Methods of modulating oocyte maturation

Abstract

A method of maturing an immature oocyte is disclosed. The method comprises contacting the immature oocyte with an agent which increases an amount and / or activity of a down- stream effector of a RANK receptor on the oocyte. Agents used for same are also disclosed.

Description

[0001] METHODS OF MODULATING OOCYTE MATURATION

[0002] RELATED APPLICATION / S

[0003] This application claims the benefit of priority of Israeli Application No. 301957, filed 4

[0004] April 2023, the contents of which are incorporated herein by reference in their entirety.

[0005] SEQUENCE LISTING STATEMENT

[0006] The XML, entitled 99783. xml, created on 4-Apr-2024 , comprising 16,574 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

[0007] FIELD AND BACKGROUND OF THE INVENTION

[0008] ’The present invention relates to the field of assisted reproductive technology (ART), particularly to methods for maturing immature human oocytes so that they are capable of being fertilized and forming a viable embryo.

[0009] Since the first successful human pregnancy by way of in vitro fertilization (IVF) was achieved in 1978, assisted reproductive therapy (ART) has helped thousands of thousands of women to overcome infertility problem.

[0010] Normally, during the follicular phase of a woman's reproductive cycle, only a single follicle grows to the preovulatory stage and releases its oocyte for potential fertilization. However, current IVF treatment requires that multiple oocytes be harvested. Therefore, women are normally pretreated for approximately two or three weeks with gonadotropin releasing hormone agonists (GnRHa). After pituitary suppression has been achieved, human menopausal gonadotropin (HMG) or purified follicle-stimulating hormone (FSH) is administered to induce de velopment of multiple follicles. Once two leading follicles have reached a diameter of at least about 18-20 mm, the patient is typically given 5,000 to 10,000 IU of human chorionic gonadotropin (HCG) to trigger final oocyte maturation. Approximately 36 hours later, a large number of oocytes (10 on average) are collected.

[0011] The harvested oocyte is then fertilized with the sperm. In many instances of human infertility, fertilization is affected by intracytoplasmic sperm injection (ICS I). Successful IVF generally requires that the oocyte have reached the rnetaphase-II (M-H) stage before fertilization is attempted. A M-II oocyte is ready to accept the sperm and be fertilized. Metaphase- II is characterized by exclusion of one polar body from the cytoplasm, and is typically detected byvisual identification under a microscope.

[0012] The harvested oocyte remains surrounded by cumulus cells that support and promote the final maturation and development of the oocyte. The cumulus cells interfere with ICS I and obscure microscopic observation of the oocyte. Therefore, prior to ICSI, it is necessary to denude the oocyte of cumulus cells to permit determination of the level of oocyte maturity. This is generally accomplished by enzymatic and mechanical stripping of the cumulus cells.

[0013] Unfortunately, not all of the oocytes have matured to the M-II stage when they are harvested in ovarian stimulation. As a result of follicular asynchrony, in general, at least 10-15% of oocytes are still immature, and are at the germinal vesicle (GV) or metaphase-I (M-I) stage, at the time of harvest. These immature oocytes, denuded of cumulus cells, are discarded at most 1VF clinics.

[0014] It would be desirable if these immature GV and M-I stage oocytes, presently wasted, could be brought to maturity, in vitro, and then successfully fertilized. Moreover, it would be desirable to be able to harvest immature oocytes from unstimulated ovaries and mature them to the M-II stage by way of in vitro maturation (IVM), thereby avoiding the entire regimen of treatment of women with different protocols including GnRHag or GnRHant and stimulation of ovaries with gonadotropin. These procedures of ovarian stimulation require frequent blood sampling and ultrasound monitoring, at considerable cost. Moreover, current IVF treatment causes substantial discomfort and, in some cases, results in ovarian hyperstimulation syndrome (OHSS) and / or even death. There is also anxiety that the long-term effects of repeated ovarian stimulation may increase the risk of ovarian, endometrial and breast cancer.

[0015] The discovery of RANK, RANKL, and the soluble decoy osteoprotegerin (OPG) in the late 1990s contributed to understanding the biology of bone homeostasis and identified it as a regulator in the adaptive immune system. Subsequently, it has been demonstrated that the RANK- RANKL-OPG system plays a key role in varied tissues and processes, e.g., organogenesis, mammary gland, brain, and cancer. Moreover, RANKL and RANK plays a role in the oncogenic process, and it has been shown that RANKL is involved in each stage of tumor development.

[0016] RANK, a type I homotrimeric transmembrane glycoprotein, is a member of the TNFR superfamily. Human RANK protein is a 616 amino acid peptide, with an N-terminal extracellular domain, a transmembrane domain with four cysteine-rich pseudo repeats, and two N glycosylation sites, a large C- terminal cytoplasmic domain added to a peptide that is cleaved. A mature RANK containing 588 amino acids is expressed. Human RANK shares 66% sequence homology with mouse RANK. Tnfrsfl la, the gene encoding the RANK protein, is located on chromosome 18q22.1.

