In vitro maturation of mammalian cumulus-oocyte complexes

Abstract

The present invention relates to compositions and methods for assisted reproductive technology in mammals. In particular, the present invention provides compositions and methods for in vitro maturation of immature cumulus-oocyte complexes (COCs) to enhance embryological outcomes.

Description

[0001] This application is a divisional application of the application filed on December 21, 2015, with international application number PCT / BE2015 / 000068, national application number 201580075261.2, and entitled "In vitro maturation of mammalian cumulus-oocyte complex". Technical Field

[0002] This invention generally relates to compositions and methods for use in mammalian assisted reproductive technologies. Specifically, this invention relates to compositions and methods for the in vitro maturation of mammalian cumulus-oocyte complexes. Background Technology

[0003] In human infertility practice, traditional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) techniques are still used after superovulation with expensive hormone therapy to obtain large follicles (i.e., at least 17 mm in diameter). The cost of hormones, the risks associated with acute and long-term complications, and the inconvenience of repeated hospital visits to monitor follicle growth motivate the development of a less costly and better-tolerated treatment for infertile couples with normal / high follicular reserves.

[0004] In vitro oocyte maturation (IVM) is a technique that allows germinal follicle (GV) stage oocytes (oocyte complexes (COCs)) enclosed in a tightly aggregated coronal cumulus oophorus. These oocytes can be obtained via ultrasound-guided needle aspiration before ovulation is triggered by administration of human chorionic gonadotropin (hCG) or earlier after hCG administration. Therefore, oocyte IVM has the potential to simplify fertility treatments or reduce the risks and costs associated with hCG stimulation in patients with normal or high ovarian follicular reserves. This follicular reserve is typically determined by measuring anti-Müllerian hormone levels and by ultrasound-guided follicle counting on day 3 of the menstrual cycle.

[0005] For oocyte IVM using ultrasound-guided needle aspiration, oocytes can be collected from small (<10 mm) follicles in minimally stimulated or unstimulated ovaries and matured in vitro. However, interfering with normal oocyte development at early stages (e.g., when follicles only reach a diameter of 10 mm or less) not only yields fewer mature oocytes but also reduces subsequent embryo development and implantation. In particular, the successful implantation rate per embryo after IVM is typically less than 10%, only half the success rate of conventional IVF or ICSI procedures. The incidence of early miscarriage appears to be variable but is generally greater than after IVF / ICSI. For human patients, the currently reduced meiotic maturation rate (50%), along with observed defects in mature oocyte embryo development, is a major bottleneck in current IVM technology (De Vos). et al.,Fertil. Steril.2011;96(4) 860-864; Guzman et al., Fertil. Steril. 2012;98(2): 503-507). Therefore, before this method can be widely accepted, the reduced embryonic developmental potential and implantation rate of IVM need to be addressed.

[0006] The cornerstone of IVM culture is providing a suitable environment for developmental capacity. This primarily requires hormonal intervention and activation of necessary signaling pathways through the use of chemical compounds that allow for the synchronization of nuclear cell and cytoplasmic maturation processes in oocytes. The principle behind extending the in vitro oocyte maturation period is to promote longer interactions between immature oocytes and well-regulated cumulus cells.

[0007] The signaling pathways between follicular cells and oocytes responsible for meiotic arrest have been extensively studied. Meiotic arrest is maintained by producing high levels of cyclic adenosine monophosphate (cAMP). The intracellular cAMP concentration is regulated by the activity of phosphodiesterases (PDEs) that degrade cAMP. Recent studies have shown that cGMP is produced in cumulus cells upon activation of the guanylate-cyclase-coupled receptor for natriuretic peptide type 2 (NPR2). NPR2 activity is induced by its ligand, precursor natriuretic peptide C (NPPC), which is primarily synthesized by parietal granulosa cells and cleaved into C-type natriuretic peptide (CNP). cGMP is then translocated to the oocyte, where it inhibits cAMP hydrolysis via the phosphodiesterase PDE3A. This inhibition maintains high cAMP concentrations, thereby preventing meiotic progression. (Tsafriri) et al. Dev. Biol. 1996, 178(2): 393-402; Conti et al., Mol. Cell. Endocrinol. 1998, 145(1-2): 9-14; Conti et al., Mol. Cell. Endocrinol. 2002, 187(1-2): 153-159).

[0008] Previously, in order to promote oocyte meiotic arrest and allow for oocyte retrieval, pharmacological interference with cAMP levels derived from in vitro mature oocytes of different species, such as mice, cattle, and humans, has been attempted (Nogueira). et al. Biol. Reprod. 2003, 69(6): 2045-2052; Thomas et al . Biol. Reprod. 2004, 71(4): 1142-1149; Shu et al. Hum. Reprod. 2008, 23(3): 504-513; Vanhoutte et al. Hum. Reprod. 2009, 24(3): 658-669). However, no significant improvement was reported in the level of embryonic developmental potential.

[0009] Recently, the CNP / NPR2 signaling pathway has been shown to be an important regulatory mechanism for maintaining meiotic arrest in oocytes of medium size and fully grown follicles (Zhang et al. J. Cell. Physiol. 2015, 230(1):71-81; Sato). et al. Mol. Endocrinol. 2012, 26: 1158-1166, Franciosi et al. Biol. Reprod. 2014, 91(3): 61, Santiquet et al. Biol. Reprod. 2014, 91(1): 16). However, the main problem in the above studies is that COC can only maintain the meiotic arrest state for a short time. Therefore, only medium to large follicles can successfully mature in vitro, while the maturation of small early antral follicles fails. Therefore, delaying the initiation of meiotic resumption in small early antral follicles and improving the IVM process is a major challenge at present. In particular, such a protocol should not affect further in vitro maturation. This challenge is even greater for such small early antral follicles, as they need to acquire or maintain the ability to complete nuclear maturation (i.e., from the non-surrounding nucleolar (NSN) to the surrounding nucleolar (SN) stage) and then enter the next step of potential development, even maintaining the interconnection between the oocyte and cumulus oophorus, allowing the transfer of nutrients (e.g., RNA cargo) from the cumulus oophorus. Summary of the Invention

[0010] This invention relates to compositions and methods for assisted reproductive technologies in mammals. In particular, the invention provides compositions and methods for in vitro maturation of immature cumulus-oocyte complexes (COCs), thereby enhancing embryological outcomes.

[0011] In the context of this invention, there is no need for prior hCG stimulation or for collecting immature mammalian COCs after hCG stimulation. In a preferred embodiment, any triggering agents, including high doses of FSH, LHRH and their agonists, recombinant LH or LH analogues, are avoided before collecting COCs in vivo.

[0012] In a first aspect, the present invention provides in vitro maturation of mammalian cumulus-oocyte complexes (COCs). Capacitated culture mediumThe culture medium contains 0.1 to 50 nM C-type natriuretic peptide (CNP), estradiol, and FSH. In a specific embodiment, the capacitation medium contains 10 to 25 nM CNP, or more particularly 25 nM CNP.

[0013] In a specific embodiment, the capacitation medium contains 1 to 1000 nM estradiol, most preferably 10 nM estradiol. In another embodiment, the capacitation medium contains 0.1 to 10 mIU / mL FSH, particularly 1 to 5 mIU / mL FSH, and even more particularly 2.5 mIU / mL FSH or 1 mIU / mL FSH. In yet another embodiment, the capacitation medium contains 25 nM CNP, 10 nM estradiol, and 2.5 or 1 mIU / mL FSH. In yet another aspect, the capacitation medium comprises an equivalent dose of recombinant FSH, an FSH analogue, or an FSH mimic molecule.

[0014] In another embodiment, the capacitation medium contains 0.1 to 10 ng / ml insulin, particularly 5 ng / ml insulin. Alternatively, the capacitation medium contains an equivalent dose of an insulin analog or insulin mimic molecule.

[0015] In a further embodiment, the capacitation medium contains oocyte secretory factors. The oocyte secretory factors used herein are selected from GDF-9, BMP-15, FGF-8, or any combination thereof; specifically, their concentrations in the capacitation medium are each from 10 to 100 ng / ml. In a specific embodiment, the oocyte secretory factors are recombinant proteins. In another aspect, the oocyte secretory factors are heterodimeric proteins.

[0016] In yet another embodiment, the capacitation medium comprises 0.1 to 50 nM CNP, most preferably 25 nM CNP; 1 to 1000 nM estradiol, most preferably 10 nM estradiol; 0.1 to 10 mIU / mL FSH, most preferably 2.5 mIU / mL FSH or 1 mIU / mL FSH; 0.1 to 10 ng / mL insulin, most preferably 5 ng / mL insulin; and oocyte secretion factors selected from GDF-9, BMP-15, FGF-8 or any equivalent or combination thereof.

[0017] This invention also provides COC for immature mammals. In vitro maturation methods .

[0018] In a first aspect, the method includes collecting immature follicle-forming cells (COCs) in a collection medium, contacting the mammalian COCs with a capacitation medium containing 0.1 to 50 nM CNP, estradiol, and FSH, and further contacting the mammalian COCs with a maturation medium. In this method, the immature COCs are typically contacted with the collection medium for at least 30 minutes and at most 2 hours. Also in this method, and after contact with the collection medium, the mammalian COCs are contacted with a capacitation medium to maintain meiotic arrest. Specifically, the COCs are contacted with the capacitation medium for a sufficient period to reach a late developmental stage, as demonstrated by chromatin reorganization of the oocytes into a condensation phase or a so-called surrounding nucleolar (SN) conformation, as observed in the GV phase. During this period, following stimulation for meiotic resumption, the oocytes will be able to undergo germinal vesicle rupture (GVBD). Even more specifically, the mammalian COCs are contacted with the capacitation medium for at least 2 hours and at most 96 hours. In another specific embodiment, the mammalian COCs are contacted with the capacitation medium for a period determined by the follicle size. More specifically, follicle size is determined upon COC retrieval, for example, by ultrasound imaging. Follicles with a diameter of 1 to 5 mm are preferably held in capacitation medium for at least 48 hours. For follicles with a diameter > 5 to 10 mm, COC is preferably held in capacitation medium for at least 24 hours, and for follicles with a diameter greater than 10 mm, it is preferably held in capacitation medium for at least 2 hours. In another embodiment, mammalian COC is contacted with a maturation medium to allow for further meiotic maturation of the oocytes.

[0019] In a specific embodiment, in the in vitro method according to the invention, the capacitation medium contains 1 to 1000 nM estradiol, more particularly 10 nM estradiol. In another embodiment, in the in vitro method according to the invention, the capacitation medium contains 0.1 to 10 mIU / mL FSH, more particularly 2.5 mIU / mL FSH or 1 mIU / mL FSH. In another aspect, the capacitation medium comprises an equal dose of recombinant FSH, an FSH analogue, or an FSH mimic molecule.

[0020] In another embodiment, in the method according to the invention, the capacitation medium contains 0.1 to 10 ng / ml insulin, more particularly 5 ng / ml insulin. In another aspect, the capacitation medium comprises an equivalent dose of an insulin analog or insulin mimic molecule.

[0021] In another embodiment, in the in vitro method according to the invention, the capacitation medium contains oocyte secretory factors. The oocyte secretory factors used herein are selected from GDF-9, BMP-15, FGF-8, or any combination thereof; particularly, their concentrations in the capacitation medium are each from 10 to 100 ng / ml. In a specific embodiment, the oocyte secretory factor is a recombinant protein. In another embodiment, the oocyte secretory factor is a heterodimeric protein.

[0022] In another embodiment, in the in vitro method according to the invention, mammalian COCs are exposed to capacitation medium for a period of at least 2 hours and up to 96 hours. This time is sufficient to reach late developmental stages, as demonstrated by the chromatin reorganization of oocytes into the condensation phase or the so-called surrounding nucleolar (SN) conformation. In another specific embodiment, mammalian COCs are exposed to capacitation medium for a period determined by follicle size. More specifically, follicle size is determined upon COC retrieval, for example by ultrasound imaging. Follicles with a diameter of 1 to 5 mm are preferably held in capacitation medium for at least 48 hours. Follicles with a diameter > 5 to 10 mm are preferably held in capacitation medium for at least 24 hours, and follicles with a diameter greater than 10 mm are preferably held in capacitation medium for at least 2 hours. The capacitation period allows the oocytes to acquire the ability to resume meiosis, which is only assessed after meiotic stimulation is triggered. In another embodiment, mammalian COCs are contacted with a maturation medium (meiotic stimulation) to allow further meiotic maturation of the oocytes, as demonstrated under an inverted microscope by germinal vesicle rupture (GVBD) and expulsion of the first polar body (PB).

[0023] In a specific embodiment, in the in vitro method according to the invention, mammalian COCs are contacted with capacitated culture medium in non-adherent or adherent culture plates, particularly in non-adherent culture plates.

[0024] In another embodiment, in the in vitro method according to the invention, the immature COC is contacted with the collection culture medium for a minimum of 30 minutes and a maximum of 2 hours.

[0025] In another embodiment, the in vitro method according to the invention is part of assisted reproductive technology. In a specific embodiment, the assisted reproductive technology includes in vitro fertilization or ICSI.

[0026] In one specific embodiment of the present invention, the mammalian COC is the human COC.

