WO2018115788A1

WO2018115788A1 - Adjuvants for cell culture media, in particular forin vitro fertilisation, or for the culture of follicles, male germ cells or embryos

Info

Publication number: WO2018115788A1
Application number: PCT/FR2017/053816
Authority: WO
Inventors: Mohamed BIDRI; Patrick Choay
Original assignee: Patrick Choay Sas
Priority date: 2016-12-23
Filing date: 2017-12-22
Publication date: 2018-06-28
Also published as: FR3061208B1; FR3061208A1

Abstract

The subject matter of the present invention is compositions for in vitro cell culture, characterized in that they comprise at least one growth factor in combination with at least one compound chosen from the group made up of a zinc salt, inositol in any one of the stereoisomeric forms thereof and camitine or a camitine derivative. The subject matter of the invention is also methods for in vitro fertilisation and methods for the maturation of follicles, of male germ cells or of embryos using such culture medium adjuvants, and also kits for carrying out these methods which comprise an abovementioned lyophilisate and an aqueous solution, containing the other constituents of said culture media.

Description

ADJUVANTS FOR CELL CULTURE MEDIA, IN PARTICULAR FOR IN VITRO FERTILIZATION, OR FOR THE CULTURE OF

FOLLICLES, GERMINAL CELLS, MALES OR EMBRYOS

The subject of the present invention is compositions which can be used as adjuvants for cell culture media, in particular for developing follicles or for the maturation of cells of the male germ line, as well as for in vitro fertilization and the development of cells. embryos, and more particularly mammalian embryos, including human embryos, possibly frozen.

After maturation of De Graaf's follicle, ovulation allows the oocyte to pass into the fallopian tube for possible fertilization. The resulting embryo remains there for 3 to 5 days to develop and reach the morula and blastocyst stages.

In the trunk, the gametes, the embryo, the morula, then the blastocyst are surrounded by the tubal fluid. The composition of the latter is complex: it combines the fundamental components of the mother's internal environment with those of the follicular fluid, and it does not seem to undergo significant qualitative changes during the first days of the post-ovulatory phase. Having reached the blastocyst stage, the embryo passes into the uterus whose mucosa has then reached a favorable state of development for the implantation and implantation of the egg.

The passage of the embryo into the uterus occurs only when both have reached the level of physiological development compatible with implantation and implantation: blastocyst stage for the embryo, endometrium close to its secretory phase for the uterus.

In the case of medical assistance to procreation, these physiological conditions suggest a unique and constant culture medium, and not sequential as it currently exists, combining all the elements necessary for the development of in vitro fertilization, possibly consecutive at ICSI (para cytoplasmic sperm injection, or intracytoplasmic sperm injection) until the embryo is transferred.

The present invention stems from the demonstration by the inventors of constant compositions that can be used both for culturing follicles or for cells of the male germline, for the fertilization of the oocyte and the development of the embryo to the blastocyst stage, and with yields comparable to or even greater than those obtained with the compositions currently on the market.

The present invention relates to compositions for in vitro culturing of cells, characterized in that they comprise:

at least one growth factor, preferably at least two growth factors, in combination with at least one compound selected from the group consisting of:

a zinc salt, preferably a water-soluble salt such as zinc chloride or zinc sulphate, in particular at a concentration of 0.1 to 3 μg / ml, preferably at a concentration of 0.5 to 1.5; μg / ml, expressed as zinc element,

inositol in any of its stereoisomeric forms, preferably myo-inositol, especially in the form of a salt, for example in the form of phosphate, preferably inositol triphosphate, in particular at a concentration of 1.8 to 18 mg / ml, preferably at a concentration of 5 to 10 mg / ml, expressed as inositol molecule,

and

carnitine in one of its stereoisomeric forms, preferably L-carnitine, a carnitine salt, preferably the hydrochloride, a carnitine derivative, preferably acetyl carnitine or a salt of a carnitine derivative; preferably in the form of hydrochloride, in particular at a concentration of 0.1 to 2 mg / ml, preferably at a concentration of 0.5 to 1 mg / ml, expressed as carnitine base, and, if appropriate, with one or more compounds selected from compounds of the family of corticosteroids involved in energy production in mammals and at least one key coenzyme of energy metabolism, such as NAD / NADH and NADP / NADPH.