[0017] RANKL is a member of the TNF superfamily and has three isoforms: RANKL 1, 2, and 3. Isoforms 1 and 2 are 317 and 270 type II homotrimeric transmembrane glycoprotein, consisting of an extracellular domain with two potential N- glycosylation sites, a transmembrane domain, and an intracellular domain. Isoform 3 is a soluble ligand, with 243 amino acids and lacks transmembrane and cytoplasmic domain. Human RANKL shares 85% sequence homology with murine RANKL.

[0018] The glycoprotein OPG is a decoy receptor for RANKL. It is highly expressed in a soluble form, lacks transmembrane and cytoplasmic domain, and exports to the extracellular space. OPG is a member of the TNFS superfamily, binds RANKL with higher affinity than RANK, and consequently prevents RANK-RANKL binding.

[0019] Inhibition of RANK-RANKL interaction is used as a treatment for osteoporosis by suppression of bone resorption. Denosumab is neutralizing antibody against the extracellular domain of RANKL, proved by the FDA and used as a treatment for osteoporosis (Honma et al., Pharmacol. Ther., vol. 218, p. 107682, 2021, doi: 10.1016 / j.pharmthera.2020.107682), and is during clinical trials as a treatment for other diseases such as cancer (Infante et al., J. Exp. Clin. Cancer Res., vol. 38, no. l, pp. 1-18, 2019, doi: 10.1186 / sl3046-018-1001-2).

[0020] Additional background art includes Yerushalmi et al., Molecular Human Reproduction, Vol.20, No.8 pp. 719-735, 2014.

[0021] SUMMARY OF THE INVENTION

[0022] According to an aspect of some embodiments of the present invention there is provided a method of maturing an immature oocyte comprising contacting the immature oocyte with an agent which increases an amount and or activity of a down-stream effector of a RANK receptor on the oocyte, thereby maturing the immature oocyte.

[0023] According to an aspect of some embodiments of the present invention there is provided an agent which increases an amount and or activity of a down-stream effector of a RANK receptor on an oocyte for use in assisted fertilization.

[0024] According to some embodiments of the invention, the agent binds to the RANK receptor.

[0025] According to some embodiments of the invention, the agent is an activating antibody.

[0026] According to some embodiments of the invention, the down-stream effector comprises tumor necrosis factor receptor-associated factor 6.

[0027] According to some embodiments of the invention, the agent increases an amount of at least one transcription factor selected from the group consisting of NFATcl, CREB, NFKB, AP-1, c-fos, and MITF.

[0028] According to some embodiments of the invention, the agent is a peptide agent. According to some embodiments of the invention, the peptide has an amino acid sequence at least 99 % identical to SEQ ID NOs: 17 or 18.

[0029] According to some embodiments of the invention, the immature oocyte is comprised in a cumulus-oocyte complex.

[0030] According to some embodiments of the invention, the contacting comprises culturing the immature oocyte in a medium comprising the agent.

[0031] According to some embodiments of the invention, the oocyte is a human oocyte.

[0032] According to some embodiments of the invention, the oocyte is retrieved from a female subject having a polycystic ovarian syndrome.

[0033] According to some embodiments of the invention, the method is an in vitro method.

[0034] According to some embodiments of the invention, the method is an in vivo method.

[0035] According to an aspect of some embodiments of the present invention there is provided a method of preparing oocytes from a human subject for in-vitro fertilization comprising:

[0036] (a) retrieving an immature oocyte from the human subject; and

[0037] (b) maturing the immature oocyte according to the method of claim 1 , thereby preparing oocytes from the human subject for in-vitro fertilization.

[0038] According to an aspect of some embodiments of the present invention there is provided an oocyte culture medium comprising an agent which increases an amount and or activity of a downstream effector of a RANK receptor on the oocyte.

[0039] According to some embodiments of the invention, the agent binds to the RANK receptor.

[0040] According to some embodiments of the invention, the down-stream effector comprises tumor necrosis factor receptor-associated factor 6.

[0041] According to some embodiments of the invention, the agent increases an amount of at least one transcription factor selected from the group consisting of NFATcl, CREB, NFKB, AP-1, c-fos, and MITF.

[0042] According to some embodiments of the invention, the agent is a peptide agent.

[0043] According to some embodiments of the invention, the peptide has an amino acid sequence at least 90 % identical to SEQ ID NO: 17 or 18.

[0044] According to some embodiments of the invention, the culture medium further comprises at least one immature oocyte.