[0027] On the other hand, the present invention provides a COC for immature mammals. In vitro maturation reagent kitThe kit comprises a capacitation medium as described herein. In a specific embodiment, in the kit according to the invention, the capacitation medium comprises 0.1 to 50 nM CNP, estradiol, and FSH. In a more specific embodiment, in the kit according to the invention, the capacitation medium comprises 10 to 25 nM CNP, preferably 25 nM CNP. In a specific embodiment, the capacitation medium in the kit comprises 1 to 1000 nM estradiol, preferably 10 nM estradiol. In another embodiment, the capacitation medium in the kit comprises 0.1 to 10 mIU / mL FSH, more particularly 2.5 mIU / mL FSH or 1 mIU / mL FSH. In another aspect, the capacitation medium comprises an equivalent dose of recombinant FSH, an FSH analog, or an FSH mimic molecule. In another embodiment, in the kit according to the invention, the capacitation medium comprises 0.1 to 10 ng / mL insulin, more particularly 5 ng / mL insulin. In another aspect, the capacitation medium comprises an equivalent dose of an insulin analog or an insulin mimic molecule. In another embodiment, in the kit according to the invention, the capacitating medium comprises 0.1 to 50 nM CNP, preferably 25 nM CNP; 1 to 1000 nM estradiol, preferably 10 nM estradiol; 0.1 to 10 mIU / mL FSH, preferably 2.5 mIU / mL FSH or 1 mIU / mL FSH; and 0.1 to 10 ng / mL insulin, preferably 5 ng / mL insulin.

[0028] In another embodiment, in the kit according to the invention, the capacitation medium contains oocyte secretory factors. The oocyte secretory factors used herein are selected from GDF-9, BMP-15, FGF-8, or any combination thereof. Specifically, the concentrations in the capacitation medium are each from 10 to 100 ng / ml. In a specific embodiment, the oocyte secretory factors used herein are recombinant proteins or heterodimeric proteins.

[0029] In another embodiment, the kit for in vitro maturation of immature mammalian COCs comprises (a) a collection medium containing a natural or synthetic chemical compound that inhibits naturally occurring phosphodiesterase or a natural inhibitor of oocyte meiosis; (b) a capacitation medium as described herein; (c) a maturation medium; (d) adherent and / or non-adherent culture plates; and (e) instructions for using the kit.

[0030] On the other hand, the present invention provides in vitro maturation of COC in immature mammals. Use of capacitated culture medium wayAs described herein, the capacitation medium comprises 0.1 to 50 nM CNP, estradiol, and FSH. In a specific embodiment, the capacitation medium comprises 1 to 1000 nM estradiol. In another embodiment, the capacitation medium comprises 0.1 to 10 mIU / mL FSH, preferably 2.5 mIU / mL FSH or 1 mIU / mL FSH. In another aspect, the capacitation medium comprises an equivalent dose of recombinant FSH, an FSH analog, or an FSH mimic molecule. In another embodiment, the capacitation medium comprises 0.1 to 10 ng / mL insulin, preferably 5 ng / mL insulin. In another aspect, the capacitation medium comprises an equivalent dose of an insulin analog or an insulin mimic molecule. In another embodiment, in the use of the capacitation medium according to the invention, the capacitation medium comprises oocyte secretory factors. The oocyte secretory factors used herein are selected from GDF-9, BMP-15, FGF-8, or any combination thereof. In a specific embodiment, the oocyte secretory factors are recombinant proteins. In another aspect, the oocyte secretory factors are heterodimeric proteins.

[0031] In a specific embodiment, in the use of the capacitation medium according to the invention, mammalian COCs are contacted with the capacitation medium for a period of at least 2 hours and up to 96 hours. More specifically, in the use of the capacitation medium, mammalian COCs are contacted with the capacitation medium to maintain oocyte meiotic arrest and allow for cytoplasmic maturation. In particular, the contact time with the COC is sufficient to achieve late development, as demonstrated by chromatin remodeling of the oocyte into the condensation phase or the so-called nucleolar (SN) conformation. In another specific embodiment, mammalian COCs are contacted with the capacitation medium for a period determined by follicle size. More specifically, follicle size is determined upon COC removal, for example by ultrasound imaging. Follicles with a diameter of 1 to 5 mm are preferably held in the capacitation medium for at least 48 hours. Follicles with a diameter > 5 to 10 mm are preferably held in the capacitation medium for at least 24 hours, and preferably follicles with a diameter greater than 10 mm are held in the capacitation medium for at least 2 hours. Oocyte maturation at the nuclear level, i.e., chromatin remodeling within the oocyte, is evaluated under a fluorescence microscope.

[0032] In a specific embodiment, in the use of the capacitation medium according to the invention, mammalian COCs are brought into contact with capacitation medium in non-adherent or adherent culture plates, particularly in non-adherent culture plates.

[0033] In another embodiment, the capacitation medium is used to maintain mammalian COC oocytes in a state of meiotic arrest. Based on the ability to maintain oocytes in meiotic arrest without affecting their further maturation, the capacitation medium of this invention creates a flexible IVM method compared to currently used IVM methods. Because the expansion of in vitro culture can be flexible, i.e., the capacitation interval—all important technical work such as microinjection of oocytes—can now be performed within normal operating time. Clearly, this is a significant practical improvement over current IVM procedures.

[0034] In a further embodiment, the capacitation medium according to the invention is used as part of assisted reproductive technology. In a specific embodiment, the assisted reproductive technology includes in vitro fertilization or ICSI.

[0035] Another application of the capacitation medium of the present invention is the improved IVM performance of COC obtained from smaller follicles compared to conventionally used methods.

[0036] By enabling smaller follicles to mature, people; - It is possible to cryopreserve mature oocytes that have developed well from small antral follicles; - It is possible to culture COCs from small antral follicles (which is important in the field of fertility preservation in the case of cancer treatment), where COCs are obtained from the dissection of smaller follicles than those obtained by ultrasound-guided anterior vaginal examination puncture.

[0037] Another aspect of the present invention is based on a capacitation medium containing mammalian COC in vitro maturation. reagent kit way The kit comprises a capacitation medium as described herein. In a specific embodiment, in the kit according to the invention, the capacitation medium comprises 0.1 to 50 nM CNP, estradiol, and FSH. In a specific embodiment, the capacitation medium in the kit comprises 1 to 1000 nM estradiol. In another embodiment, the capacitation medium in the kit comprises 0.1 to 10 mIU / mL FSH. In yet another embodiment, in the kit according to the invention, the capacitation medium comprises 0.1 to 10 ng / mL insulin. In yet another embodiment, in the kit according to the invention, the capacitation medium comprises 0.1 to 50 nM CNP, preferably 25 nM CNP; 1 to 1000 nM estradiol, preferably 10 nM estradiol; and 0.1 to 10 mIU / mL FSH, preferably 2.5 mIU / mL FSH or 1 mIU / mL FSH. In another aspect, the capacitation medium comprises an equal dose of recombinant FSH, an FSH analogue, or an FSH mimic molecule.

[0038] In another embodiment, in the kit according to the invention, the capacitation medium contains 0.1 to 10 ng / ml insulin, more particularly 5 ng / ml insulin. In another aspect, the capacitation medium contains an equivalent dose of an insulin analog or insulin mimic molecule.

[0039] In another embodiment, in the kit according to the invention, the capacitation medium contains oocyte secretory factors. The oocyte secretory factors used herein are selected from GDF-9, BMP-15, FGF-8, or any combination thereof. In a specific embodiment, the oocyte secretory factors used herein are recombinant proteins or heterodimeric proteins.

[0040] In another embodiment, the use of a kit containing a capacitation medium for in vitro maturation of immature mammalian COCs includes contacting the mammalian COCs with the capacitation medium for a period of at least 2 hours to a maximum of 96 hours. More specifically, in the use of the kit containing the capacitation medium, the mammalian COCs are contacted with the capacitation medium to induce meiotic arrest and allow maturation. In particular, the contact time with the COCs is sufficient to reach late development, as demonstrated by chromatin reorganization of the oocytes into the condensation phase or the so-called surrounding nucleolar (SN) conformation. In another specific embodiment, the mammalian COCs are contacted with the capacitation medium for a period determined by follicle size. More specifically, follicle size is determined by ultrasound imaging at the time of COC retrieval. Follicles with a diameter of 1 to 5 mm are preferably maintained in the capacitation medium for at least 48 hours. Follicles with a diameter > 5 to 10 mm are preferably maintained in the capacitation medium for at least 24 hours, and follicles with a diameter greater than 10 mm are preferably maintained in the capacitation medium for at least 2 hours. Assessment of oocyte maturation at the nuclear level under a fluorescence microscope, i.e., chromatin remodeling within the oocyte.

[0041] Another aspect of the invention is based on the use of a kit comprising (a) a collection medium containing a natural or artificial compound that inhibits naturally occurring phosphodiesterase or a natural oocyte meiosis inhibitor; (b) a capacitation medium as described herein; (c) a maturation medium; (d) adherent and / or non-adherent culture plates; and (e) instructions for using the kit.

[0042] In a particular aspect of the invention, the use of a kit for the in vitro maturation of immature mammalian cocci (COCs) includes contacting the COCs with a collection medium for at least 30 minutes to a maximum of 2 hours. In another aspect, in the use of the kit according to the invention, the mammalian COCs are contacted with a capacitation medium for a period of at least 2 hours to a maximum of 96 hours. More specifically, in the use of a kit comprising a collection medium, a capacitation medium, a maturation medium, adherent and / or non-adherent culture plates, and instructions for use, the mammalian COCs are contacted with the capacitation medium to induce meiotic arrest and allow maturation. In particular, the contact with the capacitation medium is sufficient to reach late-stage development, as demonstrated by chromatin reorganization of oocytes into the condensation phase or the so-called surrounding nucleolar (SN) conformation. In another specific embodiment, the mammalian COCs are contacted with the capacitation medium for a period determined by follicle size. More specifically, follicle size is determined upon COC removal, for example by ultrasound imaging. Follicles with a diameter of 1 to 5 mm are preferably maintained in the capacitation medium for at least 48 hours. Follicles with a diameter > 5 to 10 mm are preferably kept in capacitation medium for at least 24 hours, and follicles with a diameter > 10 mm are preferably kept in capacitation medium for at least 2 hours.

[0043] The capacitation phase enables oocytes to resume meiosis, which is only assessed after meiotic stimulation is triggered. In another embodiment, mammalian COCs are contacted with maturation medium (meiotic stimulation) to allow further meiotic maturation of the oocytes, as demonstrated under an inverted microscope by germinal vesicle rupture (GVBD) and expulsion of the first polar body (PB).

[0044] In a specific embodiment, in the use of the kit according to the invention, mammalian COCs are brought into contact with capacitated culture medium in non-adherent or adherent culture plates.

[0045] In another embodiment, in the use of the kit according to the invention, the capacitation medium maintains mammalian COC oocytes in a state of meiotic arrest.

[0046] In a further embodiment, the kit according to the invention is used to induce the maturation of mammalian COC.

[0047] In another embodiment, the kit according to the invention is used as part of assisted reproductive technology. In a specific embodiment, the assisted reproductive technology includes in vitro fertilization or ICSI.

[0048] The numbering and implementation scheme of the present invention are as follows: 1. A capacitation medium for in vitro maturation of immature mammalian cumulus-oocyte complexes, the medium comprising 0.1 to 50 nM C-type natriuretic peptide (CNP), estradiol and follicle-stimulating hormone (FSH); particularly comprising 10 to 50 nM C-type natriuretic peptide (CNP); more particularly 25 nM CNP.

[0049] 2. The capacitation medium according to claim 1, wherein the medium comprises 1 to 1000 nM estradiol.

[0050] 3. The capacitation medium according to claim 1, wherein the medium contains 0.1 to 10 mIU / mL follicle-stimulating hormone (FSH).

[0051] 4. The capacitation medium according to any one of the preceding claims, wherein the medium further comprises 0.1 to 10 ng / ml insulin.

[0052] 5. The capacitation medium according to any one of the preceding claims, wherein the medium comprises oocyte secretion factors.

[0053] 6. The capacitation medium according to claim 5, wherein the oocyte secreted factor is selected from GDF-9, BMP-15, FGF-8, their analogues or any combination thereof; particularly, the concentration of each of the factors in the capacitation medium is 10 to 100 ng / ml.

[0054] 7. A method for in vitro maturation of immature mammalian cumulus-oocyte complexes, the method comprising collecting immature cumulus-oocyte complexes in a collection medium, contacting the mammalian cumulus-oocyte complexes with a capacitating medium containing 0.1 to 50 nM CNP (particularly 10 to 50 nM CNP), estradiol and FSH, and further contacting the mammalian cumulus-oocyte complexes with a maturation medium.

[0055] 8. The method of claim 7, wherein the capacitation medium comprises 1 to 1000 nM estradiol.

[0056] 9. The method of claim 7, wherein the capacitation medium comprises 0.1 to 10 mIU / mL FSH.

[0057] 10. The method according to any one of claims 7 to 9, wherein the capacitation medium further comprises 0.1 to 10 ng / ml insulin.

[0058] 11. The method according to any one of claims 7 to 10, wherein the capacitation medium comprises oocyte secretory factors.