The present invention particularly relates to compositions for the in vitro culture of follicles being developed for the maturation of the oocytes contained in said follicles, or for the maturation of cells of the male germline, or for in vitro fertilization oocytes by the spermatozoa, or for the development of embryos, possibly after thawing of the follicles or embryos, characterized in that it comprises:

at least one growth factor, preferably at least two growth factors, in combination with a zinc salt, preferably a water-soluble salt such as zinc chloride or zinc sulphate, especially at a concentration of 0.1 to 3 μιτιΐ, preferably at a concentration of 0.5 to 1.5 μg / ml, expressed as zinc element, and at least one compound chosen from the group consisting of:

inositol in any of its stereoisomeric forms, preferably myo-inositol, especially in salt form, for example in the form of phosphate, preferably inositol triphosphate, in particular at a concentration of 1.8 to 18 mg / ml, preferably at a concentration of 5 to 10 mg / ml, expressed as inositol molecule,

and

carnitine in one of its stereoisomeric forms, preferably L-carnitine, a carnitine salt, preferably the hydrochloride, a carnitine derivative, preferably acetyl carnitine or a salt of a carnitine derivative; preferably in the form of hydrochloride, in particular at a concentration of 0.1 to 2 mg / ml, preferably at a concentration of 0.5 to 1 mg / ml, expressed as carnitine base, and, if appropriate, with one or more compounds selected from compounds of the family of corticosteroids involved in energy production in mammals and at least one key coenzyme of energy metabolism, such as NAD / NADH and NADP / NADPH. The compositions according to the invention may preferably comprise a growth factor, two growth factors or three growth factors.

The compositions of the invention can be assimilated to "adjuvants" or

"complements" or "boosters". These are compositions which, when combined with other elements, make it possible to obtain culture media.

In the compositions of the invention, the zinc salt concentrations correspond to the amount of element per volume.

Preferably, the concentrations of zinc salt in the above-mentioned compositions of the invention are from 0.1 μg / ml to 3 μg / ml, preferably from

0.5 μg / ml to 1.5 μg / ml expressed in zinc element.

The concentrations of zinc salt in the above-mentioned compositions of the invention are in particular 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9; 1.0; 1.1;

1,2; 1.3; 1.4 or 1.5 μg / ml. In the compositions of the invention, the inositol may in particular be present in the form of phosphate derivatives of inositol, such as inositol mono-, di- or tri-phosphate, or in the form of salts. Inositol concentrations correspond to the amount of element per volume.

Preferably, the inositol concentrations in the aforementioned compositions of the invention range from 1.8 mg / ml to 18 mg / ml, preferably from 5 to 10 mg / ml.

The concentrations of inositol in the aforementioned compositions of the invention are in particular 1.8; 2; 3; 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 16; 17 or 18 mg / ml.

In the compositions of the invention, the carnitine is in the form of an L enantiomer and the carnitine concentrations correspond to the amount of base per volume.

Preferably, the carnitine concentrations in the aforementioned compositions of the invention are from 0.1 mg / ml to 2 mg / ml, preferably from 0.5 mg / ml to 1 mg / ml.

The concentrations of carnitine in the aforementioned compositions of the invention are in particular 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9; 1.0; 1.1; 1,2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9 or 2.0 mg / ml.

The invention more particularly relates to the abovementioned compositions for the in vitro culture of developing follicles for the maturation of the oocytes contained in said follicles, or for the maturation of cells of the male germline, or for fertilization in vitro oocytes by spermatozoa, or for embryo culture, possibly after thawing of follicles, male cells, or embryos.

Advantageously, the growth factors contained in the compositions of the invention belong to 5 families of cytokines. Hormones and growth factors are present at balanced concentrations, compatible with physiological conditions. The compositions of the invention provide the cells in growth and differentiation regulatory molecules they are imperative. Thus, these compositions are capable of allowing the maturation of immature human oocytes cultured in the presence of follicular cells, as well as the maturation of the cells of the male germ line. They can also ensure the development of the human embryo to the blastocyst stage. Now this stage has a plus a high percentage of uterine implantation only when embryo transfer occurs at a less advanced stage, especially at the two to about eight-cell stages.

The subject of the invention is more particularly the abovementioned compositions comprising at least two growth factors chosen from:

insulin-like growth factors, also referred to as insulin-like growth factor (IGF) such as IGF-1 and / or IGF-2,

the epidermal growth factor, also referred to as EGF (epidermal growth factor) and / or HB-EGF (heparin-binding epidermal growth factor),

the alpha transformation growth factor, also referred to as TGF alpha (transforming growth factor),

beta-transformation growth factor, also called TGF beta (transforming growth factor beta), in the form of TGF-beta 1, 2 and 3, preferably TGF-beta-1,

hepatic growth factor, also known as hepatocyte growth factor (HGF),

colony stimulating factor of granulocytes and macrophages, also designated GM-CSF (granulocyte-macrophage colony stimulating factor),

growth and differentiation factors, also referred to as GDF (growth dijferentiation factor), preferably GDF-9,

- the inhibitory factor of leukemia, also called LIF (leukemia inhibitory factor).

