, in particular promoting the in vitro maturation of the oocyte, the fertilisation rate and the birth rate.

Description

USE OF A CYCLIC TRIPEPTIDE TO IMPROVE CELLULAR ENERGY METABOLISM The present invention relates to the field of medically assisted reproduction (MAR) and more generally to all medical, veterinary or other applications in which stimulation of mitochondrial, or more generally energy, activity of cells is desired, in particular during protocols including a cell culture step or ex vivo cell maintenance. More than 15% of couples use Assisted Reproductive Technology (ART) at some point in their reproductive lives. When sperm is impaired, intracytoplasmic sperm injection (ICSI) is often used to achieve fertilization. This technique is very invasive for the egg. Furthermore, in vitro fertilization (IVF) with normal sperm, unexplained fertilization failures are common in 3 to 5% of cases. In a previous patent application (WO 2005 / 051799 A2), the team of inventors described a cyclic tripeptide (Phe, Ac Glu, Ac Glu) of formula CSFEEC (SEQ ID No: I) with a cyclization linkage between the two terminal cysteines (FEEc), as well as its action in increasing the fertilizing capacity of human gametes. Equivalents of the molecule exist in various animal species with the same properties and are also described in this document. Continuing their research, the inventors demonstrated that the FEEc molecule has an impact on spermatozoa (Example 1 below). In particular, it improves sperm motility parameters as analyzed by CASA (Computer Aided Sperm Analysis). Thus, for example, linear velocity and the amplitude of lateral head movement are increased by 7% and 8%, respectively (P<0.05 and P<0.002) (Example 1). This results in an increase of nearly 30% in the percentage of hyperactivated spermatozoa (P<0.009). These hyperactivated spermatozoa are the fertilizing spermatozoa. For a spermatozoon to increase its speed of progression, it is logical to assume that it increases its ATP consumption or at least that its energy metabolism is improved, since its movement is generated by the binding of dynein arms to tubules adjacent to the axoneme. Dyneins are ATPases.By studying the mitochondrial metabolism of sperm exposed to FEEc, the inventors hypothesized that it induces an increase in mitochondrial membrane potential, indicative of either increased ATP synthesis by mitochondria or reduced ATP consumption. Thus, FEEc improves mitochondrial energy metabolism, or more generally, cellular energy metabolism in sperm, either by enhancing ATP production or by optimizing its use by the cell. The present invention therefore relates, in general, to the use of a cyclic peptide comprising a tripeptide reproducing a binding site of Fertilin beta to the integrin of the oocyte, or a tripeptide of formula EEP (SEQ ID No: 2), for use as a drug to enhance mitochondrial activity or, more generally, to improve cellular energy metabolism.The applications of such a peptide are illustrated here in two different cell types: the sperm and the oocyte, and in the development of a multicellular organism, the embryo. The results presented in the experimental section support the use of this peptide in other applications where an improvement in cellular energy metabolism is desired. In particular, this peptide can be useful for improving the yield of cell cultures, especially lymphocytes. In one specific implementation, the peptide is vectorized, meaning it is administered in association with an agent, molecule, composition, or other type of vector that facilitates its entry into cells. More generally, vectorization serves to modulate and control the distribution of an active ingredient to a target by associating it with a vector. In the foregoing, the "cyclic peptide comprising the tripeptide reproducing a binding site of fertilin beta to the integrin of the oocyte" corresponds to the peptide described in patent application WO 2005 / 051799 A2 mentioned above. As mentioned in that earlier application, particularly in Table 1, the tripeptide varies depending on the species. It can be cyclized by any means known to those skilled in the art, in particular via two cysteine ​​residues located on either side of the tripeptide. Generally, all the variations described in application WO 2005 / 051799 A2 are considered to meet the definition of the "cyclic peptide comprising the tripeptide forming a binding site of fertilin beta to the integrin of the oocyte" within the meaning of the present invention. To facilitate reading this text, this cyclic peptide, as well as the drug containing it as an active ingredient, will be referred to herein by the formula "FEEc".Those skilled in the art will fully understand that this notation also covers forms usable in species other than humans, such as, for example, the cyclic tripeptide TDE which should be used in cattle. It will be understood from reading the detailed description of the invention that follows, as well as the examples, that the scope of the present invention is not limited to applications in assisted reproductive technology (ART) but opens up real possibilities in many other fields. Thus, the invention relates more generally to the use of FEEc in medical or non-medical applications in which stimulation of cellular energy metabolism is desired. Action on sperm in Intrauterine Insemination: By improving sperm motility parameters, FEEc is also likely to improve pregnancy rates in Intrauterine Insemination (IUI). According to a first particular aspect of the invention, FEEc is used to increase the speed of sperm progression in an ART protocol.Still within the context of assisted reproductive technology (ART), FEEc can be used to improve sperm motility parameters and increase the rate of hyperactivated sperm. Furthermore, the use of FEEc is particularly advantageous in intrauterine insemination (IUI) protocols, both in humans and in non-human mammals. When implementing this aspect of the invention, the sperm are preferably incubated for one minute to three hours in the presence of 10 to 100 μM of peptide and then washed before intrauterine insemination. Action on oocyte maturation in vitro The molecule is also effective on oocytes. In vitro maturation of human oocytes arrested in the germinal vesicle stage increases from 37.71% to 59.30% (P<5.7 x 10^5) in the presence of FEEc (example 2). In patients aged 37 years and older, this rate increases from 36.96% to 68.29% (P<0.003), demonstrating that the molecule is particularly effective in this age group. Oocytes from women aged 37 to 40 are aneuploid in at least 50% and more generally in approximately 80% of cases due to a decline in mitochondrial activity. The molecule