[0059] 12. The method of claim 11, wherein the oocyte secretory factor is selected from GDF-9, BMP-15, FGF-8, their analogues or any combination thereof; particularly, the concentration of each factor in the capacitation medium is 10 to 100 ng / ml.

[0060] 13. The method according to any one of claims 7 to 12, wherein the mammalian cumulus-oocyte complex is a human cumulus-oocyte complex.

[0061] 14. The method according to any one of claims 7 to 13, wherein the mammalian cumulus-oocyte complex is in contact with the capacitation medium for a period of at least 2 hours to a maximum of 96 hours.

[0062] 15. The method according to any one of claims 7 to 14, wherein the mammalian cumulus-oocyte complex is in contact with the capacitation medium in a non-adherent or adherent culture plate, particularly a non-adherent culture plate.

[0063] 16. The method of claim 7, wherein the immature cumulus-oocyte complex is in contact with the collection culture medium for a minimum of 30 minutes and a maximum of 2 hours.

[0064] 17. The method according to any one of claims 7 to 16, wherein the method is part of assisted reproductive technology.

[0065] 18. The method of claim 17, wherein the assisted reproductive technology includes in vitro fertilization, ICSI, or fertility preservation.

[0066] 19. A kit for in vitro maturation of immature mammalian cumulus-oocyte complexes, the kit comprising a capacitation medium according to any one of claims 1 to 6.

[0067] 20. A kit for in vitro maturation of immature mammalian cumulus-oocyte complexes, the kit comprising: (a) Collect culture medium containing natural or artificial chemical compounds that inhibit naturally occurring phosphodiesterase or natural inhibitors of oocyte meiosis; (b) The capacitation medium according to any one of claims 1 to 6; (c) Maturation culture medium; (d) Non-adherent and / or adherent culture plates; and (e) Instructions for using the kit.

[0068] 21. Use of the capacitation medium according to any one of claims 1 to 6 for in vitro maturation of mammalian cumulus-oocyte complexes.

[0069] 22. Use of the capacitation medium according to any one of claims 1 to 6, wherein the mammalian cumulus-oocyte complex is in contact with the capacitation medium for a period of at least 2 hours to a maximum of 96 hours.

[0070] 23. Use of the capacitation medium according to any one of claims 1 to 6, wherein the mammalian cumulus-oocyte complex is in contact with the capacitation medium in a non-adherent or adherent culture plate; particularly a non-adherent culture plate.

[0071] 24. Use of the capacitation medium according to any one of claims 1 to 6, wherein the use is for maintaining oocytes in a mammalian cumulus-oocyte complex in a state of meiotic arrest.

[0072] 25. Use of the capacitation medium according to any one of claims 1 to 6 as part of assisted reproductive technology.

[0073] 26. Use of the capacitation culture medium according to claim 25, wherein the assisted reproductive technology includes in vitro fertilization or ICSI.

[0074] 27. Use of the capacitation medium according to any one of claims 1 to 6 in a fertility preservation method, for example in the case of cancer treatment or in the case of oocyte storage (the latter, especially for age-reserved individuals (35-40 years old)).

[0075] 28. The use according to claim 27, wherein the fertility preservation method comprises cryopreservation of small antral follicles.

[0076] 29. The use according to claim 28, wherein the capacitation medium is used for the in vitro maturation of the cryopreserved small antral follicles.

[0077] 30. Use of the capacitation medium according to any one of claims 1 to 6 in fertility preservation during cancer treatment.

[0078] 31. Use of the capacitation culture medium according to any one of claims 1 to 6 in the preservation of fertility of oocytes stored in a bank.

[0079] 32. Use of the kit according to claim 19 or 20 for in vitro maturation of mammalian cumulus-oocyte complexes.

[0080] 33. Use of the kit according to claim 20, wherein the immature cumulus-oocyte complex is contacted with the collection medium for a minimum of 30 minutes and a maximum of 2 hours.

[0081] 34. Use of the kit according to claim 19 or 20, wherein the mammalian cumulus-oocyte complex is in contact with the capacitation medium for a period of at least 2 hours to a maximum of 96 hours.

[0082] 35. Use of the kit according to claim 19 or 20, wherein the mammalian cumulus-oocyte complex is contacted with the capacitation medium in a non-adherent or adherent culture plate; particularly a non-adherent culture plate.

[0083] 36. Use of the kit according to claim 19 or 20, wherein the use is for maintaining oocytes in a mammalian cumulus-oocyte complex in a state of meiotic arrest.

[0084] 37. Use of the kit according to claim 19 or 20, wherein the use is for inducing the maturation of mammalian cumulus-oocyte complexes.

[0085] 38. Use of the kit according to claim 19 or 20 as part of assisted reproductive technology.

[0086] 39. Use of the kit according to claim 38, wherein the assisted reproductive technology includes in vitro fertilization or ICSI.

[0087] 40. Use of the kit according to claim 19 or 20 as part of a fertility preservation method; particularly fertility preservation in the context of cancer treatment. Attached Figure Description

[0088] Referring now specifically to the accompanying drawings, it is emphasized that the details shown are by way of example and are for the purpose of illustrative discussion of different embodiments of the invention only. They are provided to offer a description that is considered most useful and readily understood in relation to the principles and concepts of the invention. In this regard, no attempt is made to show structural details of the invention that are more detailed than those necessary for a substantially clear understanding of the invention. The description given with reference to the drawings is intended to guide those skilled in the art on how the invention can be practiced in several forms.

[0089] Figure 1 Dose-dependent effects of CNP-22 on meiotic maturation progression

[0090] COCs were matured in vitro for 18 hours prior to ovulation in the presence of 0 (control), 1, 10, and 100 nM CNP-22 (A), and in combination with 4 ng / mL EGF (B). Oocyte nuclear maturation was assessed after maturation. Each bar represents the mean ± SD of experimental data obtained from three replicates (at least 33 oocytes / treatment). Different letters indicate significant differences (P < 0.05).

[0091] Figure 2 CNP-22 delays EGFR-dependent meiotic restart.

[0092] (A) Preovulatory COCs were placed in cultures in the presence of 0 (control) or 25 nM CNP-22 + 4 ng / mL EGF, and early meiotic recovery was assessed at 2, 4, and 6 hours post-collection. (B) Preovulatory COCs were placed in cultures in the presence of 25 nM CNP-22 or 25 nM CNP-22 + 4 ng / mL EGF, and meiotic maturation (meiotic completion until PB expulsion) was assessed 24 hours later. Each bar represents the mean ± SD of data obtained from three replicates (at least 41 oocytes / treatment). Different letters or This indicates a significant difference (P < 0.05).

[0093] Figure 3 Effects of E2, FSH dosage and GDF9 supplementation on oocyte meiotic arrest

[0094] Immature COCs were placed in cultures in the presence of 25 nM CNP-22 alone or in combination with 10 nM E2. The potential effect of adding 2.5 mIU / mL or 5 mIU / mL FSH alone or in combination with 50 ng / mL GDF9 during a 48-hour culture period on maintaining meiotic arrest was evaluated. Each bar represents the mean ± SD of experimental data from three replicates (at least 38 oocytes / treatment). Different letters indicate significant differences (P < 0.05).

[0095] Figure 4 Effects of 48-hour culture period (pre-IVM) on chromatin conformation and diameter of GV oocytes

[0096] Immature cocci were placed in cultures in the presence of 25 nM CNP-22 + 10 nM E2. The potential complementary effects of 2.5 mIU / mL FSH or 2.5 mIU / mL FSH + 50 ng / mL GDF9 on oocyte chromatin conformation (A) or oocyte diameter (B) were assessed. Chromatin conformation was assessed as described in Materials and Methods and scored as NSN, NSN / SN (transitional), and NSN. In addition, oocyte diameter was assessed immediately after isolation and before culture (0 h). In (A), each bar represents the mean ± SD of experimental data obtained from three replicates (at least 46 oocytes / treatment). In (B), at least 31 GV oocytes / treatment were measured. Different letters indicate significant differences (P < 0.05).

[0097] Figure 5 Assess oocyte and embryo quality after pre-IVM + IVM

[0098] Following a 48-hour pre-IVM period and an 18-hour IVM period, oocytes were fertilized in vitro, and embryos were cultured for up to 5 days. The assessment parameters were the 2-cell rate (fertilization) (A) and blastocyst formation on day 5 (day 5 blastocyst / 2-cell) (B). Each bar in (A) and (B) represents experimental data obtained from four replicates (at least 64 oocytes / treatment); results are shown as mean ± SD.

[0099] (C) Shows the 2-cell rate and day 5 blastocyst rate (day 5 blastocyst / 2-cell) for two reference controls. Immature cocci were obtained from small antral follicles of 20-day-old mice and matured in vitro for 18 hours in the presence of 100 ng / mL EREG [20 do (IVM)]. Similarly, gold standard, in vivo-grown oocytes (controls) were obtained from 25–27-day-old mice after 48 hours of eCG priming followed by 14 hours of hCG. Data for these controls were from two replicates (at least 56 oocytes / treatment) (P < 0.05).

[0100] Figure 6 The effect of CNP on the junction of oocytes in vitro

[0101] Transzonal projection (TZP) in mouse cocci after 48 hours of culture with either a PDE3 inhibitor or CNP. The left image shows the TZP in the presence of a PDE3 inhibitor, and the right image shows the TZP in the presence of CNP. The zona pellucida surrounding the oocyte is depicted and indicated by arrows. In the presence of a PDE3 inhibitor, the zona pellucida is black, indicating almost no transzonal projection. Unexpectedly, in the presence of CNP, the zona pellucida is filled with transzonal projections, clearly demonstrating cumulus-oocyte junctions.

[0102] Figure 7 Staining of actin filaments in the zona pellucida of cumulus oocytes cultured in vitro.

[0103] Average pixel intensity on the COC zona pellucida exposed to CNP or PDE3 inhibitors. In (A), Org9935 was used as a PDE3 inhibitor, and actin filaments were confirmed with phalloidin bound to Texas red; while in (B), cyclohexylquinamide was used as a PDE3 inhibitor, and actin filaments were confirmed with Actin green. TM Confirmed. (n = number of COCs analyzed). The Mann-Whitney test was used to statistically compare the CNP and PDE3i groups, with P = 0.0082 for Figure A and < 0.0001 for Figure B.

[0104] Figure 8The differential effect of the presence of CNP and PDE3I on the developmental ability of cumulus-closed oocytes from early antral follicles during capacitation culture.

[0105] Fertilization rate (A) and blastocyst formation (B) following capacitation culture followed by IVM. In both cases, data were included from two reference controls: 1) IVM control without prior capacitation culture & 2) standard in vivo control (fully mature oocytes).

[0106] Figure 9 Meiosis resumed 18 hours later. The dose-dependent effect of CNP-22 on meiotic maturation progression. Preovulatory COCs were cultured at CNP doses ranging from 0.1 nM to 1 μM. Control conditions (basal medium without CNP) are included. Each bar represents the mean ± SD (mean 54 oocytes / treatment) of experimental data obtained from three replicates. An asterisk indicates a significant difference relative to the control condition without CNP (P < 0.01). Figure A provides the percentage (%) of oocytes with intact germinal vesicles (GVs), where the presence of intact GVs indicates meiotic arrest. Figure B provides the percentage of oocytes at germinal vesicle rupture (GVBD) stage, which is expected to be low in the case of meiotic arrest, and Figure C provides the percentage of oocytes extruding the first polar body (PB), again expected to be low in the case of meiotic arrest.

[0107] Figure 10 Meiosis resumed 18 hours later. The dose-dependent effect of CNP-53 on meiotic maturation progression. Preovulatory COCs were cultured at CNP doses ranging from 0.1 nM to 1 μM. Control conditions were included (basal medium without CNP and a positive control containing 25 nM CNP-22). Each bar represents the mean ± SD (mean 54 oocytes / treatment) of experimental data obtained from three replicates. An asterisk indicates a significant difference relative to the control condition without CNP (P < 0.01). Figure A shows the percentage of oocytes with intact germinal vesicles (GV). Figure B shows the percentage of oocytes at the germinal vesicle rupture (GVBD) stage. Figure C shows the percentage of oocytes expelling the first polar body (PB).

[0108] Figure 11The maturity rate, fertilization rate, and number of high-quality embryos (GQE) expressed as per number of fertilized eggs (2PN) or per initial number of cocci (COC). Black bars (N = 374 patients): Results from the “Conventional IVF (ICSI) field”: Reflecting current ICSI practices in Europe. These data were obtained from the most commonly used stimulation therapy (GnRH antagonist + HP-hMG): Embryological data from the published “MEGASET” data: a multicenter, multinational study in Europe by Ferring Pharmaceuticals, in which all embryos were cultured to the blastocyst stage. Gray bars (N = 413 patients): Results obtained using conventional IVM (Origio Kit) when oocytes were obtained from non-HCG-triggered cycles. White bars (N = 15): Results from oocytes from 15 patients (who also provided COCs for capacitation culture) using the “new capacitation culture” step. Striped white bars (N = 15 patients): These results are from conventional IVM (Origio® method) of a subset of oocytes from 15 patients who obtained COCs for capacitation culture (i.e., “siblings” associated with the Green group).