Advantageously, the compositions of the invention contain at least three growth factors chosen from those listed above.

Particularly preferred compositions contain at least IGF-1 and / or IGF-2.

More preferably, the aforementioned compositions of the invention comprise all of the aforementioned growth factors.

Preferably, the concentrations of growth and differentiation factors in the aforementioned compositions of the invention are nanomolar, and especially between about 0.25 and about 100 μg / L, preferably between 0.25 and about 60 μg / L. or between about 0.5 μg / L and about 50 μg / L.

Preferably, the concentrations of growth and differentiation factors in the aforementioned compositions of the invention are nanomolar, and especially from 0.25 to 100 μg / L, preferably from 0.25 μg / L to 60 or from 0.5 to 50 μg / L.

Preferably, the concentrations of growth and differentiation factors in the aforementioned compositions of the invention are 0.5; 1; 2; 3; 4; 5; 6; 7; 8; 9; 10; 12.5; 20; 25; 30 ; 40; 50; 60; 70; 80; 90 or 100 μg / L.

Advantageously, the concentrations of the growth and differentiation factors contained in the compositions of the invention are such that:

the concentration of EGF is from approximately 10 μg / l to approximately 100 μg / l, in particular from approximately 40 μg / l to approximately 60 μg / l, in particular approximately 50 μg / l,

the concentration of IGF-1 is from approximately 10 μg / l to approximately 100 μg / l, in particular from approximately 12.5 μg / l to approximately 25 μg / l, in particular approximately 15 μg / l,

the concentration of IGF-2 is from approximately 10 μg / l to approximately 100 μg / l, in particular from approximately 12.5 μg / l to approximately 25 μg / l, in particular approximately 15 μg / l,

the concentration of TGFα is from approximately 10 μg / l to approximately 50 μg / l, in particular from approximately 20 μg / l to approximately 30 μg / l, in particular approximately 25 μg / l,

the concentration of HGF is from approximately 10 μg / l to approximately 100 μg / l, in particular from approximately 40 μg / l to approximately 60 μg / l, in particular approximately 50 μg / l,

the concentration of GM-CSF is from approximately 1 μg / l to approximately 5 μg / l, in particular from approximately 1.25 μg / l to approximately 2.5 μg / l, especially around 2 μg / l ,

the concentration of GDF-9 is from approximately 2 μg / l to approximately 10 μg / l, in particular from approximately 4 μg / l to approximately 6 μg / l, in particular approximately 5 μg / l,

the concentration of TGF-beta-1 is from approximately 10 μg / l to approximately 100 μg / l, in particular approximately 50 μg / l,

the concentration of LIF is from approximately 10 μg / l to approximately 100 μg / l, in particular approximately 50 μg / l,

the Zinc concentration, particularly in the form of chloride or Zinc sulphate, is approximately 1 μg / ml,

the concentration of Inositol, particularly in the form of myo-inositol, is approximately 5 mg / ml,

the concentration of L-carnitine, particularly in base form or hydrochloride, is about 0.6 mg / ml, expressed as carnitine base.

A preferred form of composition according to the invention comprises approximately 12.5 μg / L of IGF-1, 50 μg / L of EGF, 1 μg / ml of Zinc, 5 mg / ml of Inositol, 0.6 mg / ml ml of L-carnitine base. The invention also relates to the abovementioned compositions for in vitro culture, comprising a compound of the family of corticoids as defined above, and preferably hydrocortisone.

The subject of the invention is also the abovementioned compositions for in vitro culture, comprising at least one compound of the family of corticosteroids involved in energy production in mammals, preferably hydrocortisone in the form of a water-soluble salt such as hemisuccinate. hydrocortisone, hydrocortisone sodium succinate and hydrocortisone sodium phosphate.

The subject of the invention is also the abovementioned compositions for in vitro culture, comprising at least one zinc salt in hydrated or anhydrous form chosen from the following salts: zinc chloride, zinc sulphate, zinc gluconate, zinc picolinate, citrate zinc, zinc acetate, zinc lactate, zinc stearate, preferably a water-soluble salt such as zinc sulfate or zinc chloride.

The invention also relates to the abovementioned compositions characterized in that the at least one zinc salt is in hydrated or anhydrous form and is chosen from the following salts: zinc chloride, zinc sulphate, zinc gluconate, zinc picolinate, zinc citrate, zinc acetate, zinc lactate, zinc stearate, preferably a water-soluble salt such as zinc sulfate or zinc chloride.