is therefore likely to improve the ploidy of oocytes and, consequently, that of embryos, whose developmental potential and implantation capacity also depend on the mitochondrial activity of the fertilized oocyte.The significant increase in pregnancy rates among women under 37 years of age, as observed in the "Fertiline" clinical trial described below, demonstrates that this beneficial effect occurs on all oocytes, particularly during fertilization and early embryonic development. Based on these results, it is hypothesized that Fertiline can improve oocyte ploidy. Supplementing culture media and in vitro fertilization media, and embryo incubation media, with the molecule therefore makes it possible to improve oocyte and embryo maturation (especially for women under 30 and 37 and over) and the fertilization rate in conventional IVF, as well as in IVF with ICSI (intracytoplasmic sperm injection). Supplementing culture media during in vitro fertilization, with or without micromanipulation, with the FEEc molecule improves pregnancy and live birth rates, particularly in women under 37 years of age under the experimental conditions used. Action in In Vitro Oocyte Maturation (IVM) protocols. The FEEc molecule is also likely to be effective in in vitro maturation protocols of oocytes for fertility preservation (MIV). In an in vitro fertilization (IVF) protocol, oocyte maturation is generally complete by the time of egg retrieval. However, some oocytes may still be immature. Furthermore, some women have ovarian dysfunction or a clinical condition that makes ovarian stimulation difficult. In these cases, egg retrieval is intentionally performed at an immature stage to facilitate in vitro maturation. Peptides could effectively aid in this maturation process (in vitro maturation for fertility preservation). The completely immature oocyte has a large nucleus called the germinal vesicle (GV). The mature oocyte is characterized by the presence of the first polar body (PB) in the perivitelline space (between the surface of the oocyte and the zona pellucida). Only mature oocytes are capable of fertilization. The inventors demonstrated that FEEc improves oocyte maturation in vitro. This is explained by the effect of the tripeptide on mitochondrial activity or on the energy metabolism of the oocyte. Indeed, mitochondrial activity, or more generally energy metabolism, decreases with age, and oocyte maturation involves several highly energy-intensive stages: - Rupture of the Germinal Vesicle - Chromosome Condensation - Formation of the Metaphase Plate - Spindle Formation - Synthesis of Checkpoint proteins - Telophase - GP expulsion. These oocyte maturation disorders, which worsen with age, are corrected by microinjection of mitochondria from young cells (oocytes from young donors or oogonal stem cells). This strengthens the likelihood that FEEc effectively corrects the cellular defect linked to mitochondrial or, more generally, energy insufficiency. According to another aspect, the present invention relates to the use of FEEc to improve the in vitro maturation of an oocyte. The improved quality of meiosis is likely responsible for the lower miscarriage rate observed even in younger women, making this aspect of the invention also relevant for improving the in vitro maturation of oocytes from women under 37, or even under 30. In implementing this aspect of the invention, the oocyte is incubated for a period of between 1 hour and 4 days, particularly up to 3 days or 24 hours, in the presence of 10 to 100 μM of peptide. Action on the activation of the fertilized oocyte The inventors also demonstrated an increase in sperm head decondensation after fertilization. This reflects improved oocyte activation during fertilization, which is linked to the oocyte's mitochondrial activity. This has an effect on blastocyst formation. The inventors also demonstrated (in mice) that FEEc improves blastocyst formation. This can also be attributed to the effect of the tripeptide on mitochondrial activity or, more generally, on cell metabolism. Indeed, it is known that mitochondrial DNA replication does not occur during preimplantation embryogenesis. During the first week of development, the zygote (or egg) divides by successive mitoses, starting with 2, then 4 cells, passing through the morula stage until reaching the blastocyst stage, preferentially using mitochondria initially present in the oocyte. A mitochondrial deficiency (in number or output) can therefore lead to chromosomal instability of the blastomeres during meiosis and mitosis, and result in arrest of zygote development, embryonic development, or even pregnancy.This is a common cause of spontaneous miscarriage after natural fertilization or after embryo transfer during IVF. The present invention therefore also relates to the use of FEEc to improve blastomere ploidy during the first week of zygote development. Consequently, the invention relates to the use of FEEc to reduce the number of miscarriages. The invention also relates to the use of FEEc to reduce the risk of aneuploidy, particularly trisomy. In implementing this aspect of the invention, the embryo is incubated for a period of between 24 hours and 6 or 7 days in the presence of 10 to 100 μM of peptide. The uses described above are particularly beneficial in in vitro fertilization (IVF) protocols. In humans, they allow women of all ages to have children through assisted reproductive technology (ART) using their own oocytes without resorting to mitochondrial injections as described in the literature (the most documented major effect to date). They also reduce the risk of miscarriage. The present invention also relates to the use of FEEc to reduce the risk of miscarriage, as previously mentioned and illustrated in the experimental part below. Reduced risk of Down syndrome The present invention also relates to the use of FEEc to reduce the risks of trisomy, or more generally aneuploidy, during IVF, in all women, particularly in women aged 35, 36, 37, 38, 39 or 40 and over. Improvement of in vitro embryo development kinetics The present invention also relates to the use of FEEc to improve pre-implantation embryonic development in vitro. In one particular embodiment, pre-implantation development is achieved under prolonged culture conditions. Improving the kinetics of embryonic development leads to an improved live birth rate. Action in natural reproduction Although the effects of FEEc on the oocyte and zygote have been demonstrated by the inventors in an IVF context, it is evident that these effects could also be obtained during natural fertilizations, for example by administering FEEc vaginally at the time of ovulation, the tripeptide being