[0045] According to an aspect of some embodiments of the present invention there is provided a non-hormonal contraceptive composition comprising an agent that reduces the amount of a downstream effector from a RANK receptor of an oocyte and a pharmaceutically acceptable carrier, with the proviso that when the agent is osteoprotegerin, the osteoprotegerin is present in an amount which reduces the risk of pregnancy and / or the composition is formulated for local delivery.

[0046] According to some embodiments of the invention, the contraceptive is formulated for oral delivery.

[0047] According to some embodiments of the invention, the local delivery comprises intra-uterine delivery.

[0048] According to some embodiments of the invention, the agent binds to and sequesters RANK.

[0049] According to some embodiments of the invention, the agent is a neutralizing antibody which binds to RANKL or the RANK receptor.

[0050] According to some embodiments of the invention, the agent binds to the extracellular domain of the RANKL.

[0051] According to some embodiments of the invention, the neutralizing antibody is Denosumab.

[0052] According to an aspect of some embodiments of the present invention there is provided an article of manufacture having a plurality of separate dosage units of an agent that reduces the amount of a down-stream effector from a RANK receptor of an oocyte, each of the dosage units, formulated for oral administration.

[0053] According to some embodiments of the invention, the plurality of separate dosage units comprises at least 20.

[0054] According to some embodiments of the invention, the agent binds to and sequesters RANK.

[0055] According to some embodiments of the invention, the agent is a neutralizing antibody which binds to RANKL or the RANK receptor.

[0056] According to some embodiments of the invention, the agent binds to the extracellular domain of the RANKL.

[0057] According to some embodiments of the invention, the neutralizing antibody is Denosumab.

[0058] According to an aspect of some embodiments of the present invention there is provided a method of reducing the risk of pregnancy in a female subject comprising administering to the subject a preventatively effective amount of an agent that reduces the amount of a down-stream effector from a RANK receptor of an oocyte, thereby reducing the risk of pregnancy in the subject.

[0059] According to some embodiments of the invention, the agent binds to and sequesters RANK.

[0060] According to some embodiments of the invention, the agent is osteoprotegerin.

[0061] According to some embodiments of the invention, the agent is a neutralizing antibody which binds to RANKL or the RANK receptor.

[0062] According to some embodiments of the invention, the agent binds to the extracellular domain of the RANKL. According to some embodiments of the invention, the neutralizing antibody is Denosumab.

[0063] According to some embodiments of the invention, the administering comprises daily administering.

[0064] According to some embodiments of the invention, a dosage of the agent alternates according to the menstrual cycle of the subject.

[0065] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0066] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0067] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0068] In the drawings:

[0069] FIG. 1. RANK expression in mouse oocytes. mRNA (15 oocytes at each experimental sample) was extracted from GV and Mil oocytes and subjected to qPCR analysis for detection of RANK mRNA expression. Bars are mean ± SEM of three independent experiments.

[0070] FIGs. 2A-B. RANKL and OPG expression in MGCs and CCs. MGCs and CCs were obtained during IVF. RANKL (A), OPG (B), and RANK were quantified by qPCR and normalized to b-actin expression. Results are means ± SEM of five independent experiments. Each sample contains mix of cells from 1-5 patients. RANK was not detected.

[0071] FIG. 2C. RANK expression in human oocyte but not in granulosa cells. MGCs, CCs, and GV oocytes were obtained during IVF. RANK was quantified by special qPCR kit and normalized to b-actin expression.

[0072] FIGs. 3A-B. RANKL and OPG expression in CCs according to the cumulus-oocyte complex (COC) maturation stage. CCs were obtained according to the maturational stage of the denuded oocyte (GV or Mil). Each sample contains CCs from 3-5 COCs. RANKL (A), OPG (B), and RANK were quantified by qPCR and normalized to b-actin expression. Results are means ± SEM of five independent experiments. RANK was not detected.

[0073] FIGs. 4A-B. RANKL and OPG expression during the ovulatory interval of cultured MGCs. MGCs were seeded in culture. Cells were treated with hCG and harvested at the indicated time points: Oh, 3h, 6h, 9h, 12h, 24h, and 36h. RANKL (A) and OPG (B) were quantified by qPCR and normalized to b-actin expression. The results are expressed as fold change for control that was set to 1. Results are means ± SEM of four independent experiments. RANK was not detected.

[0074] FIG. 5. hCG induces RANKL protein expression in MGCs. MGCs were seeded in culture for 4 days with a daily medium exchange and then treated with lU / ml hCG. Cells were harvested after 16h and protein levels of RANKL expression were determined by western blotting. Tubulin was used as control (n=2).