Advantageously, the above-mentioned hydrocortisone is in the form of a water-soluble salt, especially in the form of hydrocortisone hemisuccinate. Preferably, the concentration of the hydrocortisone salt in the abovementioned compositions is between about 5 × 10 ^-7 M and about 10 ^-6 M, and is especially about 7 × 10 ^-7 M, ie about 350 μg / L for hydrocortisone hemisuccinate.

Advantageously, the compositions defined above contain key coenzymes of the aforementioned energy metabolism, in particular at concentrations such that:

the concentration of NAD / NADH is approximately 1 mg / L,

the concentration of NADP / NADPH is approximately 1 mg / l.

The subject of the invention is also the abovementioned compositions for in vitro culture, comprising:

- at least two growth factors, at least one water-soluble zinc salt selected from zinc sulphate or zinc chloride and / or myo-inositol and / or carnitine or a carnitine salt,

hydrocortisone hemisuccinate, and

NAD / NADH and / or NADP / NADPH.

The invention also relates to the compositions defined above which are in the form of a lyophilizate, namely in the form of compositions whose different constituents are brought to the dry state, and are capable of being put back in solution while keeping their physical properties. -chemical and biological.

As such, the invention more particularly relates to the aforementioned lyophilizates, containing:

at least one growth factor as defined above, or all of these factors,

at least one compound selected from the group consisting of:

a zinc salt, especially at a concentration of 0.1 to 3 μg / ml, preferably at a concentration of 0.5 to 1.5 μg / ml, expressed as zinc element

inositol in any of its stereoisomeric forms, preferably myo-inositol, especially at a concentration of 1.8 to 18 mg / ml, preferably at a concentration of 5 to 10 mg / ml, expressed in inositol molecule,

and

carnitine in one of its stereoisomeric forms, preferably L-carnitine, a carnitine salt, preferably the hydrochloride, a carnitine derivative, preferably racetyl carnitine, or a salt of a carnitine derivative, in particular at a concentration of 0.1 to 2 mg / ml, preferably 0.5 to 1 mg / ml, expressed as carnitine base,

and eventually

at least one compound of the family of corticosteroids defined above, such as hydrocortisone hemisuccinate,

and / or at least one key coenzyme of the energetic metabolism defined above, such as NAD / NADH and NADP / NADPH.

The subject of the invention is also the application of the abovementioned compositions or lyophilizates as effector adjuvants for the preparation of in vitro culture media of follicles under development for the maturation of the oocytes contained in said follicles, or for the maturation of male germ line cells, or for in vitro fertilization of oocytes by spermatozoa, or for embryo culture, optionally after thawing follicles, male cells, or embryos, said in vitro culture media comprising a composition as defined above, in combination with the elements conventionally used in the context of in vitro fertilization, or culturing follicles, male cells, or embryos, said elements being chosen in particular from human or bovine serum albumin, where appropriate recombinant, and / or mineral salts, and / or energetic molecules such as glucose, pyruvate , citrate and lactate, and / or amino acids for the biosynthesis of proteins, and / or purine and pyrimidine bases for the biosynthesis of nucleic acids, and / or phospholipids or cholesterol for the formation of cell membranes, and / or vitamins, such as vitamins of group B, including vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vita B9 mine, vitamin B12 and / or vitamin C and / or vitamin E, and optionally insulin and / or at least one other substance contributing to the process, in particular a growth factor and in particular GM-CSF and / or at least one antibiotic.

The aforementioned inorganic salts can be chosen from KCl, NaCl, MgSO ₄ , NaHCO ₃ , Na ₂ HPO ₄ , KH ₂ PO ₄ .

The subject of the invention is also the application of the abovementioned compositions or lyophilisates as effector adjuvants for the preparation of in vitro culture media of follicles under development for the maturation of the oocytes contained in said follicles, or for maturation of male germ line cells, or for in vitro fertilization of oocytes by spermatozoa, or for embryo culture, optionally after thawing of follicles, male cells, or embryos, said in vitro culture media comprising a composition as defined above, in combination with insulin.

The invention also relates to a process for the preparation of adjuvants of in vitro culture media defined above, characterized in that it comprises a step of mixing a composition as defined above, if appropriate after dissolution. in a suitable volume of an above-mentioned composition in freeze-dried form, with a solution containing the elements conventionally used in the context of in vitro fertilization, or the culture of follicles, male cells, or embryos, said elements being as defined above. The invention more particularly relates to in vitro culture media of follicles being developed for the maturation of the oocytes contained in said follicles, or for the maturation of cells of the male germline, or for in vitro fertilization oocytes by the spermatozoa, or for the culture of embryos, possibly after thawing the follicles, male cells, or embryos, said media being characterized in that they comprise a composition as defined above, in association with the elements above mentioned conventionally used in the context of in vitro fertilization, or the culture of follicles, male cells, or embryos.