coupled with means capable of vectoring it to the oocyte. Action during the cryopreservation of gametes and embryos It has also been shown that the survival rate of cryopreserved oocytes depends, among other things, on their mitochondrial activity. It is likely that the same is true for embryos. The FEEc peptide is therefore likely to improve the survival rates and / or quality of cryopreserved gametes and embryos upon thawing. Action on other cell types The cyclic tripeptide was designed to bind to Pintegrin α6β1 on the oocyte. It is capable of binding to the cytoplasmic membrane of various other cell types because this integrin is highly ubiquitous. This molecule is therefore likely capable of increasing the energy activity of many cell types. It thus has potential applications beyond IVF. Since the molecule's mode of action likely occurs via integrin α6β1, FEEc is likely to have effects on many other cell types. This molecule could be used to improve the yield of any cell culture. As mentioned above, FEEc may act via the integrin αόβΐ or another receptor present on many cell types. It can therefore be used to enhance mitochondrial activity or energy metabolism in any cell expressing this integrin or other receptor. An immediate application of this property is the improvement of the yield of any cell culture. The present invention therefore also relates to a method for enhancing mitochondrial or, more generally, energy activity in cells in vitro, comprising a step of exposing the cells in question to FEEc. This method can advantageously be implemented on primary cells cultured ex vivo for administration to a patient as part of cell therapy.Non-limiting examples of cell cultures that could benefit from this process include skin cultures for skin grafts, lymphocyte cultures for cell immunotherapies, etc. The invention relates to the use of an FEEc peptide for the purpose of promoting ex vivo culture of all cell types expressing an FEEc receptor, in medical applications (such as cell therapy) or non-medical applications (such as maintaining cells in culture for experimental purposes or protein production). Action in the animal world of mammals The molecule exhibits species specificity. Its isoforms are available for use in all animals, domestic or otherwise, including farm animals, some species of which have difficulty reproducing (racehorses, Holstein cows). Action on mitochondrial pathologies and against aging The properties of FEEc as a molecule that stimulates mitochondrial activity, or more generally energy metabolism, can also be used in vivo in any type of pathology linked to impaired mitochondrial activity. In this regard, age-related diseases can be cited as a general example. The relationship between mitochondrial dysfunction and neurodegenerative diseases, for instance, has been established by several research teams. Therefore, FEEc could be used as a drug to treat neurodegenerative diseases such as Alzheimer's or Parkinson's. A relationship has also been shown between telomere length and mitochondrial activity in the cell. Telomere shortening is linked to the aging process. It is therefore possible that by stimulating mitochondrial or energy activity in the cell, it may be possible to delay the effects of aging. Mitochondrial diseases present a varied clinical picture but frequently involve ocular manifestations such as retinitis pigmentosa or ophthalmoplegia. For the latter, topical administration of FEEc in the eye, for example in eye drops, could improve symptoms related to mitochondrial or energy deficiency. Another object of the invention is therefore an eye drop comprising a cyclic peptide including the tripeptide capable of forming a binding site for fertilin beta to the integrin of the oocyte. Such an eye drop may comprise, in addition to FEEc, another agent such as a thickening agent, an antiseptic, an antibiotic, or any other compound usable for this type of product. The media described in application WO 2005 / 051799 A2 are, of course, excluded from the definition of the term "eye drops" as used in the present invention. Action in cosmetics The present invention also relates to the use of FEEc in a cosmetic or therapeutic composition intended for topical application. Examples include the use of FEEc to stimulate fibroblasts for collagen production, or to stimulate hair follicles to promote hair growth, for example, to prevent or slow alopecia. The present invention therefore also relates to a cosmetic or dermatological composition comprising FEEc as an active ingredient. "Cosmetic or dermatological composition" herein means a composition which, in addition to FEEc, comprises ingredients commonly used in the field of cosmetology. According to the invention, the cosmetic or dermatological composition can be in any form known to those skilled in the art.This may include, for example, an oil-in-water emulsion, a water-in-oil emulsion, a water-in-silicone emulsion, a multiple emulsion, a microemulsion, a nanoemulsion, a solid emulsion, an aqueous or hydroalcoholic gel, a cream, a milk, a lotion, an ointment, an oil, a balm, an ointment, a mask, a powder, an impregnated carrier, for example a transdermal patch, an aqueous or hydroalcoholic lotion and / or a wax, a makeup product, for example a foundation, a shampoo, a conditioner, a mask, a serum for topical application, a hair lotion. The media described in application WO 2005 / 051799 A2 are of course excluded from the definition of cosmetic or dermatological compositions within the meaning of the present invention. According to the invention, the cosmetic or dermatological composition can be, for example, a composition for the care of the face, body, hair, for example compositions for the face and / or body and / or hair. The following examples illustrate the invention without, however, limiting its scope. Figure captions Figure 1: Variation in mitochondrial membrane potential in the presence of FEEc (right) versus the control "scramble" peptide (left). Note the increase in membrane potential in exposed spermatozoa in the majority of patients. Figure 2: Counting after UV excitation of human spermatozoa fused with uncoated human oocytes, incubated in the absence (A) or in the presence of FEEc at 100 μM (B). Note the increase in the number of fused spermatozoa and the more rapid decondensation of their heads. Figure 3: Proportion of mature oocytes at day 1 of In Vitro Maturation (IVM). Schematic representation of the proportions of human oocytes in metaphase II (M1) from the VG stage after IVM in control medium or medium supplemented with 100μM FEEc. *ρ=0.02. **p=0.003. Day 1=24h of IVM. Figure 4: Proportion of atretic oocytes at day 1 of In Vitro