[0109] Figure 12 : Day 5-6 blastocyst formation, expressed as per number of fertilized eggs (2PN), per MII oocyte, or per initial COC (COC). Black bars (N = 374 patients) are from results in the conventional IVF (ICSI) field: reflecting current ICSI practices in Europe. These data were obtained from the most commonly used stimulation therapy (GnRH antagonist + HP-hMG): embryological data from the published "MEGASET" data: a multicenter, multinational study in Europe by Ferring Pharmaceuticals, in which all embryos were cultured to the blastocyst stage. Gray bars (N = 98 patients): results from a subgroup of 98 patients whose embryos could only be further cultured to blastocyst medium if they had 4 or more good day 3 embryos. White bars (N = 15): results from oocytes from 15 patients (who also provided COCs for capacitation culture) using the "new capacitation culture" step. Striped white bars (N = 5 patients): results from a subgroup of 5 patients whose embryos could only be further cultured to blastocyst medium if they had 4 or more "good" day 3 embryos. Note: This is the most common "bias" in conventional IVMs. Dotted white streaks (N = 7): Results from a subgroup of 7 sibling patients in the “newly capacitated culture” step; in cases where they had 4 or more “high-quality” day 3 embryos, their development was only considered in blastocyst medium. Note: This subgroup analysis was performed to provide an ideal comparison of blastocyst culture strategies in conventional IVM (dotted white streaks). Detailed Implementation

[0110] This invention relates to compositions and methods for assisted reproductive technologies in mammals. Specifically, it relates to compositions and methods for the in vitro maturation of mammalian cumulus-oocyte complexes (COCs). The invention is based on the discovery that a combination of low doses of CNP, estradiol, and FSH successfully allows for the in vitro maturation of early antral follicles, particularly small early antral follicles with a diameter less than 9 mm.

[0111] As used in this article, the term "follicle" refers to the ovarian follicle, which is the basic unit of female reproductive biology and consists of a roughly spherical aggregate of cells found in the ovary. A follicle contains a single oocyte. Follicles periodically begin to grow and develop, eventually ovulating, usually with a single competent oocyte. The cells of an ovarian follicle are the oocyte, granulosa cells, and cells of the inner and outer thecae.

[0112] As used herein, the term "oocyte" includes a single oocyte or an oocyte associated with one or more other cells, such as an oocyte that is part of the oocyte complex (COC). The oocyte nucleus is called a germinal vesicle.

[0113] As used in this article, the term "cumulus cell" refers to a cell in a developing ovarian follicle that is directly adjacent to or very close to the oocyte. Cumulus cells are involved in providing some of the nutrients, energy, and / or other requirements necessary for the oocyte to produce a usable embryo at the time of fertilization.

[0114] As used in this article, the term "cumulus-oocyte complex" refers to at least one oocyte and at least one cumulus cell that are physically joined together. Typically, the oocyte is surrounded by a tightly packed layer of cumulus cells, thus forming the cumulus-oocyte complex.

[0115] The assisted reproductive technologies or ARTs used in this article encompass all fertility treatments that involve both female gametes (oocytes) and male gametes (sperm). In vitro fertilization (IVF) is one of several assisted reproductive technologies used to help infertile couples conceive. IVF refers to the process of retrieving oocytes from a female's ovary and fertilizing them with sperm in a laboratory setting.

[0116] In conventional ART, gonadotropins are used to stimulate the ovaries to produce numerous large follicles with mature oocytes. IVM, on the other hand, primarily aims to avoid the side effects of ovarian stimulation by retrieving immature oocytes from small follicles (<12 mm in diameter) from unstimulated or minimally stimulated ovaries. However, compared to conventional ART, the intrinsic developmental capacity of oocytes is reduced after IVM.

[0117] Nuclear maturation of oocytes involves the process of reversing meiotic arrest in prophase I and stimulating meiosis to proceed to metaphase II (MII), where fertilization usually occurs. Oocytes arrested in prophase I exhibit the so-called germinal vesicle (GV- stage), in which the nuclear membrane and nucleolus are visible under a microscope. Nuclear maturation becomes apparent when the oocyte undergoes the so-called GV rupture (GVBD stage), enters MII, and extrudes the first polar body (PB). Cytoplasmic maturation refers to the process preparing the oocyte for activation, forming a pronucleus, and the developmental pathway that continues until implantation is complete. The ability of oocytes to undergo nuclear and cytoplasmic maturation during GV is typically acquired in a stepwise manner.

[0118] In vitro oocyte maturation (IVM) is a technique that allows GV-stage oocytes (such as COCs) enclosed in a tightly packed coronal cumulus cell layer to mature. These oocytes can be obtained via ultrasound-guided needle aspiration before or after ovulation is triggered by the administration of human chorionic gonadotropin (hCG). Oocyte IVM has the potential to simplify fertility treatments or reduce the risks and costs associated with hCG stimulation in patients with normal or high ovarian follicular reserves. This follicular reserve is typically determined by measuring anti-Müllerian hormone (AMH) levels and by ultrasound-guided follicle counting on day 3 of the menstrual cycle.

[0119] The success of ART and IVM largely depends on the maturity of the oocytes before fertilization. Oocytes harvested from antral follicles upon placement in the culture undergo spontaneous resumption of meiosis, i.e., nuclear maturation. This nuclear maturation often occurs before the oocyte has undergone complete cytoplasmic maturation. This is thought to ultimately affect fertilization success and, potentially, subsequent embryonic development and implantation. Therefore, a major challenge of IVM is the synchronization of nuclear cytoplasmic maturation processes within the oocyte. A prolonged oocyte maturation period will promote longer interactions between immature oocytes and well-regulated cumulus cells. Furthermore, IVM with small follicles appears to be more challenging. It is known from the literature that small human follicles do not express sufficient amounts of LH receptors and / or EGF and EGF-like factor receptor systems. Therefore, the major cascade of EGF-like factors that induce maturation cannot be activated.

[0120] In this invention, immature mammalian COCs are collected without prior hCG stimulation or only after hCG stimulation. In a preferred embodiment, no hCG stimulation occurs prior to COC collection. In an even more preferred embodiment, any triggering agents, including high doses of FSH, LHRH, recombinant LH, or LH analogues, must be avoided before COC collection in vivo.

[0121] This invention is based on the finding that specific ranges of “low” doses of C-type natriuretic peptide (CNP) in combination with estradiol and FSH significantly improve the IVM process in mammalian cocci, particularly those from small early antral follicles less than 9 mm in diameter. As will be further seen in the examples below, CNP exhibits a bell-shaped dose curve, distinctly different from the use of PDE3 inhibitors to maintain cocci in meiotic arrest. Surprisingly, not only very low doses, such as 0.1 nM, but also very high doses, such as 1 μM, have proven to be suboptimal for maintaining oocyte arrest in the GV phase. This difference in the dose curve implies a different underlying mechanism, suggesting that CNP treatment does more than simply delay the onset of meiotic resumption in small early antral follicles, as detailed below.

[0122] Therefore, a first aspect of the present invention is to provide in vitro maturation of mammalian COCs. Capacitated culture mediumThe capacitation medium contains 0.1 to 50 nM C-type natriuretic peptide (CNP), estradiol, and FSH. In a specific embodiment, the capacitation medium contains 1 to 1000 nM estradiol. In another embodiment, the capacitation medium contains 0.1-10 mIU / mL of FSH. In yet another aspect, the capacitation medium contains an equivalent dose of recombinant FSH, an FSH analog, or an FSH mimic molecule. In yet another embodiment, the capacitation medium contains 0.1-10 ng / mL insulin. In yet another aspect, the capacitation medium comprises an equivalent dose of an insulin analog or an insulin mimic molecule. In another embodiment, the capacitation medium comprises 0.1 to 50 nM CNP, preferably 10-50 nM CNP, most preferably 10-25 nM CNP, and even more preferably 25 nM CNP; 1 to 1000 nM estradiol, most preferably 10 nM estradiol; 0.1 to 10 mIU / mL FSH, most preferably 2.5 or 1 mIU / mL FSH; and 0.1 to 10 ng / mL insulin, most preferably 5 ng / mL insulin. Furthermore, in some embodiments of the invention, the capacitation medium comprises oocyte secretory factors or combinations of oocyte secretory factors. The oocyte secretory factors used herein are selected from GDF-9, BMP-15, FGF-8, or any combination thereof. In a specific embodiment, the oocyte secretory factor is a recombinant protein. In another aspect, the oocyte secretory factor is a heterodimeric protein. In yet another embodiment, the capacitation medium preferably contains 0.1 to 50 nM CNP, more preferably 10-50 nM CNP, most preferably 10 to 25 nM CNP, even more preferably 25 nM CNP; 1 to 1000 nM estradiol, even more preferably 10 nM estradiol; 0.1 to 10 mIU / mL FSH, even more preferably 2.5 or 1 mIU / mL FSH; 0.1 to 10 ng / mL insulin, even more preferably 5 ng / mL insulin; and oocyte secretion factors selected from GDF-9, BMP-15, FGF-8 or any equivalent or combination thereof.

[0123] Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) are the most studied members of the natriuretic peptide family. CNP is encoded by the precursor natriuretic peptide C (NPPC) gene, which is expressed in different cell types, where the precursor NPPC protein is cleaved into the 22-amino acid peptide CNP. CNP activates its homologous receptor guanylate cyclase B (GC-B), also known as natriuretic peptide receptor B (NPRB), while ANP and BNP stimulate guanylate cyclase A (GC-A), also known as natriuretic peptide receptor A (NPRA). GC-A and GC-B are membrane-anchored guanylate cyclases that signal through the production of the second messenger cGMP. CNP functions in an autocrine / paracrine manner to induce vasodilation and vascular remodeling, and regulate bone growth.

[0124] CNP is 22 amino acid residues long (CNP-22), and an N-terminal elongated form with 53 amino acid residues has also been described (CNP-53). ANP, BNP, and CNP are highly homologous to a 17-residue loop structure formed by intramolecular disulfide bonds. The genetic sequence of the NPPC gene can be obtained from GenBank at locus NM_024409. ANP and BNP are primarily cardiac hormones produced by the atria and ventricles. CNP is thought to be primarily expressed in the brain. However, other studies have shown that CNP is produced by cultured endothelial cells and in vivo blood vessels, and that CNP production is increased by various cytokines and growth factors. Studies have reported ovarian expression of NPPC and NPRB and their regulation by gonadotropins. Recent studies have shown the expression of NPPC mRNA in granulosa cells and the ability of CNP to stimulate cGMP production in cumulus cells. cGMP then diffuses from cumulus cells to oocytes via gap junctions and prevents phosphodiesterase type 3 (PDE3)-dependent cAMP degradation, thus maintaining oocyte meiotic arrest.

[0125] The following examples illustrate that a combination of low-dose CNP with estradiol and FSH is crucial for prolonging CNP-induced meiotic arrest. CNP effectively maintains meiotic arrest for at least 24 hours in COCs, but CNP alone is insufficient to maintain it for 48 hours. Therefore, CNP alone does not allow for IVMs in small early antral follicles, and a prolonged in vitro culture period is essential for successful maturation. However, while a combination of low-dose CNP and estradiol allows COCs to maintain meiotic arrest for an extended period, the final embryo quality after IVF is insufficient. Furthermore, in addition to CNP and estradiol, supplementing the capacitated medium with FSH can also maintain COCs in a prolonged state of meiotic arrest. Additionally, FSH supplementation improves meiotic recovery, increases oocyte diameter, and enhances embryo quality after IVF.

[0126] FSH is a hormone synthesized and secreted by gonadotropic cells in the anterior pituitary gland. FSH regulates human development, growth, puberty maturation, and reproductive processes. FSH and luteinizing hormone (LH) work synergistically in reproduction. In the ovary, FSH stimulates the growth and maturation of immature follicles. As the follicle grows, it releases inhibin, which blocks FSH production. FSH is a diglycoprotein. The α-subunit of LH, FSH, TSH, and hCG is identical and contains 92 amino acids. FSH has a β-subunit of 118 amino acids, which confers its specific biological function and is responsible for interacting with FSH receptors.

[0127] For clinical use, various formulations are available. It is commonly used in the treatment of infertility to stimulate follicle development. FSH can be available in pure forms such as Pergonal or Menopur (which are urinary purified gonadotropins) and as such as Gonal-F, Gonal-f RFF, Gonal-f RFF Pen with LH or hCG. FSH analogues are also clinically useful and include all bioactive mutant forms (e.g., altered one, two, three or more amino acids from the natural form), pegylated FSH, single-chain bifunctional mutants, FSH-CTP, etc. Long-acting FSH therapies have also been developed, including FSH-CTP (Corifollitropin alfa, where the β subunit of FSH is linked via the C-terminal peptide (CTP) portion of hCG) such as Elonva.

[0128] Insulin is a hormone that plays a crucial role in regulating carbohydrate and fat metabolism in the body. In target cells, insulin initiates signal transduction by activating membrane receptors with tyrosine kinase activity. This signal transduction leads to increased glucose uptake and storage. Similarly, in oocytes, the insulin signaling cascade is active. Insulin works synergistically with FSH to promote granulosa cell differentiation and function. Recombinant insulin is also available for clinical use. Insulin is used in clinics as part of synthetic serum replacement (SSR). SSR is a component of various culture media widely used in embryo culture.