The subject of the invention is also a process for maturing the developing mammalian follicles with a view to the maturation of the oocytes contained in said follicles, and more particularly of human follicles, where appropriate after thawing previously frozen follicles, characterized in that it comprises a step of culturing in vitro follicles taken from the female, and more particularly from women, in a culture medium as defined above according to the invention, advantageously for about 24 to 48 hours .

The invention also relates to a method for maturation of cells of the male germ line of mammals, and more particularly human, where appropriate after thawing of previously frozen cells, characterized in that it comprises a step of culturing said cells in vitro. cells, in a culture medium as defined above according to the invention, especially for several jouis, preferably from 1 to 5 days.

The subject of the invention is also a process for the in vitro fertilization of oocytes by spermatozoa, and more particularly of oocytes and spermatozoa of human origin, where appropriate after thawing of previously frozen spermatozoa, characterized in that it comprises a step of culturing in vitro the oocytes and spermatozoa mentioned above, in a culture medium as defined above according to the invention, advantageously for about 1 day to about 2 days.

The invention also relates to a method for developing mammalian embryos, and more particularly human embryos, where appropriate after thawing previously frozen embryos, characterized in that it comprises a step of culturing in vitro said embryos, in a culture medium as defined above according to the invention, preferably during the first 6 days following the in vitro fertilization, where appropriate after thawing, advantageously to obtain embryos at the blastocyst stage.

In a nonlimiting manner, the invention relates in particular to the methods as defined above in which the mammals are chosen from:

- primates, including humans,

- sheep,

- cattle.

Advantageously, the compositions of the invention can be used both for the maturation of the oocytes or that of the cells of the male germ line, the in vitro fertilization of the oocytes, and the maturation of the embryos resulting from said fertilization, in particular until blastocyst stage, possibly after thawing of follicles, male cells, or embryos.

The subject of the invention is also a kit (or kit) for the extemporaneous preparation of a culture medium as defined above according to the invention, especially in the context of the implementation of a method mentioned above, kit comprising:

a composition as described above, or

a composition in the form of a lyophilizate as described above,

and, if appropriate, an aqueous solution containing the elements conventionally used in the context of in vitro fertilization, or the culture of follicles, male cells, or embryos, such as the elements defined above, in particular a commercial medium .

The present invention will be further illustrated by the following detailed description of examples of culture media prepared according to the invention.

As an aqueous solution as defined above, a culture medium prepared from the compositions according to the invention may comprise mainly the following compounds:

inorganic salts: KCl, NaCl, MgSO ₄ , NaHCO ₃ , Na ₂ HPO ₄ , KH ₂ PO ₄ ,

essential amino acids, as well as other amino acids such as glutamic acid, glycine, taurine, cysteine and glutamine,

- monosaccharides and metabolic derivatives, such as glucose, pyruvate, lactate, acetate,

vitamins, especially vitamins of group B and vitamin C,

purine and pyrimidine bases,

- albumin, phospholipids,

- cholesterol,

insulin and / or at least one other substance contributing to the process, in particular a growth factor,

antibiotics, such as penicillin G, streptomycin, gentamycin or other aminoglycoside.

EXPERIMENTAL PART :

In all the following experiments, the active ingredients are used at the following concentrations:

IGF-1 = 12.5 μg / l

TGF-α = 25 μg / l

EGF = 50 μg / l

Zinc expressed as element = 1 μg / ml

Inositol expressed in molecule = 5 mg / ml

Carnitine expressed as a base - 0.6 mg / ml are described below compositions of the effector adjuvant object of the present invention that can be used for the preparation of in vitro culture media of follicles in the course of development for the maturation of the oocytes contained in said follicles, or for the maturation of cells of the male germline, or for the in vitro fertilization of oocytes by the spermatozoa, or for the culture of embryos, possibly after thawing of the follicles , male cells, or embryos, said in vitro culture media.

All operations are performed with sterile raw materials and in sterile medium.

Composition 1 of Reference

A freshly prepared aqueous solution containing two growth factors is distributed in 1 ml vials, such that each vial contains 500 ng EGF and 125 ng IGF-1. This solution is to be diluted at ^1/10 in culture medium during the production processes. Composition 2 of Reference

An aqueous solution prepared extemporaneously containing two growth factors is distributed in 1 ml vials, such that each vial contains 500 ng of EGF and 125 ng of IGF-1 and 10 μg of zinc element.

This solution is to be diluted at ^1/10 in culture medium during the production processes.