Maturation (IVM). Schematic representation of the proportions of atretic human oocytes. Results observed at day 1 from the VG stage after in vitro maturation (IVM) in control medium or medium supplemented with FEEc at 100 μM. Day 1 = 24h of IVM. Figure 5: Meiotic spindle labeling obtained from a human oocyte in MIL after IVM in standard medium (24 h). Spindle labeling with an anti-α-tubulin antibody and chromosomes with DAPI. Image obtained by confocal microscopy. A; whole oocyte. B; magnification of the metaphase plate. Figure 6: Comparison of the fertilization rate at Jl between young and old mice, in the presence or absence of fertiline, which corresponds to the QDEc peptide. Young: 7-week-old B6CBAF 1 mice; Older: 7-month-old B6CBAF1 mice Figure 7: Comparison of the average percentages of cleaved embryos at day 2 and day 4 between young and old mice, in the presence or absence of the QDEc peptide. Young: 7-week-old B6CBAF1 mice (n=108 oocytes, 56 controls, 52 QDEc); Older: 7-month-old B6CBAF1 mice (n=128 oocytes, 65 controls, 63 QDEc),*; p=0.02. **p=0.008. ***p=0.01. Figure 8: Comparison of mean percentages of atresia (ATR) at day 2 between young and old mice, in the presence or absence of the QDEc peptide. Young: 7-week-old B6CBAFI mice (n=108 oocytes, 56 controls, 52 QDEc); Older: 7-month-old B6CBAF1 mice (n=128 oocytes, 65 controls, 63 QDEc). Figure 9: Schematic representation of the clinical study methodology carried out in example 5. Figure 10: Preliminary results of the primary and secondary endpoints in the clinical study. Pregnancy rate per fresh or frozen embryo transfer (control group, n=17 transfers / FEEc group, n=3 transfers). Percentage of "top embryos" (control group, n=75 cleaved embryos / FEEc group, n=72). Fertilization rate (control group, n=259 Mil / FEEc group, n=246 Mil) Figure 11: Pregnancy rates obtained in the clinical study. Figure 12: Improvement in the maturation rate of oocytes arrested in the ovary after incubation in the presence of FEEc. This study was carried out considering one oocyte per woman and incubating it in the presence of FEEc or the control peptide. Figure 13: Effects of FEEc on the maturation of human oocytes arrested in VG by age group. Figure 14: Stimulation of pre-implantation embryonic development in young mice by QDEc. (*P<0.00532; **P<0.00374; ***P<0.00933; ****P<0.068; ( ) number of embryos) Materials and Methods The experimental examples presented below were obtained using the following materials and methods: Observation of sperm motility parameters The sperm samples studied were each divided into two aliquots, one of which was incubated with FEEc and the other with a scramble peptide containing the same amino acids but in a random order. The inventors incubated human spermatozoa for 3 hours at 37°C in the presence of 100 μM of either the FEEc peptide or the scramble peptide, then observed sperm motility parameters using computer-assisted sperm analysis (CASA). The sperm parameters tested were: smoothed VAP, VSL, VCL, and ALH. These correspond respectively to velocity along the mean trajectory, straight-line velocity, curvilinear velocity, and lateral head displacement. The study showed a significant increase in the percentage of hyperactivated sperm (according to the criteria of Mortimer et al.). This likely explains the increased fertilization rates observed in the presence of the peptide. Measurement of mitochondrial membrane potential The inventors incubated human sperm for 3 hours at 37°C in the presence of either the FEEc peptide or the "scramble" peptide, which contains the same amino acids in a random order and thus constitutes the control group. After washing, the sperm were labeled with a fluorescent lipophilic dye, DIOC6. The mitochondrial membrane potential (proton gradient at the inner mitochondrial membrane) was then measured by flow cytometry. It was found to be increased in sperm after exposure to FEEc. Measurement of the fertilization index De-coated human oocytes were incubated with human sperm in the absence or presence of FEEc at 100 μM. Fused sperm were counted after UV excitation. Sperm were considered fused when the nucleus was labeled with Hoechst 33342. Furthermore, the heads of sperm that penetrated the oocyte in the presence of the FEEc peptide were not only more numerous but also exhibited a blurred appearance, indicative of decondensation of their sperm heads. This decondensation is one of the first steps in oocyte activation after sperm penetration. We can therefore conclude that FEEc not only improves sperm fertilization but also activates the fertilized oocyte. Collection of human oocytes for In Vitro Maturation (IVM) Immature human oocytes donated for research were collected at the In Vitro Fertilization (IVF) laboratory of the Assisted Reproductive Technology (ART) Center at Cochin Hospital (Paris, France). Two hours after oocyte retrieval, the oocytes intended for microinjection were decoronized with hyaluronidase (ORIGIO, Limonest, France). After observation under an inverted microscope (Hoffman), the immature oocytes at the germinal vesicle (GV) stage were selected for further experiments. In Vitro Maturation (MIV) of Immature Human Oocytes Immature human oocytes at stage VG were randomized to either the control culture medium (Global, JCD, La Mulatière, France) (n=203) or the same medium supplemented with 100 μM of FEEc (n=193). The immature oocytes were divided into two groups: those from women under 37 years of age and those from women 37 years of age and older. On the day of follicular aspiration, both groups of VG oocytes were incubated in 20 μM oil-covered drops and maintained at 37°C under 5% CO2 for observation using an inverted (Hoffman) microscope on day 1 (24 h incubation) and day 2 (48 h incubation). The oocytes were classified as metaphase II (1st polar body in the perivitelline space), germinal vesicle (GV), metaphase I (rupture of the germinal vesicle without expulsion of the polar body) or atretic. Collection of murine oocytes for In Vitro Maturation (IVM) B6CBAF1 females (aged between 5 and 8 weeks) provided by the Charles River laboratory (L'Arbresie, France) were stimulated by injection of 10 IU of PMSG (Pregnant Mare Serum Gonadotropin) (SIGMA-ALDRICH, Saint-Quentin Fallavier, France) without ovulation induction. Immature oocytes were retrieved from the ovaries 48 hours after this last injection, then decapitated using hyaluronidase and washed three times in M2 culture medium. Only oocytes classified as VG were retained for subsequent experiments. In Vitro Maturation (MIV) of Immature Mouse Oocytes Mouse oocytes were randomly incubated in either standard medium or medium supplemented with 100 μM QDEc. Culture