[0129] Oocyte-secreting factors are paracrine factors secreted by oocytes and are essential for the normal function of granulosa cells and membrane cells. As used herein, the term "oocyte-secreting factor" should be understood as a factor secreted by oocytes that acts on granulosa cells to regulate key functions such as proliferation, differentiation, glucose metabolism, and cholesterol biosynthesis. Oocyte-secreting factors are selected from GDF-9, BMP-15, FGF-8, or any equivalent thereof. GDF-9 is a member of the transforming growth factor β superfamily. GDF-9 expression in oocytes begins in the primary follicular phase and continues through ovulation. As used herein, "GDF-9" refers to the GDF-9 protein, its single subunit, a multimer of its single subunit, a functional fragment or portion of GDF-9, functional equivalents of GDF-9, and / or analogs. Functional equivalents or fragments of "GDF-9" as defined herein include modified GDF-9 proteins such that the resulting GDF-9 product has activity similar to GDF-9.

[0130] BMP-15 is a member of the transforming growth factor β (TGF-β) superfamily. It is synthesized as a prepropeptide, cleaved, and then processed into a dimer protein. BMP-15 can form homodimers or heterodimers with GDF-9. As used herein, "BMP-15" refers to the BMP-15 protein, its single subunit, multimers of its single subunit, functional fragments or portions of BMP-15, and functional equivalents and / or analogs of BMP-15. Functional equivalents or fragments of "BMP-15" as defined herein include modified BMP-15 proteins, resulting in BMP-15 products with activity similar to BMP-15.

[0131] FGF-8 is a member of the fibroblast growth factor (FGF) family. In the adult ovary, FGF-8 is expressed in oocytes, and FGF receptor expression has been reported in granulosa cells. During oocyte maturation, FGF-8, along with other oocyte-secreted factors, promotes glycolysis in cumulus cells. As used herein, “FGF-8” refers to the FGF-8 protein, its single subunit, multimers of its single subunit, functional fragments or portions of FGF-8, and functional equivalents and / or analogs of FGF-8. Functional equivalents or fragments of “FGF-8” as defined herein include modified FGF-8 proteins such that the resulting FGF-8 products possess FGF-8-like activities.

[0132] In another embodiment, the capacitation medium comprises CNP, estradiol, and FSH, or any combination thereof, in combination with oocyte-secreting factors selected from GDF-9, BMP-15, FGF-8, or combinations thereof. As demonstrated in the examples described below, this combination of growth factors and hormones significantly improves the developmental capacity of oocytes from small antral follicles and the final embryo quality. In particular, the addition of GDF-9 to the capacitation medium promotes oocyte chromatin conformation remodeling into the condensation phase, oocyte diameter, and oocyte and embryo quality.

[0133] Another aspect of the present invention is the in vitro maturation of immature mammalian COCs as described above. Capacity Training Uses of culture medium The intended use includes exposing mammalian COCs to capacitation medium for a period of at least 2 hours and up to 96 hours. Specifically, the exposure of COCs to capacitation medium is sufficient to reach late-stage development, as demonstrated by chromatin reorganization of oocytes into the condensation phase or the so-called surrounding nucleolus (SN) conformation. In another specific embodiment, mammalian COCs are exposed to capacitation medium for a period determined by follicle size.

[0134] Meiotic arrest is induced in immature mammalian cocci using a capacitation medium containing CNP, estradiol, and FSH. Typically, and as can be seen from the examples below, the use of a capacitation medium containing CNP, estradiol, and FSH allows oocytes to undergo extended capacitation culture for up to 96 hours. As a result, this extended period of meiotic arrest allows oocytes from small follicles to survive, grow, and enter further late developmental stages with the aim of achieving greater reproductive capacity. In vitro maturation of these small follicles has been challenging until now because the culture conditions and time available for oocytes to survive under meiotic arrest are too short to achieve adequate nuclear and developmental capacity. As further shown in the examples, the number of follicular oocytes that spontaneously resume meiosis is minimal in the absence of CNP. In contrast, almost 100% of oocytes can be maintained in a state of meiotic arrest when treated with 1 nM, 10 nM, 25 nM, or 50 nM CNP.

[0135] The capacitation medium of the present invention can not only prolong the culture of oocytes, but the presence of CNP also maintains the cumulus-oocyte connection, which significantly improves the developmental capacity of oocytes, especially when compared with the PDE3 inhibitor used in the present invention to maintain COC in a state of meiotic arrest.

[0136] After capacitation using the culture medium described herein, oocytes are able to reach the late developmental stage. Chromatin conformation of GV oocytes was evaluated by Hoechst staining and analysis under a fluorescence microscope. Chromatin conformation was classified into non-nucleolar (NSN), nucleolar (SN), or transitional (NSN / SN) stages based on the pattern of chromatin aggregation around the nucleolus. Oocyte diameters before Hoechst staining were also recorded. Furthermore, meiotic capacity can be analyzed after capacitation by inducing and assessing nuclear maturation under an inverted microscope. Oocyte nuclear maturation is graded as GV (germinal vesicle) stage, GVBD (germinal vesicle rupture) stage, and MII (metaphase) – or PB (polar body). As oocytes grow, they acquire the ability to restart meiosis, a process also known as meiotic resumption. During this stage, the oocyte undergoes rupture of the germinal vesicle membrane and separation of chromosomes from each other.

[0137] In this invention, the contact time between COC and capacitation medium is sufficient to reach the late developmental stage, as demonstrated by, for example, by the chromatin reorganization of oocytes into the condensation phase or SN conformation. Furthermore, the contact time between mammalian COC and capacitation medium is determined by follicle size. Follicle size is determined, for example, by ultrasound imaging upon COC retrieval. Follicles with a diameter of 1 to 5 mm are preferably maintained in capacitation medium for at least 48 hours. Follicles with a diameter >5 to 10 mm are preferably maintained in capacitation medium for at least 24 hours, and follicles with a diameter greater than 10 mm are preferably maintained in capacitation medium for at least 2 hours.

[0138] In another embodiment, the use of capacitation culture medium is part of assisted reproductive technologies, particularly in vitro fertilization and ICSI.

[0139] This invention also provides COC for immature mammals. In vitro maturation methods In this method, immature COCs are collected in a collection medium and contacted with the collection medium for a minimum of 30 minutes and a maximum of 2 hours. Also in this method, and after contact with the collection medium, the mammalian COCs are contacted with a capacitation medium as described above to maintain meiotic arrest and promote cumulus-oocyte interactions. Finally, the mammalian COCs are further contacted with a maturation medium to allow the oocytes to mature through meiosis.

[0140] In a specific implementation, in the method according to the invention, the immature COC is contacted with the collection culture medium for a minimum of 30 minutes and a maximum of 2 hours.

[0141] The collection medium contains a natural or synthetic pharmaceutical compound that inhibits naturally occurring phosphodiesterase, or a natural inhibitor of oocyte meiosis resumption commonly known as an arrester. This natural inhibitor may be selected from CNP, hypoxanthine, or analogues thereof. Using this collection medium allows COCs to be retrieved from very small antral follicles without requiring mandatory ovarian stimulation at any time during estrus or the menstrual cycle. The purpose of the meiotic inhibitor in the collection medium is to prevent the reduction of cAMP from the collected COCs, particularly to prevent the restart of meiosis in those with this ability—those with maturated nuclei—and to maintain the “correct” conditions of the connection between the oocyte and cumulus. The collection medium is used during COC collection as the medium in which the COCs are transferred from the puncture needle. In this collection medium, freshly isolated COCs are preferably held for a minimum of 30 minutes and a maximum of 2 hours. The primary purpose of this collection medium is to maintain the COCs in optimal functional condition while separating them from follicular fluid and other contaminating cells. The COCs are separated as separate entities to be transferred to a “capacitation medium.”

[0142] In another embodiment of the in vitro method according to the invention, mammalian COCs are contacted with a capacitation medium as described above. In this method, the mammalian COCs are contacted with a capacitation medium containing CNP, estradiol, and FSH for a period of at least 2 hours and at most 96 hours. Specifically, in the in vitro maturation method, the contact time between the COCs and the capacitation medium is sufficient to reach late developmental stages, as demonstrated by chromatin reorganization of the oocytes into the condensation phase or the so-called surrounding nucleolus (SN) conformation. More specifically, in the in vitro maturation method, the restart of meiosis is analyzed by assessing nuclear maturation under an inverted microscope. Nuclear maturation of the oocytes is graded as GV (germinal vesicle) phase, GVBD (germinal vesicle rupture) phase, and MII (metaphase) or PB (polar body) phase. In another specific embodiment, the mammalian COCs are contacted with the capacitation medium for a period determined by follicle size. As already described herein, follicle size is determined upon COC retrieval, for example by ultrasound imaging. Follicles with a diameter of 1 to 5 mm are preferably maintained in the capacitation medium for at least 48 hours. Follicles with a diameter >5 to 10 mm are preferably kept in capacitation medium for at least 24 hours, and follicles with a diameter >10 mm are preferably kept in capacitation medium for at least 2 hours.

[0143] In a specific implementation, mammalian COCs are contacted with capacitated medium in both adherent and non-adherent culture plates. As will become apparent from the following examples, the combination of low-dose CNP with estradiol and FSH is crucial for prolonging CNP-mediated meiotic arrest. CNP effectively maintains meiotic arrest in COCs for at least 24 hours, but CNP alone is insufficient to maintain meiotic arrest for 48 hours. However, while the combination of low-dose CNP with estradiol allows COCs to maintain meiotic arrest for an extended period, the final embryo quality of the oocytes after IVF is not sufficiently high. In addition to CNP and estradiol, supplementing the capacitated medium with FSH also maintains COCs in meiotic arrest for a longer period, but also increases oocyte diameter and improves embryo quality after IVF.

[0144] In another embodiment, in the in vitro method according to the invention, oocyte-secreting factors such as GDF-9, BMP-15, FGF-8, or any combination thereof, are added to a capacitation medium containing CNP, estradiol, and FSH. Similar to FSH, these oocyte-secreting factors improve the developmental capacity of small antral follicle oocytes and their final embryo quality.

[0145] As previously described, in the in vitro method of the present invention, COCs are further contacted with a maturation medium. During this period, and when given the capacitation medium according to the present invention, capacitated GV-stage oocytes with an arbitrary basic composition can be matured in a maturation medium supplemented with selected growth factors. These growth factors are selected from, but are not limited to, EGF-like factors, such as bimodalin or epithelial regulatory proteins, FSH, LH, cAMP regulators, or combinations thereof. Therefore, in another embodiment, the in vitro maturation method of the present invention comprises contacting COCs with the capacitation medium described herein, and then contacting COCs with a maturation medium, characterized in that the maturation medium is supplemented with selected growth factors as described herein.

[0146] Another object of the present invention is to provide an in vitro maturation method for immature mammalian COCs. Reagent test kit The kit comprises a capacitation medium as described herein, containing 0.1-50 nM CNP, estradiol, and FSH. In a specific embodiment, the kit according to the invention comprises a capacitation medium containing 1 to 1000 nM estradiol. In another embodiment, the kit comprises 0.1 to 10 mIU / mL FSH. In yet another aspect, the capacitation medium comprises an equivalent dose of recombinant FSH, an FSH analog, or an FSH mimic molecule. In yet another embodiment, in the kit according to the invention, the capacitation medium comprises 0.1 to 10 ng / mL insulin. In another aspect, the capacitation medium comprises an equivalent dose of an insulin analog or an insulin mimic molecule. In another embodiment, in the kit according to the invention, the capacitating medium comprises 0.1 to 50 nM CNP, preferably 10 to 25 nM CNP, even more preferably 25 nM CNP; 1 to 1000 nM estradiol, preferably 10 nM estradiol; 0.1 to 10 mIU / mL FSH, preferably 2.5 or 1 mIU / mL FSH; and 0.1 to 10 ng / mL insulin.

[0147] In another embodiment, in the kit according to the invention, the capacitation medium contains oocyte secretory factors. The oocyte secretory factors used herein are selected from GDF-9, BMP-15, FGF-8, or any combination thereof. In a specific embodiment, the oocyte secretory factors used herein are recombinant proteins or heterodimeric proteins. In another embodiment, the kit for in vitro maturation of immature mammalian COCs comprises (a) a collection medium containing a natural or synthetic chemical compound that inhibits naturally occurring phosphodiesterase or a natural inhibitor of oocyte meiosis, (b) the capacitation medium as described above, (c) a maturation medium, (d) adherent and non-adherent culture plates, and (e) instructions for using the kit. As described herein, the kit can be used to perform the methods described herein and the instructions for performing the methods contained in the kit.