Composition 3 of Reference

An extemporaneously prepared aqueous solution containing two growth factors is distributed in 1 ml vials, such that each vial contains 500 ng of EGF and 125 ng of IGF-1 and 50 mg of inositol.

This solution is diluted at1 / ^10th in a culture medium during the production processes.

Composition 4

An aqueous solution prepared extemporaneously containing two growth factors is distributed in 1 ml vials, such that each vial contains 500 ng of EGF and 125 ng of IGF-1, 50 mg of inositol and 10 μg of zinc element.

Composition 5 of Reference

An extemporaneously prepared aqueous solution containing two growth factors is distributed in 1 ml vials, such that each vial contains 500 ng of EGF and 125 ng of IGF-1 and 6 mg of L-carnitine base.

Composition 6

An extemporaneously prepared aqueous solution containing two growth factors is distributed in 1 ml flasks, such that each flask contains 500 ng of EGF and 125 ng of IGF-1, 6 mg of L-carnitine base and 10 μg zinc element Composition 7 of Reference

An extemporaneously prepared aqueous solution containing two growth factors is distributed in 1 ml vials, such that each vial contains 500 ng of EGF and 125 ng of IGF-1, 50 mg of inositol and 6 mg of L -carnitine base.

Composition 8

An extemporaneously prepared aqueous solution containing two growth factors is distributed in 1 ml vials, such that each vial contains 500 ng of EGF and 125 ng of IGF-1, 50 mg of inositol, 6 mg of L -carnitine base and 10 μg of zinc element.

Composition 9 of Reference

An aqueous solution prepared extemporaneously containing a growth factor is distributed in 1 ml vials, such that each vial contains 500 ng of EGF.

Composition 10 of Reference

A freshly prepared aqueous solution containing a growth factor is distributed in 1 ml bottles, such that each flask contains 500 ng of EGF and 10 μg of zinc element.

This solution is to be diluted at ^1/10 in culture medium during the production of procédésr

Composition 11 of Reference

An extemporaneously prepared aqueous solution containing a growth factor is distributed in 1 ml vials, such that each vial contains 500 ng of EGF and 50 mg of inositol. This solution is to be diluted 1/10 in a culture medium during the realization of the processes.

Composition 12

An aqueous solution prepared extemporaneously containing a growth factor is distributed in 1 ml bottles, such that each flask contains 500 ng of EGF, 50 mg of inositol and 10 μg of zinc element.

Composition 13 of Reference

A freshly prepared aqueous solution containing a growth factor is distributed in 1 ml vials, such that each vial contains 500 ng EGF and 6 mg L-carnitine base.

Composition 14

An aqueous solution prepared extemporaneously containing a growth factor is distributed in 1 ml vials, such that each vial contains 500 ng of EGF, 6 mg of L-carnitine base and 10 microgram of zinc element.

This solution is diluted 1:10 ^6th in a culture medium during the production processes. Composition 15 of Reference

A freshly prepared aqueous solution containing a growth factor is distributed in 1 ml vials so that each vial contains 500 ng of EGF, 50 mg of inositol and 6 mg of L-carnitine base.

Composition 16 A freshly prepared aqueous solution containing a growth factor is distributed in 1 ml vials, such that each vial contains 500 ng of EGF, 50 mg of inositol, 6 mg of L-carnitine base and 10 μg of zinc element.

All vials are immediately lyophilized and the lyophilisate is stored at + 4 ° C. All assays are performed in duplicate with both a growth factor (ie EGF) on the one hand and with 2 growth factors EGF, and IGF-1 on the other hand.

According to the invention, it is also possible to prepare the composition comprising GM-CSF with or without EGF, and compositions containing three growth factors.

In vitro maturation of ovocytes of women.

The purpose of this study is to establish the kinetics of maturation of supernumerary immature oocytes under different conditions. According to the Bioethics Law, the patients gave their consent that the supernumerary oocytes could be used for research.

Depending on the stage of oocyte maturation, the "oocytes at the stage of the germinal vesicle" or the "oocytes at the stage of metaphase I" are cultured in a conventional medium of in vitro maturation supplemented with one or more substances.

Oocyte maturation may be carried out on animal oocytes, for example the maturation of mutine or bovine oocytes.

Test la); Ovocvtes at the stage of the germinal vesicle (VG)

The number of VG oocytes that evolve at the metaphase I (MI) or metaphase II (MI1) stage is determined.

In this experiment, 9 groups are used and the tests are performed once with two growth factors and once with a single growth factor. Group No. 1 (control), 10 oocytes VG are placed in wells containing only the culture medium

Group No. 2, 10 VG oocytes are placed in wells containing the culture medium plus 2 growth factors (IGF-1, EGF).