plates were prepared the day before and incubated at 37°C under 5% CO₂. Oocytes were observed on day 0 (8 hours post-sacrifice) and day 1 (24 hours). Immunofluorescence Human oocytes from in vitro maturation (IVM) were fixed in 2% paraformaldehyde (PFA) for 1 h at room temperature and then washed in PBS containing 0.5% BSA. Permeabilization was achieved by incubating the oocytes in a solution containing 0.5% BSA, 0.1% Triton X-100, 0.05% Tween-20, and 5% normal goat serum. The oocytes were then washed in 0.5% PBS-BSA before being incubated overnight in a 1 / 200 dilution of anti-human α-tubulin antibody (SIGMA-ALDRICH) in PBS containing 0.5% BSA. The oocytes were then incubated for 1 h in the presence of an IgG secondary antibody conjugated to Alexa Fluor (LIFE TECHNOLOGIES, Alfortville, France). After a washing step, the oocytes were incubated for 10 min in DAPI (diluted to 1 / 1000) before being mounted on a slide and observed by confocal microscopy in the dark.For spindle analysis, oocytes with distinct and well-organized microtubule fibers associated with perfect chromosome alignment at the metaphase plate are identified as normal. Stimulation and mating of mice Young B6CBAF1 females aged 7 weeks and 7 months were mated with C57N males after superovulation, the latter consisting of injecting PMSG at 10 IU (SIGMA-ALDRICH) followed by ovulation induction via hCG administration (Human Chorionic Gonadotropin) at 10 IU (SIGMA-ALDRICH), 46-48h later. The day after mating, mice with a vaginal plug were sacrificed. Oocytes were collected from the oviducts 15-16h after hCG injection and the fertilization rate was assessed by the presence of the second polar body in the perivitelline space. Incubation of fertilized mouse oocytes Fertilized mouse oocytes collected after mating were randomized into four groups (young mice exposed to cyclic QDE peptide (QDEc), young controls, older mice exposed to QDEc, and older controls) and placed in 20 μL drops of culture medium (KSOM) for the controls and supplemented with 100 μM of QDEc for the exposed group. Exposure lasted from day 1 to day 4 after in vivo mating (day 0). QDEc is equivalent to human FEEc. The culture dishes were incubated at 37°C under 5% CO2 and covered with mineral oil. The oocytes were observed daily under a binocular microscope to assess signs of embryonic development. Normal development kinetics corresponds, at a minimum, to a cleaved 2-cell embryo on day 2 and to a morula or blastocyst embryo on day 5. Prospective randomized study in human IVF A clinical trial began on September 8, 2014, at the IVF laboratory of the Cochin Assisted Reproductive Technology Center, involving 66 couples. The mean age of the women was 34.3 ± 4.2 years, and the mean age of their partners was 37.0 ± 5.2 years. This was a prospective, single-center, randomized study of In Vitro Fertilization (IVF) performed with or without FEEc. Oocytes, retrieved from their cumulus clouds, were divided into two groups, alternating between them according to the order of retrieval. Once all oocytes had been retrieved, a technician who had not participated in the cumulus cloud collection randomly determined which of the two groups would be inseminated with FEEc and which would serve as the control. Part of the oocytes is incubated in the standard culture medium (Global, JCD), the other part in the same medium supplemented with FEEc 1000M. The methodology of this study, illustrated in Figure 9, is presented below. Human oocytes from women aged 18 to 43 years were retrieved by ovarian puncture after hormonal stimulation by gynecologists. They were randomly divided into two groups: oocytes incubated in either a standard culture medium supplemented with 100 μM FEEc or a standard culture medium (Global, JCD) and then placed in an incubator at 37°C under a 5% CO2 atmosphere. Partner sperm were retrieved in the laboratory, and the most motile sperm were selected according to a standard preparation method used in assisted reproductive technology (ART) centers. IVF consists of combining the sperm and oocytes in the insemination medium at a concentration of 10⁵ selected spermatozoa / ml in 20 microliter drops in oil. Insemination takes place in an incubator at 37°C under 5% CO2 for 18 hours.Eighteen hours after insemination (Day 1), the oocytes are decocted. The fertilized oocytes are washed and transferred to a fresh drop of medium and cultured for an additional 24 hours. During intrauterine transfer, they are washed three times, then placed in transfer medium and positioned in the uterine cavity. Embryos are transferred according to their apparent quality, regardless of their blood group of origin. One or more embryos are transferred depending on the age of the couple, the indication for IVF, the number of attempts, and the quality of the embryos obtained, with the consent of the couples. Some embryos may undergo extended culture for 5 days (at the blastocyst stage), either immediately or after embryo transfer on Day 2. The main evaluation criterion is the clinical pregnancy rate per fresh or frozen embryo transfer and the miscarriage rate taking into account the 3 groups: homogeneous transfers (control and treated) and mixed (mixture of both). The secondary criteria are: Fertilization rates, i.e., the ratio of the number of zygotes having two pronuclei in the cytoplasm, 18 hours after insemination, to the number of oocytes in metaphase 2 in the cohort. The percentage of good-quality embryos (Le) is defined as embryos whose cleavage sequence corresponds to the ideal sequence: 4 to 5 cells on day 2 and 8 to 9 cells on day 3, and whose blastomere fragmentation is type A (when the volume occupied by the fragments is less than 10% of the embryonic volume) or type B (when the volume occupied by the fragments is between 10% and 30% of the total embryo volume). These "Top" embryos are thus represented by the ratio of the number of embryos of each type to the total number of embryos in each group. This protocol received a favorable opinion from the Ethics Committee (CPP) West VI on December 13, 2012. The study was authorized by the French Biomedicine Agency on July 8, 2013. IVF is performed in strict compliance with Good Clinical Practice. Each couple agreeing to participate in the study has signed a free and informed consent form. Statistical analysis Quantitative variables were analyzed using their frequencies, mean, and standard deviation. Data were compared between the exposed and unexposed groups using an appropriate test (Student's t-test or Wilcoxon signed-rank test) for quantitative variables. Comparisons of percentages were performed using a chi-square (χ²) test or Fisher's exact test. Differences between