[0148] Another aspect of the present invention discloses the in vitro maturation of mammalian COCs as described above. reagent kit way In specific embodiments, the use of a kit containing capacitation medium as described above is disclosed. This use includes contacting mammalian COCs with capacitation medium for a period of at least 2 hours to a maximum of 96 hours. More specifically, in the use of the kit containing capacitation medium, mammalian COCs are contacted with capacitation medium to maintain meiotic arrest and allow nuclear and cytoplasmic maturation. In particular, the contact time with capacitation medium is sufficient to achieve late development, as demonstrated by chromatin remodeling to surround the nucleolus (SN). In another specific embodiment, mammalian COCs are contacted with capacitation medium for a period determined by follicle size. As already described herein, follicle size is determined upon COC retrieval, for example by ultrasound imaging. Follicles with a diameter of 1 to 5 mm are preferably maintained in capacitation medium for at least 48 hours. Follicles with a diameter > 5 to 10 mm are preferably maintained in capacitation medium for at least 24 hours, and follicles with a diameter greater than 10 mm are preferably maintained in capacitation medium for at least 2 hours.

[0149] The capacitation phase enables oocytes to acquire the ability to resume meiosis, which can only be assessed after meiotic stimulation is triggered (e.g., contact with maturation medium). Assessment of oocyte nuclear maturation is performed under an inverted microscope and demonstrated by germinal vesicle rupture (GVBD) and further expulsion of the first polar body (PB).

[0150] In another embodiment, the use of a kit comprising: (a) a collection medium containing a natural or synthetic chemical compound that inhibits naturally occurring phosphodiesterase or a natural inhibitor of oocyte meiosis; (b) a capacitation medium as described above; (c) a maturation medium; (d) adherent and / or non-adherent culture plates; and (e) instructions for using the kit. Typically, the use of a kit for in vitro maturation of immature mammalian COCs involves contacting the COCs with the collection medium for at least 30 minutes to a maximum of 2 hours. In another aspect, in the use of the kit according to the invention, the mammalian COCs are contacted with the capacitation medium for a period of at least 2 hours to a maximum of 96 hours. More particularly, in the use of a kit comprising a collection medium, a capacitation medium, a maturation medium, adherent and / or non-adherent culture plates, and instructions for use, the mammalian COCs are contacted with the capacitation medium to maintain meiotic arrest and allow maturation. Specifically, the contact time with the capacitation medium is sufficient to reach the GVBD phase and restart meiosis, thereby reaching a late developmental stage, as demonstrated by the surrounding nucleolus (SN). In addition, mammalian COCs are exposed to capacitation medium for a period of time determined by follicle size. As already described herein, follicle size is determined upon COC retrieval, for example by ultrasound imaging. Follicles with a diameter of 1 to 5 mm are preferably held in capacitation medium for at least 48 hours. Follicles with a diameter >5 to 10 mm are preferably held in capacitation medium for at least 24 hours, and follicles with a diameter greater than 10 mm are preferably held in capacitation medium for at least 2 hours.

[0151] Capacitation enables oocytes to acquire the ability to resume meiosis, which is assessed only after meiotic stimulation is triggered (e.g., contact with maturation medium). Assessment of oocyte nuclear maturation is performed under an inverted microscope and is demonstrated by germinal vesicle rupture (GVBD) and further expulsion of the first polar body (PB).

[0152] In different embodiments of the present invention, the mammalian COC is the human COC.

[0153] Other aspects of the invention are well known in the art. For example, methods for obtaining oocytes from a subject, tools and apparatus for manipulating oocytes, cryopreservation methods, IVF methods, in vitro embryo culture, and methods for placing embryos in a patient's reproductive tract are well known in the art, and any such suitable methods known in the art can be used in combination with the methods and compositions of the present invention.

[0154] The invention can be further illustrated by the following non-limiting embodiments.

[0155] Example

[0156] Example 1

[0157] Materials and methods

[0158] animal models

[0159] The animals used in this study were CBAB6F1 (an F1 hybrid of C57Bl / 6j and CBA / ca). These animals were kept and fed in captivity in accordance with national regulations and with the consent of the Ethics Committee of the Vrije Universiteit Brussel (Project No.: 09-216-1).

[0160] Collection of immature cumulus-oocyte complex (COC) from small antral follicles and ovulation from large antral follicles. Former Clash of Clans

[0161] To collect immature follicular follicles (COCs), dense COCs from the first wave of follicular development were collected from small antral follicles of prepubertal mice (19–21 days old) without prior gonadotropin administration. To collect pre-ovulatory COCs (control), dense COCs were collected from large antral follicles of prepubertal female mice (25–27 days old) by puncture 48 hours after stimulation with 2.5 IU of equine chorionic gonadotropin (eCG, Folligon, Intervet, Oss, The Netherlands). The collection medium consisted of Leibovitz L-15 containing 10% heat-inactivated fetal bovine serum (FBS), 100 IU / ml penicillin, and 100 μg / ml streptomycin (all from Life Technologies, Gent, Belgium), supplemented with 200 μM 3-isobutyl-1-methylxanthine (IBMX; Sigma, Schnelldorf, Germany) to prevent meiotic restart during collection and pre-culture treatment.

[0162] COC culture

[0163] The basal medium used to culture COCs (pre-IVM and IVM phases) consisted of α-MEM, 2.5% FBS (both from Life Technologies, Gent, Belgium), and 5 ng / mL insulin, 5 μg / mL Apo-transferrin, and 5 ng / mL sodium selenite (all from Sigma, Schnelldorf, Germany).

[0164] For the pre-IVM experiments, CNP-22 was obtained from Phoenix Europe (Karlsruhe, Germany), 17-β-estradiol from Sigma (Schnelldorf, Germany), and growth and differentiation factor 9 (GDF9) from R&D systems Europe (Oxon, United Kingdom).

[0165] For experiments involving IVM, recombinant epidermal growth factor (r-EGF) (Roche; Mannheim, Germany) and recombinant mouse epithelial regulatory protein (EREG) (R&D systems Europe; Oxon, United Kingdom) were used as ovulation stimulants, and the culture was carried out for 18 hours.

[0166] The text mentions adding recombinant follicle-stimulating hormone (FSH) (Merck-Serono, Geneva, Switzerland) to pre-IVM and IVM culture media.

[0167] Evaluation of meiosis restart

[0168] At specified time points, oocytes were mechanically freed from dense or expanded cumulus cells using a controlled opening via a fine-well glass pipette. Meiotic restart was analyzed by assessing nuclear maturation under an inverted microscope equipped with a Hoffman modulated contrast system (Nikon, Tokyo, Japan). Nuclear maturation was scored as GV (germinal vesicle stage), GVBD (when GV is not visible), PB (first polar body observed in the perivitelline space), or DEG (when the oocyte degenerates).

[0169] Assess the chromatin conformation of oocytes

[0170] Chromatin conformation of oocytes was evaluated before and after pre-IVM culture in germinal follicle oocytes. Briefly, GV oocytes were stained with 10 μg / mL Hoechst 33258 (Sigma; Schnelldorf, Germany) for 5 min after assessment of meiotic restart. Nucleolar chromatin conformation was analyzed under a fluorescence microscope (IX70; Olympus). Chromatin conformation was classified into non-nucleolar (NSN), nucleolar (SN), or transitional (NSN / SN) phases based on the pattern of chromatin aggregation around the nucleolus [27-29]. The diameter of some oocytes in these oocytes was recorded prior to Hoechst staining.

[0171] In vitro fertilization (IVF) procedure

[0172] In the final test to assess oocyte developmental capacity, in vitro fertilization (IVF) was performed after a pre-IVM + IVM culture period, followed by embryo culture to the blastocyst stage. For this experiment, 100 ng / mL EREG was used as a trigger for meiotic resumption.

[0173] The culture medium used for IVF consists of M16 medium, 3% bovine serum albumin fraction V (BSA) (both from Sigma, Schnelldorf, Germany), and non-essential amino acids (Life Technologies, Gent, Belgium). The embryo culture medium consists of M16 medium and essential and non-essential amino acids (Life Technologies, Gent, Belgium).

[0174] Cumulo-oocyte complexes were collected under different conditions and washed once in IVF medium. Capacitated sperm (2 × 10⁻⁶) obtained from male CBAB6F1 cells were used. 6 In vitro fertilization was performed in IVF medium at a final dilution of sperm / mL. After co-incubation at 37°C, 5% CO2, 5% O2, and 100% humidity for 3.5 hours, the presumed zygotes were excised, washed twice, and cultured in groups of 10–15 fertilized eggs in 20 μL of oil-coated embryo culture medium at 37°C, 5% CO2, 5% O2, and 100% humidity for embryo culture (Irvine Scientific, Alere; Sint Denijs Westrem, Belgium). The division (2-cell) rate was scored 24 hours post-IVF. Blastocyst development and hatching were recorded on day 5.

[0175] The ability of immature antral follicles to form cocoons was also assessed in 20-day-old mice that had undergone 18 hours of IVM with 100 ng / mL EREG.

[0176] In vivo oocytes (control) were obtained from 25-27 day old female mice treated with 2.5 IU of equine chorionic gonadotropin (eCG, Folligon®) for 14 hours and then stimulated with 2.5 IU of hCG (Chorulon®) for 48 hours (both from Intervet, The Netherlands). These oocytes were fertilized with the same sperm samples and cultured under the exact same conditions as IVM oocytes.

[0177] Statistical analysis

[0178] Unless otherwise stated, results are presented as mean ± SD. Differences in oocyte meiotic recovery rate (per meiotic phase), chromatin conformation, and embryonic development after IVF under different in vitro conditions were assessed by ANOVA followed by Tukey's multiple comparison test, p < 0.05. When comparing two conditions, unpaired t-tests were used to compare meiotic recovery rates. Data were converted to percentages (arcsine) before statistical analysis.

[0179] result

[0180] CNP effectively maintains meiotic arrest and delays EGFR-dependent meiotic recovery.

[0181] In the presence of 0 (control), 1, 10, and 100 nM CNP-22 and bound to 4 ng / mL EGF, preovulatory COCs taken from gonadotropin-stimulated mice (26-27 days old) were cultured for 18 hours.

[0182] CNP-22 has a dose-dependent effect on maintaining meiotic arrest. At doses of 100 and 10 nM, the GV rate at the end of culture was significantly higher than that at 1 nM and the control (96%, 93%, 48%, and 0%, respectively). Figure 1 A)

[0183] In the presence of EGF, a dose-dependent effect of CNP-22 on PB rate was observed. At doses of 10 and 100 nM CNP-22, many oocytes remained in the GVBD phase, thus maintaining significantly lower PB rates (35% and 15%, respectively) compared to the control group and 1 nM CNP-22 (98% and 92%, respectively). Figure 1 B). Due to the higher proportion of GVBD oocytes observed after treatment with 10 and 100 nM CNP, follow-up experiments were conducted to explore the possibility of a slower meiotic recovery process.

[0184] Preovulatory cocci (COCs) from gonadotropin-stimulated mice were cultured in the absence of (control) or in the presence of 25 nM CNP-22 + 4 ng / mL EGF, and meiotic maturation was assessed at 2, 4, and 6 hours. Meiotic recovery was increased by 2 to 6 hours in the control group, but within the same timeframe, the CNP-22 + EGF treatment group induced less GVBD (≤11%). Figure 2 A). Furthermore, COC cultured in CNP-22 + EGF medium for 24 hours had a high incidence of PB oocytes (93%), a figure significantly higher than that of COC cultured solely in CNP-22 for the same period. Figure 2 B).

[0185] In summary, these data indicate that CNP-22 can maintain meiotic arrest for at least 24 hours, and EGFR signaling can induce meiotic recovery in COCs with meiotic arrest maintained by CNP, but at a slower rate (vs. 6 hours delay in control).

[0186] Prolonged meiotic arrest in CNP-induced immature COCs depends on the presence of estradiol in the culture environment.

[0187] Similar to the studies described above, a replication study was conducted with immature follicle-forming cells (high meiosis / underdevelopment). Such follicle-forming cells (as described in Materials and Methods) were extracted from small antral follicles and cultured for 48 hours. Results showed that CNP-22 could maintain meiotic arrest for 24 hours but not 48 hours (data not shown). Therefore, a study was designed to investigate the responsiveness of CNP-22 to a medium supplemented with E2, FSH, and GDF9.

[0188] Immature cocci were removed from small antral follicles of 19-20 day old female mice and cultured for 48 hours in the presence of 25 nM CNP-22 and with or without 10 nM 17-β-estradiol; the effects of further addition of 2.5 or 5 mIU / mL FSH and / or 50 ng / mL GDF9 were also evaluated.

[0189] Oocytes from cocci cultured in the presence of 25 nM CNP (alone) primarily failed to maintain meiotic arrest; therefore, after 48 hours of culture, the GV rate was only 26% of the total number of cocci. In contrast, most oocytes from cocci cultured in the presence of 25 nM CNP-22 plus 10 nM 17-β-estradiol (E2) effectively maintained GV phase (≥89%). Figure 3 In ), regardless of whether FSH or GDF9 is used as a supplement.

[0190] FSH and GDF9 supplementation affect oocyte chromatin condensation, oocyte diameter, and COC in response to EGFR ovulation signals. The effect of conduction reactivity

[0191] Immature cocci (COCs) were extracted from small antral follicles of female mice (19-20 days old) in the presence of 25 nM CNP-22 and 10 nM 17-β-estradiol and cultured for 48 hours (pre-IVM conditions) with the addition of 2.5 mIU / mL FSH or 2.5 mIU / mL LFSH + 50 ng / mL GDF9. These pretreatments were followed by ovulation stimulation for 18 hours in media containing EGF (IVM conditions).