Group 3, 10 VG oocytes are placed in wells containing culture medium plus 2 growth factors (IGF-1, EGF) and zinc

Group No. 4, 10 oocytes VG are placed in wells containing the culture medium plus 2 growth factors (IGF-1, EGF) and inositol.

Group No. 5, 10 oocytes VG are placed in wells containing the culture medium plus the 2 growth factors (IGF-1, EGF) of zinc and inositol.

Group No. 6, 10 VG oocytes are placed in wells containing the culture medium plus 2 growth factors (IGF-1, EGF) and L-carnitine.

Group No. 7, 10 oocytes VG are placed in wells containing the culture medium plus 2 growth factors (IGF-1, EGF), zinc and L-carnitine.

Group No. 8, 10 VG oocytes are placed in wells containing the culture medium plus 2 growth factors (IGF-1, EGF), inositol and L-carnitine.

Group No. 9, 10 oocytes VG are placed in wells containing the culture medium plus 2 growth factors (IGF-1, EGF), inositol, L-carnitine and zinc. The same tests are then repeated as previously described using a growth factor namely EGF.

The oocytes are cultured in the same oven at 37 ° C in an atmosphere containing 5% CO2. Results

It is observed that the addition of zinc to the 2 growth factors IGF-1, EGF and inositol results in an increase in the maturation of VG oocytes at the MI and M1 stage after 12H and 24H of culture compared to the VG oocytes placed in culture in the medium containing only 2 growth factors and inositol.

In addition, an improvement is also observed when the same comparative test is carried out with 1 growth factor EGF.

The same improvements in maturation are observed with the addition of zinc to L-carnitine in addition to the 2 growth factors. In addition, improvements are also observed when comparing with a growth factor EGF.

. Test lb) Metaphase I stage ovocvts

In this experiment, the number of oocytes which evolves in M II is determined. The same protocol as above is used by selecting oocytes at the ML stage

Results

Milk oocytes maturing at Mil are observed to improve with the addition of zinc to both growth factors and inositol and / or L-carnitine.

In addition, improvements are also observed when performing the comparative tests with a growth factor EGF.

Conclusion

The addition of zinc to both growth factors (IGF-1 and EGF) and inositol and / or L-carnitine provides a beneficial effect on in vitro maturation of oocytes in both the VG and MI stages. compared to oocytes cultured in the presence of these substances without zinc.

Improvements are also observed when performing the comparative tests with a growth factor EGF.

Test 2) In vitro fertilization

To evaluate the influence of the addition of zinc to the various substances (inositol, L-carnitine) on in vitro fertilization (IVF), the experiments are carried out in animals, for example in mice or cattle.

To do this, the most mature eggs and the most competent spermatozoa are selected to facilitate fertilization and the number of embryos formed after 48 hours of culture is evaluated.

As for the evaluation of in vitro maturation, in this experiment 9 groups are used.

In each group, 20 eggs are placed in the presence of 10 ⁶ competent spermatozoa in a culture medium to which are added the 2 growth factors IGF-1 and EGF, zinc and at least one compound selected from the group consisting of inositol and L-carnitine. The same tests were carried out in the presence of a growth factor, namely EGF.

Results

The addition of zinc to one or two growth factors and inositol and / or L-carnitine improves the percentage of fertilized eggs compared to the non-zinc group.

Trial 3) Early embryonic development

To evaluate the effects of the addition of zinc to the various substances (inositol, L-carnitine) on early embryonic development, the mouse strain CF-1 is used, since the number of embryos evolving at the blastocyst stage in a medium of Standard culture is low and around 30%. This model will make it possible to appreciate the beneficial effects of these substances on early embryonic development.

It is known that in the majority of other mouse strains used for early embryonic development, the rate of spontaneous blastocyst stage embryos in a conventional medium is close to 90%.

In this experiment, the same protocol is used: 9 groups of 20 1-cell embryos cultured under different conditions.

To better evaluate the effects of zinc addition to growth factors and inositol and / or L-carnitine on early embryonic development, bovine embryos or those of other animals can also be used.

Results

It is observed that the addition of zinc to growth factors and inositol and / or L-carnitine leads to an improvement in the development of embryos at different stages compared to the group containing only 1 or 2 growth factors and the inositol and / or L-carnitine. Indeed, more embryos evolving at more advanced stages such as the "blastocyst" stage, expanded blastocyst "Expanded blastocysts" and hatching blastocyst hatching blastocysts.