the compared data were considered statistically significant when the p-value (significance threshold) was less than 0.05. Example 1: Improvement of sperm motility parameters and the percentage of hyperactivated sperm in men Preliminary experiments have shown that in an 18-hour survival test of sperm incubated in the presence of the FEE peptide, survival is significantly improved in the group treated with FEE at a concentration of 100μM compared to the control. Example 1: Automated analysis of sperm motility parameters after incubation in the presence of FEEc and a scramble peptide as a control. The results presented in Table 1 below show an increase in smoothed VAP (p=0.008), VSL (p=0.048), VCL (ρ0,000), and ALH (p=0.002), resulting in a 29% increase in hyperactivated sperm (p=0.009) compared to the control group. This improvement in the percentage of hyperactivated sperm explains the improvement in their fusogenic capacity and the increased fertilization rates observed in mice with intact cumulus oocyte complexes. Table 1: Automated analysis of sperm motility parameters compared between control groups and after incubation in the presence of FEEc. Example lb: Measurement of mitochondrial membrane potential The mitochondrial membrane potential is found to be increased by 21% in the presence of the FEEc peptide compared to the "scramble" peptide (pO.001) (Figure 1). FEEc therefore improves sperm motility parameters by increasing sperm mitochondrial membrane potential. Example: Study of the fertilization index The results presented in Figure 2 show that not only are more sperm fused with uncoiled oocytes in the presence of the FEEc peptide, but the latter are also decondensed unlike those in the control group. In control oocytes, an average of 19.0 ± 4.6 spermatozoa were found in the cytoplasm, whereas an increase to 36.9 ± 11.7 fused spermatozoa per oocyte was reported after incubation with 100 μM FEEc (p<0.001). This phenomenon suggests improved sperm fertilization and oocyte activation mediated by the FEEc peptide. Sperm motility parameters from 37 patients were analyzed in the presence and absence of FEEc (3-hour incubation). There was a significant increase in the percentage of hyperactivated spermatozoa according to Mortimer's criteria, which explains the increase in their fertilization (see Table 1 above). Example 2: Improving the in vitro maturation percentages of immature human oocytes A significant increase in oocyte maturation under FEEc was observed at day 1 (J1) for all human oocytes tested. The results were as follows: 42.3% (69 / 163) of oocytes in metaphase II (M1) under FEEc versus 30.0% (52 / 173) in the control group, p=0.02 (Figure 3). This increase in maturation was even more pronounced for oocytes from women aged 37 years and older at J1. The results were as follows: 47.9% (23 / 48) of oocytes in M1 under FEEc versus 20.4% (11 / 54) in the control group, (p=0.003). Although small for oocytes from women under 37 years of age, the improvement in oocyte MIV in humans is very significant for oocytes from older women since the same maturation rate as for oocytes from younger women was obtained (47.9% of oocytes in metaphase II in the presence of FEEc versus 20.4% in the control group, p=0.003). The results obtained with the 336 human oocytes at the VG stage, incubated randomly in the presence or absence of the FEEc peptide, show that the presence of FEEc improves the maturation rate of human oocytes. No significant difference in oocyte atresia rates was detected between the two groups (16.5% in the presence of the peptide versus 16.8% for the third control group) (Figure 4). In the sample of women aged 37 years and older, a non-significant trend toward a decrease in the atresia rate was observed in the presence of the FEEc peptide (16.7%, 8 / 48) compared to the control group (24.1%, 13 / 54) p>0.05. The study was continued and further results were obtained. These results confirm the findings described above and highlight other effects of the FEEc peptide, as described below. A total of 600 oocytes were placed in vitro for maturation. For the analysis, only one oocyte per woman was included in the study to ensure that all events were independent. In the presence of Fertiline in the medium, the maturation rate increased from 38.3% to 59.0% (P<1.6 × 10⁻⁴) (Figure 12). When the analysis was performed according to the age of the woman from whom the oocytes were obtained, the improvement in maturation was relatively modest for women under 37 years of age (42.6% to 51.8%, P<0.2), but much greater in oocytes from women aged 37 years and older (35.1% to 65.9%, P<3.1 × 10⁻⁵). Fertiline is therefore able to stimulate the in vitro maturation of the decoronized oocyte and the expulsion of the first polar body. She apparently does this all the better when the age-related energy deficit in the oocyte is greater.This study also highlighted an increase in the maturation rate, particularly of oocytes in the left ventricle (VG) of women over 37 years of age and under 30 years of age (Figure 13). Example 3: Organization of the meiotic spindle of human oocytes matured in vitro Figure 5 shows the incomplete and aberrant organization of chromosome alignment on the metaphase plate with misaligned chromosomes. The image was obtained from a human oocyte in MH after 24 h of IVM in the presence of control medium. Example 4: Improving fertilization rates and early embryonic development in mice At J1, the fertilization rate remains unchanged in young mice, while it increases from 39% to 51% in older mice (p<0.03) (Figure 6). At day 2, among young mice, 50.0% (26 / 52) of oocytes were cleaved in the QDEc group versus 32.4% (18 / 56) in the control group (p=0.02) (Figure 7). In older mice, there were significantly more cleaved embryos at day 2 in the presence of QDEc (58.7%, 37 / 63) compared to the control (35.4%, 23 / 65), p=0.008. At day 34, the proportion of cleaved embryos from young mice reaching the morula or blastocyst stage was 34.6% (7 / 16) for the control group versus 63.0% (17 / 26) for the QDEc-supplemented medium, p<0.03. This proportion reached 86.3% (63 / 73) for older mice in the presence of the peptide against 28.3% (15 / 53) in the control medium, p=0.001. Interestingly, blastocyst formation in mice is enhanced in the presence of the QDEc peptide. The percentages of atretic embryos were not significantly different between the two groups among young mice: 17.3% (9 / 52) in the presence of QDEc and 30.4% (17 / 56) in the control group (p=0.1). Compared to young mice, the percentage of atresia was higher in the older mouse group, with similar rates between the QDEc (38.1%, 24 / 63) and control (49.2%, 32 / 65) groups (p=0.2) (Figure 8). To study preimplantation embryonic development in more detail, the following protocol was implemented: Mice were mated on day 0. On day 1, oocytes were retrieved by laceration of the fubar ampullae. They were then randomly divided into two groups and cultured with or without QDEc before fertilization. The results show an increase in morulas on day 3, blastocysts on day 4, and among them, an increase in expanded blastocysts when follicles were incubated in the presence of QDEc (31.1% vs. 45.9%, p < 0.009) (Figure 14). Furthermore, after transfer of these blastocysts into pseudopregnant females, there was a significant increase in the number of offspring obtained per transferred embryo, especially for embryos from young mice (Table 2 below). Control 109 37 34% Youth 0.05 : :;:F rttiir>ë;::. '. 