[0192] Chromatin condensation analysis of oocytes before and after pre-IVM treatment showed that during 48 hours of culture, the chromatin of oocytes changed from a predominantly non-surrounding nucleolar (NSN) dispersed conformation to a surrounding nucleolar (SN) condensed conformation. In fact, prior to pre-IVM, 34% of oocytes had a transitional NSN / SN conformation (66% NSN), while prior to pre-IVM, ≥68% of oocytes showed an SN pattern under each condition. Figure 4 A). In oocytes cultured in the presence of 2.5 mIU / mL FSH + 50 ng / mL GDF9, the SN pattern was not statistically significant but the absolute number was high (86%).

[0193] Furthermore, the average diameter of oocytes obtained after pre-IVM culture in FSH-containing medium (w / o GDF9) was significantly larger than that of oocytes cultured in FSH-free medium. Therefore, immediately after follicle separation (before pre-IVM), the oocyte diameter was 71.9 ± 2.1 μm, while after 48 hours of culture, the oocyte diameters were as follows: CNP + E2, CNP + E2 + FSH, and CNP + E2 + FSH + GDF9 were 72.1 ± 1.7 μm; 73.5 ± 1.7 μm; and 73.3 ± 1.4 μm, respectively. Figure 4 B).

[0194] Following pre-IVM, some COC meiotic restart was stimulated with EGF, and its PB ratio was assessed after 18 hours. Meiotic restart was delayed in the presence of CNPs in IVM medium (see previous experiments). CNP effectively maintains meiosis arrest. And delay EGFR-dependent meiotic recovery (For practical reasons, CNPs were omitted from the IVM medium in this experiment.) Oocytes from the three culture conditions showed high meiotic recovery rates: PB rates of 79%, 78%, and 82% for CNP + E2, CNP + E2 + FSH, and CNP + E2 + FSH + GDF9, respectively.

[0195] Pre-IVM improves oocyte and embryo quality in the presence of CNP, FSH, and GDF9.

[0196] The developmental capacity of oocytes that underwent pre-IVM followed by IVM culture was investigated. Oocytes were fertilized in vitro, and embryos were cultured until day 5.

[0197] For this experiment, EREG was used as a meiotic trigger during the IVM phase, and only CNP and GDF9 were added to the pre-IVM medium and omitted from the IVM medium.

[0198] Following the pre-IVM and IVM culture periods, cumulus cells from all treatments exhibited significant expansion and mucinization in response to EREG.

[0199] There was no difference in 2-cell (fertilization) rates among the different treatments (CNP + E2, CNP + E2 + FSH, and CNP + E2 + FSH + GDF9 were 60%, 54%, and 56%, respectively). Compared to the CNP + E2 condition, oocytes cultured in media containing FSH or FSH + GDF9 had a higher blastocyst / 2-cell ratio on day 5; the latter was significantly higher (…). Figure 5 ).

[0200] For reference, the ability of 1) immature COCs that have undergone IVM (without pre-IVM culture) and 2) oocytes that have undergone ovarian hyperstimulation with eCG followed by hCG to grow in vivo shows... Figure 5 C.

[0201] Example 2

[0202] The above experiments demonstrate that the use of the capacitation medium according to the invention not only maintains oocytes under meiotic arrest but also does so without affecting the ability of these cocci to undergo IVM. However, the effect of CNP cannot be considered solely as a result of its action on phosphodiesterase (PDE, the enzyme responsible for the degradation of cAMP within the oocyte during meiotic recovery); because oocyte developmental capacity is greatly enhanced during capacitation culture. Therefore, it is hypothesized that at “low” doses, CNP (especially at concentrations ranging from 1 nM to 50 nM) has a further (additional) effect on improving oocyte developmental capacity by maintaining good communication between oocytes and cumulus cells.

[0203] In the presence of cAMP regulators (especially in pre-maturation cultures using PDE3 inhibitors), the problem of disconnection between the cumulus-corona and oocytes during long-term culture of COCs has been clearly reported as a limitation of the PDE inhibitor approach (Nogueira et al., 2003, Nogueira et al., 2006, Vanhoutte et al., 2009a).

[0204] To demonstrate that capacitation medium containing CNP actually maintains oocyte junctions, oocyte complexes (COCs) from early antral follicles of CBAB6F1 mice (as above) were placed in basal medium used to culture COCs and exposed to CNPs and two well-known phosphodiesterase inhibitors, PDE3-I (Org9935 and cyclohexylquinamide), as comparisons.

[0205] Materials and methods

[0206] animal models

[0207] The animals used in this study were CBAB6F1 (an F1 hybrid of C57Bl / 6j and CBA / ca). These animals were kept and fed in captivity in accordance with national regulations and with the consent of the Ethics Committee of the Vrije Universiteit Brussel (Project No.: 14-216-1).

[0208] COC culture

[0209] The basal medium used for culturing COCs (pre-IVM and IVM phases) consisted of α-MEM, 2.5% FBS (both from Life Technologies, Gent, Belgium), and 5 ng / mL insulin, 5 μg / mL Apo-transferrin, and 5 ng / mL sodium selenite (all from Sigma, Schnelldorf, Germany). The capacitation medium consisted of basal medium supplemented with 25 nM CNP, 1 μM Org9935, or 1 μM cyclohexylquinamide in combination with 10 nM E2 17-β-estradiol. CNP was obtained from Phoenix Europe (Karlsruhe, Germany), while cyclohexylquinamide was obtained from Enzo Life Sciences (Antwerpen, Belgium). Because the purpose of the experiment was to understand the potential differences between CNP and PDE3 inhibitors, avoiding potential interference from FSH was crucial; therefore, the latter was omitted from the capacitation medium. When tested in the presence of FSH, the latter masks the effect of CNP on the connection between the oocyte and the surrounding cumulus cell layer, which itself contributes to the connection, even in the presence of PDE3 inhibitors (data not shown).

[0210] Staining and Image Analysis

[0211] By using Texas Red-Phallotoxin or Actin Green TM Fluorescently labeled F-actin was used to demonstrate transregional protrusions (TZPs, membranous extensions of granulosa cells connected to oocytes) and were shown as filaments (arrows) traversing the zona pellucida.

[0212] result

[0213] Although the three compounds were able to maintain oocyte meiotic arrest, it was unexpected that CNP was a factor of the transregional protrusions necessary to maintain bidirectional communication between the oocyte and the surrounding cumulus cell layer. Figure 6 and Figure 7 ).exist Figure 7In this study, average pixel intensity was used as a tool to quantify positive actin staining (TZP) across the zona pellucida. Image analysis was performed using ImageJ and included calculating the average pixel intensity on a generalized region of interest (ROI) between the oocyte and cumulus cells, within which the zona pellucida lies. Based on the image analysis, COCs exposed to the CNP were observed to maintain better connectivity between the oocyte and cumulus cells via transregional protrusions (TZPs). Figure 7 In (A), Org9935 was used as a PDE3 inhibitor, and actin filaments were confirmed using phalloidin bound to Texas red; while... Figure 7 In (B), cyclohexylquinamide was used as an inhibitor of PDE3, and Actin green was used. TM Actin filaments were confirmed. The Mann-Whitney test was used to statistically compare the CNP and PDE3i groups, with P-values ​​of 0.0082 in Figure A and <0.0001 in Figure B.

[0214] Example 3

[0215] If the above results indicate that CNP enhances connectivity between oocytes and cumulus cells in a coccus, the following results aim to determine whether this affects the developmental capacity of such coccuses cultured from early antral follicles. In this study, the differential effects of CNP and PDE3I on the developmental capacity of mouse cumulus-closed oocytes from early antral follicles were assessed during capacitation culture. Since the aim of the experiment was to understand the potential differences between CNP and PDE3 inhibitors, avoiding potential interference from FSH was crucial; therefore, the latter was omitted from the capacitation medium in this experiment.

[0216] set up: Similar to Example 2; immature cumulus-oocyte complexes were isolated from early antral follicles (unstimulated 19-20 day old mice). COCs were placed in basal medium for 48 hours in the presence of CNP or cyclohexylquinamide (a PDE3 inhibitor).

[0217] Includes two reference comparisons: 1) Under control conditions, no capacitation culture was performed before IVM. 2) Standard in vivo control, derived from fully mature oocytes from 26-27 day old mice. These COCs were obtained by administering PMSG followed by hCG to 23-24 day old mice (IVF protocol).

[0218] Following capacitation culture, COCs mature in the presence of epidermal growth factor (EGF) and in vitro fertilization. Post-fertilization embryonic development is assessed.

[0219] The acquisition of oocyte capacity under each condition is assessed by its ability to mature, fertilize (2-cell rate), and produce high-quality blastocysts (through day 5 embryo culture).

[0220] Statistical analysis

[0221] The differences in fertilization rate and blastocyst formation rate between the CNP and cyclohexylquinamide groups were assessed using the chi-square test.

[0222] result

[0223] Although no difference was observed in fertilization rates between the two treatments (see [link to treatment]). Figure 8 (A)), but after capacitation culture in the presence of CNP, the number of blastocysts formed per fertilized egg was significantly higher (see [A]). Figure 8 (B)).

[0224] Conclusions of Examples 2 and 3

[0225] The unexpected differences in oocyte and embryo quality observed in these two complementary studies comparing PDE inhibitors and CNP can be attributed to CNP. These results suggest that CNP has a greater role than those mediated by type 3 phosphodiesterase. In addition to inhibiting PDE3, CNP increases physical connectivity between oocytes and cumulus cells, thereby enhancing the acquisition of factors necessary for the final development of oocytes (cytoplasmic maturation).

[0226] Example 4

[0227] In Example 1, CNP-22 was demonstrated to have a dose-dependent effect on maintaining meiotic arrest. Additional experiments, encompassing a broader range of CNPs, were conducted in the following studies.

[0228] set up: The materials and methods used in this further embodiment are correspondingly the same as those used in Example 1. Cumulooocyte complexes (COCs) from 24-26 day old mice were isolated from antral follicles grown in vivo after 48 hours of stimulation with equine chorionic gonadotropin (eCG, Folligon, Intervet, Oss, The Netherlands). When the mice were 22-24 days old, they were injected with 2.5 IU of eCG. Intact COCs with at least two layers of cumulus cells were cultured for 18 hours in the presence of the following doses of CNP: - Control group (without CNP)

[0229] - 0.1nM

[0230] - 1nM

[0231] - 10nM

[0232] - 50nM

[0233] - 100nM

[0234] - 1µM Control conditions, in which CNP is not present in the culture medium.

[0235] New test doses of 0.1 nM, 50 nM and 1 µM (included in the claims).

[0236] COCs were collected and cultured in mice according to the previous dose experiment provided in Example 1.

[0237] Statistical analysis

[0238] Differences in oocyte meiotic recovery rates (per meiotic phase) were assessed using ANOVA and Tukey's multiple comparison test, with p < 0.01. Data were converted to percentages (arcsine) before statistical analysis.

[0239] result: In summary, the supplementary experiments of this invention confirm that CNP-22 has a dose-dependent effect on maintaining meiotic arrest. The presence of intact germinal vesicles (GVs) in oocytes under an inverted microscope confirms meiotic arrest.

[0240] Figure 9 A showed that doses of 1 nM, 10 nM, 50 nM, and 100 nM maintained oocyte meiotic arrest at rates ≥80% (80%, 98%, 94%, and 87%, respectively). However, only the 10 nM and 50 nM doses were significantly higher than the control condition without CNP (98% and 94% vs 50%, respectively, p < 0.01).

[0241] No significant difference was recorded in the percentage of oocytes during the germinal follicle rupture (GVBD) phase. Figure 9 B).

[0242] At CNP doses of 1 nM, 10 nM, 50 nM, and 100 nM, the proportion of oocytes extruding the first polar body was low or absent (PB ratio). Figure 9 (C) (The last three doses were significantly different from the control condition without CNP, p<0.001), which matched the finding that CNP showed a more effective effect in maintaining oocyte meiotic arrest at these doses.

[0243] Surprisingly, not only very low doses such as 0.1 nM, but also very high doses such as 1 μM, have proven to be suboptimal for maintaining oocyte arrest during the GV phase.

[0244] in conclusion: In summary, these data indicate that CNP-22 can maintain meiotic arrest in preovulatory oocytes at a rate of over 80% at dose intervals of 1 nM to 100 nM, consistent with the recommended CNP dose for maintaining oocyte arrest during the GV phase, preferably 10-50 nM, more preferably 10-25 nM. Therefore, this behavior differs from the use of PDE3 inhibitors, where increasing the dose does indeed lead to sustained oocyte arrest.

[0245] Example 5

[0246] To support the claim that the effects of CNP are not limited to CNP-22 itself, additional experiments were conducted to test whether these effects could be reproduced by CNP analogs. Further experiments were performed to test the effects of the CNP analog CNP-53. Similar to CNP-22, CNP-53 is one of the major endogenous forms of C-type natriuretic peptide, containing a 53-amino acid sequence.