Claims

A composition for in vitro culturing of follicles under development for the maturation of oocytes contained in said follicles, or for maturation of cells of the male germ line, or for in vitro fertilization of oocytes by spermatozoa, or for the development of embryos,

optionally after thawing the follicles or embryos, characterized in that it comprises:

at least one growth factor, preferably at least two growth factors, in combination with a zinc salt, preferably a water-soluble salt such as zinc chloride or zinc sulphate, especially at a concentration of from 0.1 to 3 μg / ml, preferably at a concentration of 0.5 to 1.5 μ ^ ιηΐ, expressed as zinc element, and at least one compound selected from the group consisting of:

and

2. Composition according to claim 1, characterized in that it comprises at least two growth factors chosen from:

insulin-like growth factors, also referred to as insulin-like growth factor (IGF) such as IGF-1 and / or IGF-2, the epidermal growth factor, also referred to as EGF (epidermal growth factor) and / or HB-EGF (heparin-binding epidermal growth factor),

the beta transformation growth factor, also designated TGF beta

transforming growth factor beta, in the form of TGF-beta 1, 2 and 3, preferably TGF-beta-1,

hepatic growth factor, also known as hepatocyte growth factor (HGF),

growth and differentiation factors, also referred to as GDF (growth differentiation factor), preferably GDF-9,

3. Composition according to claim 1 or 2 characterized in that it comprises at least one compound of the family of corticosteroids involved in energy production in mammals, preferably hydrocortisone in the form of water-soluble salt such as hemisuccinate hydrocortisone, hydrocortisone sodium succinate and hydrocortisone sodium phosphate.

4. Composition according to claim 1, 2 or 3 characterized in that the at least one zinc salt is in hydrated or anhydrous form and is selected from the following salts: zinc chloride, zinc sulfate, zinc gluconate, picolinate zinc, zinc citrate, zinc acetate, zinc lactate, zinc stearate, preferably a water-soluble salt such as zinc sulfate or zinc chloride.

5. Composition according to one of claims 1 to 4 characterized in that the concentrations of growth factors are in particular between about 0.25μg / L and 100 μg / L, and preferably between about 0.5 μg / L and about 50 ug / L.

6. Composition according to one of claims 1 to 5 characterized in that it contains:

- at least two growth factors,

at least one water-soluble zinc salt selected from zinc sulphate or zinc chloride and / or myo-inositol and / or carnitine or a carnitine salt, hydrocortisone hemisuccinate, and

NAD / NADH and / or NADP / NADPH.

7. Composition according to one of claims 1 to 6 characterized in that it is in the form of lyophilisate.

8. Application of the compositions according to one of claims 1 to 7, as effector adjuvants for the preparation of in vitro culture media follicles under development for the maturation oocytes contained in said follicles, or for maturation of male germ line cells, or for in vitro fertilization of oocytes by spermatozoa, or for embryo culture, possibly after thawing of follicles, male cells, or embryos, said in vitro culture media comprising a composition according to one of claims 1 to 6, in combination with the elements conventionally used in the context of in vitro fertilization, or the culture of follicles, male cells, or embryos, said elements being selected from the human serum albumin or bovine, optionally recombinant, and / or mineral salts, and / or energetic molecules such as glucose, pyruvate, citrate and lactate, and / or amino acids for protein biosynthesis, and / or purine and pyrimidine bases for the biosynthesis of nucleic acids, and / or phospholipids or cholesterol for the formation of cell membranes, and / or vitamins, such as B vitamins, including vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B9, vitamin B12, and / or vitamin C and / or vitamin E, and optionally insulin and / or at least one other substance contributing to the process and / or at least one antibiotic.

9. In vitro culture media of follicles being developed for the maturation of oocytes contained in said follicles, or for maturation of male germ line cells, or for in vitro fertilization of oocytes by spermatozoa, where for the embryo culture, optionally after thawing follicles, male cells, or embryos, said media being characterized in that they comprise a composition according to one of claims 1 to 6, in association with the elements conventionally used in the framework of in vitro fertilization, or culturing follicles, male cells, or embryos, said elements being as defined in claim 8.

10. In vitro method of maturation of developing mammalian follicles for the maturation of the oocytes contained in said follicles and more particularly of human follicles, where appropriate after thawing the previously frozen follicles, characterized in that it comprises a step of culturing in vitro follicles taken from the female and more particularly from women, in a culture medium according to claim 9, advantageously for about 24 to 48 hours.

11. Kit for the extemporaneous preparation of a medium according to claim 9, especially in the context of the implementation of a method according to claim 10, characterized in that it comprises:

a composition according to any one of claims 1 to 6, or

a composition in the form of a lyophilizate according to claim 7,

and, if appropriate, an aqueous solution containing the elements conventionally used in the context of the culture of follicles, male cells, or embryos, as defined in claim 8.