46% Aged lliilllï Fertiline ¾¾¾¾ 40% Witness 167 Total Fertiline ¾' - i.73;¾ 46% Table 2: Birth rates after transfer of control and QDEc-treated embryos in mice Example 5: Increase in clinical pregnancy rates in In Vitro Fertilization The results presented below were obtained in the "Fertiline" study. Initially, 56 couples were included. The mean age was 33.9 ± 4.1 years for the women and 36.7 ± 5.3 years for the partners. No significant differences were observed in fertilization rates or the percentage of top-quality embryos between the two FEEc groups versus the control group. A non-significant trend toward increased pregnancy rates per transfer was observed in the presence of the peptide compared to the control and mixed groups: 46.1% (6 / 13) versus 29.4% (5 / 17) and 40% (2 / 5), respectively (Figure 10 and Table 2 below). One miscarriage was reported after the transfer of an embryo from the control group. No adverse events have been reported to date. To date, 66 couples have been included in the study. 54 transfers have been performed: 26 with control embryos, 22 with embryos from the FEE group, and 6 with embryos from both groups (mixed transfers). The cumulative pregnancy rate was 34.6%, 45.5%, and 33.3% in the three groups, respectively. The spontaneous miscarriage rates were 33.3%, 10%, and 0%, respectively. The clinical pregnancy rates were therefore 23%, 41%, and 33.3%, respectively. Crucially, when the clinical pregnancy rate is adjusted for patient age, it is observed that in younger patients, the rate of ongoing clinical pregnancy (i.e., pregnancy reaching term) is significantly increased, from 20% to 57.1% (P<0.03). Table 3: Pregnancy rate per transfer (for 66 patients) The results also show a 21% increase in clinical pregnancies after transfer of an embryo from the FEEc group: 40.0% (8 / 20) versus 33.3% (8 / 24) in the control group (p>0.05). Fertilization rates were 66.2% among the controls versus 67.6% in the FEEc-exposed group (p>0.05). The early spontaneous miscarriage rate reached 37.5% (3 / 8) in the control group versus 11.1% (1 / 9) when the embryo was exposed to FEEc (p>0.05) (Figure 11). For women under 37 years of age, after exposure to FEEc, fertilization rates were 70.9% compared to 68.3% in the control group. When the oocyte was exposed to FEEc, pregnancy rates reached 57.1% compared to 20.0% in the control group (Table 4 below). Age woman Number of couples (n) Fertilization rate (year) Pregnancy rate (year) Control (%) FEEc (%) Control (%) FEEc (%) 26-36 47 68.3% 70.9% 20.0% 57.1%* 37-43 19 60.2% 60.3% 33.0% 20.0% Table 4: Fertilization and pregnancy rates according to partner age (>37 or <37 years) * P<0.03) Of the first 66 couples included in the study to date, 51 transfers have been performed, the others being deferred, and the results show that of these 51 transfers: 21 were produced from embryos in the control group; 18 from the group in the presence of the FEEc; 12 with embryos from both groups. The pregnancy rate is higher and miscarriages are less frequent for the group in the presence of FEEc compared with the other groups (33% miscarriages versus 9% for embryos in the presence of FEEc) (Tables 5 and 6 below). Pregnancy rates for young women increased from 20% to 57.1% (p<0.03) in the presence of FEEc compared to the control. Table 5: Pregnancy rates per transfer (for 66 patients) Table 6; Pregnancy rate per transfer according to the age of the women. The continuation of the Fertilme study yielded additional results. A total of 66 couples were included in the study. The results are reported in the tables below. The overall data from the attempts are reported in Table 7. Overall fertilization rates were virtually unchanged. However, the percentage of attempts with low fertilization rates (less than 20%) decreased from 37.9% to 27.3% in the presence of FEEc, suggesting improved gamete fertilization in the presence of Fertiline. Similarly, polyspermia was of the same order of magnitude in the Fertiline group as in the control group (4.0% vs. 5.2%), showing that the normal mechanism blocking polyspermia was not altered. Witness Ferîilme Woman's Age UM2 592 (91,4?·,,) 302 f <J2.9 "■<■-} fl (90.0 'Ό) :iiiiiiiii ¾ 1 6 p4Ucife tsdat!t>n 25 (37.9%) 1 » ¾ ' ,) Failed to found. :'::;¾¾û ; '.··'·: 6 (9Λ%) BSai>toi rmatkm utile 70 (35.0%) Table 7: Overall results of the Fertiline study on 66 patients The results of all couples who had an embryo transfer are reported in Table 8. This table excludes couples who had an unexplained fertilization failure in both groups of oocytes with or without Fertiline (n=6). Table 8: Results obtained in the 66 couples of the Fertiline study As shown in Table 8, for patients who underwent embryo transfer, the fertilization rate increased from 69.5% to 78.6%, a 13% improvement, but this increase was not statistically significant in the present cohort. Embryonic development up to the blastocyst stage was unchanged, as was the implantation rate of embryos in the uterus. However, the miscarriage rate decreased by nearly 50% in the Fertiline group (15.4% vs. 30.5%). The results for couples who underwent embryo transfer and in whom the woman was under 37 years of age are reported in Table 9. If we consider only couples where the woman is under 37 years old (n=47), representing more than 70% of the patients treated, we observe a significant improvement in the fertilization rate, increasing from 70.9% to 83.3% (P<0.05). The miscarriage rate decreases from 36.3% for patients who received an embryo from the Control group to 9.1%, four times lower, for those who received an embryo from the Fertiline group. In fact, the rate of ongoing pregnancies resulting in a live birth increases from 28.6% in the Control embryo group to 41.6% in the Fertiline embryo group. Table 9: Results obtained in couples where the woman is under 37 years old in the Fertiline study.