[0247] set up: Except for the alternative use of CNP-53, the materials and methods used in this further embodiment are correspondingly the same as those used in Example 4. Cumulooocyte complexes from 24-25 day old mice were isolated from antral follicles grown in vivo after 48 hours of eCG stimulation. When the mice were 22-23 days old, they were injected with 2.5 IU of eCG. Intact COCs were cultured for 18 hours in the presence of the following doses of CNP-53: - Control group (without CNP)

[0248] - 0.1 nM

[0249] - 1 nM

[0250] - 10 nM

[0251] - 50 nM

[0252] - 100 nM

[0253] - 1 µM

[0254] -Comparison with 25nM CNP-22 Includes two control conditions: 1) 25 nM CNP-22 (standard dose known and used in previous experiments to maintain) Meiosis arrest), 2) Control conditions, in which CNP is not present in the culture medium.

[0255] Statistical analysis

[0256] The differences in oocyte meiotic recovery rates (per meiotic phase) were assessed using ANOVA and Tukey's multiple comparison test, with p < 0.001. Data were converted to percentages (arcsine) before statistical analysis.

[0257] result: Similar to the results found in supplemental experiments using CNP-22, CNP-53 demonstrated a surprisingly (maximum) dose-dependent effect on maintaining oocyte meiotic arrest.

[0258] Figure 10 A showed that, compared with the control condition without CNP, CNP-53 doses of 1 nM, 10 nM, 50 nM and 100 nM maintained meiotic arrest in oocytes at significantly higher rates (66%, 98%, 72% and 69% vs. 18%, respectively, p < 0.001), comparable to the CNP-22 control (75%, p < 0.01).

[0259] No significant difference was recorded in the percentage of oocytes during the germinal follicle rupture (GVBD) phase. Figure 10 B).

[0260] Compared to the control condition without CNP, the proportion of oocytes expelled from the first polar body (PB stage) was significantly different at doses of 1 nM, 10 nM, 50 nM, and 100 nM (p < 0.001), and this matched the finding that these specific doses of CNP-53 showed a more effective effect in maintaining oocyte meiotic arrest. Figure 10 C).

[0261] Surprisingly, not only very low doses such as 0.1 nM, but also very high doses such as 1 μM, have proven to be suboptimal for maintaining oocyte arrest during the GV phase.

[0262] in conclusion: Overall, these data indicate that, comparable to CNP-22, CNP-53 is able to maintain meiotic arrest in preovulatory oocytes at a high rate. Furthermore, the effective doses of CNP-53 are similar to those of CNP-22: 1 nM, 10 nM, 50 nM, and 100 nM, with varying expression levels within the 1 nM to 100 nM CNP range; the optimal effect in maintaining meiotic arrest in preovulatory oocytes is observed within the 10 nM to 50 nM dose range.

[0263] Example 6

[0264] Preclinical results demonstrate that the capacitation medium of this invention does indeed have an unexpected effect on the maturation of oocytes derived from small follicles. In this study, the IVM method using the capacitation medium of this application has been evaluated to assess whether it affects oocyte developmental potential, whether it produces fertilizable oocytes, and whether it increases their blastocyst formation potential.

[0265] Patient group: Patients participating in this study underwent IVM treatment (N = 15), agreed to donate a subset of oocytes for the production of study embryos, and had the following characteristics: age <37 years; clinical history of polycystic ovary syndrome (PCO or PCOS) according to the Rotterdam criteria (Rotterdam ESHRE / ASRM-Sponsored PCOS consensus workshop Group, 2004).

[0266] If a patient has 30 or more follicles in the last ultrasound scan prior to oocyte retrieval, a portion of them (usually 5-10) are assigned to a new IVM procedure; the remainder are assigned to the routine clinical IVM procedure as part of the patient’s treatment.

[0267] All patients received a personalized stimulation protocol consisting of a cumulative dose of 600 IU of HP-hMG (a highly purified human menopausal gonadotropin from Ferring Pharmaceuticals SA).

[0268] Once the patient has at least one dominant follicle with an average diameter of 10-12 mm on ultrasound scan, live oocyte retrieval (OPU) is scheduled 42 hours after the last HP-hMG injection.

[0269] This evidence-based study recruited 15 sibling cases: Experimental treatment = COC capacitation (using the capacitation medium according to the invention) + IVM Routine clinical arm = Routine IVM (Origio® IVM methodology, adapted from VUB)

[0270] Immature oocytes were retrieved, capacitated, cultured, and subjected to IVM and ICSI.

[0271] Cumulo-oocyte complexes (COCs) were retrieved from follicles measuring 2-10 mm using a 17-gauge single-inner-cavity needle at an aspiration pressure of 70 mmHg and collected in a 50 μM IBMX (Sigma) “collection medium” supplemented with 25 IU / ml heparin (Heparin Leo, Leo Pharma, Belgium).

[0272] The collected follicle aspirate was immediately diluted in collection medium (3 ml pre-filled collection medium per tube). The contents of the collection tube were filtered from contaminated blood cells (Falcon cell filter; 70 mm sieve), and COCs were collected from the culture dish and held in the collection medium for a maximum of 1 hour. The COCs were then washed and cultured in air at 37°C and 6% CO2, grouped into 4-well IVF culture dishes (Nunc; Thermo Fisher Scientific; Denmark), with up to 10 COCs per well, each well containing 500 μl of "new capacitation medium" (i.e., the capacitation medium according to the invention) with 25 nM CNPs.

[0273] After 22-26 hours of capacitation culture, the COC was thoroughly washed and transferred to IVM medium containing 100 ng / ml human recombinant bimodal protein (rhAREG) and 100 mIU / ml recombinant FSH (Gonal-F), and incubated for 30 hours under the same culture conditions that allow in vitro meiotic maturation.

[0274] After 30 hours of IVM culture, oocytes were mechanically and enzymatically freed from the cumulus layer using hyaluronidase (Cook Medical) under a stereomicroscope, and oocyte maturation was assessed under an inverted microscope.

[0275] Mature oocytes (exposed to PB) involved in the study were microinjected with sperm from a common donor, and embryonic development was assessed from ICSI to day 5 (final day 6).

[0276] Fertilization and embryonic development were recorded at the standard assessment time point. Embryos with good morphology and considered transferable on day 3 [based on the number of blastomeres (at least 5 cells), fragmentation rate (maximum 20%), and no evidence of blastomere polynucleation and / or early compaction] were classified as “GQE” (Good Quality Embryos).

[0277] result: Figure 11 and 12The results of “newly capacitated culture + IVM” were compared with “conventional IVM” for sibling oocytes, based on European ICSI data (from normally stimulated cycles) and the routine application of IVM in UZBrussel (2014-2015).

[0278] The first dataset (conventional ICSI (Megaset)) published conventional ICSI results in 374 patients who underwent HP-hMG superovulation and collected oocytes from large follicles (Megaset® Study: (Source: Devroey et al. FertilSteril 2012 Mar; 97(3):561-71)

[0279] The second dataset (Conventional IVM (Origio)) is UZBrussel data from 413 patients who received conventional IVM treatment using Origio® IVM medium.

[0280] The final and third datasets (COC capacitation + IVM) are the results of “new capacitation culture + IVM” (i.e., the method of this patent application) compared with sibling oocytes treated with Origio® IVM medium.

[0281] The application of capacitation culture (i.e., the capacitation medium according to the present invention) to the culture of immature COCs prior to human IVM has shown significant benefits: higher nuclear maturation rate and higher yield of high-quality day 3 embryos and blastocysts.

[0282] Figure 11 Arrow (1) in the figure shows that the oocyte maturation rate is greatly improved compared with conventional IVM. Figure 11 Arrow (2) in the image shows the fertilization rate compared to a conventional ICSI cycle or conventional IVM. equal . Figure 11 Arrow (3) in the figure shows that, comparable to the rate of high-quality embryos obtained from mature oocytes in large follicle ICSI cycles, the yield of high-quality embryos on day 3 (day 3 GQE) per initial COC number is almost twice that of conventional IVM.

[0283] Figure 12 Arrow (4) indicates the yield of high-quality blastocysts on day 5 or 6 of culture, expressed as per number of fertilized eggs (2PN) or per MII oocyte. Results for “capacitation culture” are omitted. Higher than Conventional IVM. However, the fact that conventional IVM blastocysts can only grow further if the embryology is favorable on day 3 (4 or more high-quality embryos on day 3) positively influences these results. Therefore, when the same approach is applied to CNP (dotted white streaks), the results are far superior to any other group.

[0284] Figure 12 Arrow (5) in the diagram refers to the yield of high-quality blastocysts on day 5 or day 6 of culture, expressed as per initial COC number in the CNP group. Higher than The standard IVM (although this group had a positive selection bias) and the standard ICSI cycle were more comparable. Again, the strategy of applying ≥4 GQE on day 3 to the CNP group produced the best results across all groups.

[0285] in conclusion

[0286] As part of IVM treatment, the application of the "new capacitation culture" step enhances the maturation of follicles from small follicles to a level similar to that of oocytes grown in vivo (obtained from large follicles after stimulation). This enhanced maturation is durable during early embryogenesis, resulting in twice the number of high-quality embryos compared to conventional IVM. Achieving such durable results during early embryogenesis exceeds expectations and creates a more flexible IVM approach compared to currently used methods.

Claims

1. An in vitro method for capacitation and maturation of immature human cumulus-oocyte complexes, the method comprising: - Contact the immature human cumulus-oocyte complex with the collection medium for at least 30 minutes; - Subsequently, immature human cumulus-oocyte complexes were exposed to capacitation medium containing 10 to 50 nM C-type natriuretic peptide (CNP), estradiol and follicle-stimulating hormone (FSH) for a period of 22 to 26 hours to maintain meiotic arrest in immature human cumulus-oocyte complexes during the contact period. and - The immature human cumulus-oocyte complex was then contacted with a maturation culture medium containing EGF-like factors.

2. The in vitro method according to claim 1, wherein the capacitation medium does not contain a reagent that triggers meiotic restart.

3. The in vitro method according to claim 2, wherein the reagent that triggers meiosis restart is an EGF-like factor.

4. The in vitro method according to any one of claims 1 to 3, wherein the capacitation medium comprises 1 to 1000 nM estradiol; more particularly 10 nM estradiol.

5. The in vitro method according to any one of claims 1 to 4, wherein the capacitation medium comprises 1 to 10 mIU / ml FSH; more particularly 2.5 mIU / ml FSH or 1 mIU / ml FSH.

6. The in vitro method according to any one of claims 1 to 5, wherein the capacitation medium further comprises 0.1 to 10 ng / ml insulin; more particularly 5 ng / ml insulin.

7. The in vitro method according to any one of claims 1 to 6, wherein the capacitation medium comprises 1 mIU / ml FSH, 5 ng / ml insulin, 10 nM estradiol and 25 nM CNP.

8. The in vitro method according to any one of claims 1 to 7, wherein the EGF-like factor is selected from bimodal proteins, epithelial regulatory proteins, FSH, LH, cAMP regulators or combinations thereof; particularly bimodal proteins.

9. The in vitro method according to any one of claims 1 to 8, wherein the immature human cumulus-oocyte complex is contacted with capacitated medium in non-adherent or adherent culture plates, particularly in non-adherent culture plates.

10. The in vitro method according to any one of claims 1 to 9, wherein the immature human cumulus-oocyte complex is derived from a person with polycystic ovary syndrome (PCOS) or with high ovarian follicular reserve.

11. A kit for in vitro capacitation and maturation of immature human cumulus-oocyte complexes, the kit comprising: (a) Collecting the culture medium; (b) Capacitating medium containing 10 to 50 nM C-type natriuretic peptide (CNP), estradiol and follicle-stimulating hormone (FSH). (c) Maturation culture medium containing EGF-like factors; (d) Non-adherent or adherent culture plates; and (e) Instructions for use of the kit.

12. The kit of claim 11, wherein the instructions for use specify a time period of 22 to 26 hours for contacting the immature human cumulus-oocyte complex with the capacitation medium.

13. The kit according to any one of claims 11 or 12, wherein the capacitating medium comprises: 10 to 50 nM C-type natriuretic peptide (CNP); 1 to 1000 nM estradiol; 1 to 10 mIU / ml FSH; 0.1 to 10 ng / ml insulin.

14. The kit according to any one of claims 11 or 12, wherein the capacitation medium comprises 1 mIU / ml FSH, 5 ng / ml insulin, 10 nM estradiol and 25 nM CNP.

15. The kit according to any one of claims 11 to 14, wherein the EGF-like factor is selected from bimodal proteins, epithelial regulatory proteins, FSH, LH, cAMP regulators or combinations thereof; particularly bimodal proteins.

16. The kit according to any one of claims 11 to 15, wherein the maturation medium comprises 100 ng / ml bimodal protein and 100 mIU / ml FSH.

17. A capacitation medium for in vitro capacitation culture of immature human cumulus-oocyte complexes, the capacitation medium comprising: 10 to 50 nM C-type natriuretic peptide (CNP); 1 to 1000 nM estradiol; 1 to 10 mIU / ml FSH; 0.1 to 10 ng / ml insulin.

18. The capacitation medium according to claim 17, comprising 1 mIU / ml FSH, 5 ng / ml insulin, 10 nM estradiol and 25 nM CNP.

19. A maturation medium for in vitro maturation of immature human cumulus-oocyte complexes, wherein the medium comprises 100 ng / ml bimodalin and 100 mIU / ml FSH.

Images