Claims

DEMANDS 1. Cyclic peptide comprising the tripeptide reproducing a binding site of fertilin beta to integrin of the oocyte, for use in medical or non-medical applications in which stimulation of cellular energy metabolism is desired.

2. Peptide according to claim 1, for its use according to claim 1, characterized in that the drug increases the speed of sperm progression and their fertilization in a medically assisted reproduction (MAR) protocol, 3. Peptide according to claim 1, for its use according to claim 1, characterized in that the drug increases the rate of hyperactivated sperm in an ART protocol.

4. Peptide according to any one of claims 1 to 3, for use according to any one of claims 1 to 3, in an intrauterine insemination (IUI) protocol.

5. Peptide according to any one of claims 2 to 4, for use according to any one of claims 2 to 4, characterized in that the spermatozoa are incubated for 1 minute to 3 hours in the presence of 1 to 100 μM of peptide before intrauterine insemination.

6. Peptide according to claim 1, for its use according to claim 1, characterized in that the drug improves the in vitro maturation of oocytes.

7. Peptide according to claim 6, for its use according to claim 6, to improve the in vitro maturation of human oocytes.

8. Peptide according to any one of claims 6 or 7, for its use according to any one of claims 6 or 7, to enhance fertilization and oocyte activation.

9. Peptide according to any one of claims 6 to 8, for use according to any one of claims 6 to 8, characterized in that oocytes are incubated for a period of between 1 minute and 4 days in the presence of 1 to 100 μM of peptide.

10. Peptide according to any one of claims 6 to 9, for its use according to claim in claims 6 to 9, characterized in that the drug improves blastomere ploidy during the first week of zygote development.

1. Peptide according to claim 1, for its use according to claim 1, characterized in that the drug improves pre-implantation embryonic development.

12. Peptide according to claim 11, for its use according to claim 11, characterized in that the drug is used under prolonged culture conditions.

13. Peptide according to claim 1, for its use in improving the cryopreservation of gametes and embryos.

14. Peptide according to any one of the preceding claims, for use according to any one of the preceding claims, in an in vitro fertilization (IVF) protocol.

15. Peptide according to claim 10, for its use according to claim 10, characterized in that the drug increases the chances of pregnancy.

16. Peptide according to claim 10, for its use according to claim 10, characterized in that the drug reduces the risk of miscarriage.

17. Peptide according to claim 10, for its use according to claim 10, characterized in that the drug reduces the risks of aneuploidy, in particular trisomy.

18. Peptide according to claim 1, for its use according to claim 1, characterized in that the drug is used to promote ex vivo cell culture, 19. Peptide according to claim 18, for its use according to claim 18, characterized in that the cells are lymphocytes.

20. Peptide according to claim 1, for its use according to claim 1, characterized in that the drug is administered into the eye.

21. Peptide according to claim 1, for its use according to claim 1, characterized in that the drug is vectorized.

22. Peptide according to claim 1, for use in a cosmetic or therapeutic composition intended for topical application.

23. Cosmetic or dermatological composition comprising a cyclic peptide including the tripeptide reproducing a binding site of fertilin beta to integrin of Povocyte.

24. Eye drops comprising a cyclic peptide including the tripeptide reproducing a binding site of fertilin beta to integrin of the oocyte.