[0075] FIGs. 6A-B. The effect of PGE2 on RANKL and OPG expression in MGCs. MGCs were seeded in culture for 4 days with a daily medium exchange and then treated with hCG or PGE2 at different concentrations (Ing / ml, lOng / ml, lOOng / ml) or both. Cells were harvested after 8h with hCG / PGE2. RANKL (A) and OPG (B) were quantified by qPCR and normalized to b-actin expression. Results are means ± SEM of three independent experiments. RANK was not detected.

[0076] FIG. 7 Effect of PGE2 inhibition on RANKL expression in MGCs. MGCs were seeded in culture for 4 days with a daily medium exchange and then treated with hCG (lU / ml) or PGE2 (Ing / ml) with or without indomethacin (250pM). Cells were harvested after 8h and RANKL was quantified by qPCR and normalized to b-actin expression. Results are means ± SEM of four independent experiments.

[0077] FIGs. 8A-B. COCs were harvest from mice ovaries after PMSG treatment. Two groups of COCs at time 0 were present, complete compact (left) and partial compact (right). The COCs were divided randomly to IVM medium or IVM with RANKL peptide (100 ng / ml). After 16h each oocyte was examined for the presences of polar body (a sign of maturity).

[0078] Figs 9A-B. Effect of OPG on ovulation and oocytes maturation. Super ovulated mice were injected with hCG (n=18), hCG+OPG (n=5) for RANKL inhibition, and hCG+OPG+RANKL (n=3) for rescue. Ovulation (A) was assessed by No. of oocytes per mouse, and maturation rate (B) was assessed by polar body identification. Bars are means ± SEM of independent experiments as indicated.

[0079] FIG. 10. Effect of RANKL peptide on human oocyte maturation. GV oocytes were incubated in one step medium (Sage) with or without RANKL peptide (100 ng / ml) for 16h. Polar- body formation was the sign of oocyte maturation. DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0080] The present invention relates to the field of assisted reproductive technology (ART), particularly to methods for maturing immature human oocytes so that they are capable of being fertilized and forming a viable embryo.

[0081] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0082] According to an aspect of the invention there is provided a method of preparing oocytes from a human subject for in-vitro fertilization comprising:

[0083] (a) retrieving an immature oocyte from the human subject; and

[0084] (b) maturing the immature oocyte according by contacting the immature oocyte with an agent which increases an amount and / or activity of a down- stream effector of a RANK receptor on the oocyte, thereby preparing oocytes from the human subject for in-vitro fertilization.

[0085] As used herein, die term “'immature human oocyte” refers to a human GV stage oocyte.

[0086] The oocytes may be retrieved form any female human subject who possesses viable oocytes and is a candidate for in vitro maturation (IVM) therapy. In one embodiment, the subject suffers from some form of infertility. For instance, the subject may experience normal oocyte production but have an impediment to fertilization, as in, e.g. polycystic ovarian syndrome (PCOS) or PCOS -like ovaries. IVM is also useful in women infertility with endometriosis, blockage of either or both fallopian tubes, etc. In typical PCOS, numerous follicles mature simultaneously in the ovaries, without the appearance of a dominant follicle. The subject’s menstrual cycle may be regular, irregular or non-existent. IVM may be especially useful in women who are susceptible to or suffer from ovarian hyperstimulation syndrome (OHSS) and are therefore not suitable candidates for traditional in vitro fertilization techniques involving an ovarian stimulation protocol. Alternatively, the subject may be an individual who does not suffer from infertility or have any reproductive impediment whatsoever, but for whom an in vitro fertilization method nevertheless remains desirable, as in, e.g. cases of male factor infertility.

[0087] Unlike in conventional in vitro fertilization methods, IVM in accordance with the invention does not necessarily involve an ovarian stimulation protocol. One of two “ovarian stimulation protocols” is usually used in conventional IVF, a “long protocol” or a “short protocol.” The long protocol has two steps. The first step involves pre-treating the subject for two or three weeks with GnRHa to down-regulate pituitary activity. Once pituitary suppression has been achieved, in the second step, HMG or FSH is administered to induce the development of multiple follicles. The short protocol involves only the ovarian stimulation step, but not the down-regulation step.

[0088] Ovarian stimulation is required in conventional IVF techniques to permit the oocytes to mature to the M-II stage prior to harvest. In the present invention, wherein oocytes are harvested at an immature stage, i.e. prior to reaching the M-II stage, ovarian stimulation need not be used. That is, the subject need not be pre-treated with GnRHa’s, HMG and / or FSH.

[0089] Prior to retrieving immature oocytes, an increase in endogenous levels of LH may be induced in the subject (also described herein as “priming”). This may be accomplished by e.g. administering to the subject an effective amount of human chorionic gonadotropin (HCG) (Profasi Serono, Oakville, Ontario, Canada) or LH, either of which stimulate endogenous LH production. Baseline concentration of LH in the plasma of premenopausal women is usually about 2-4 mIU / ml, and at midcycle peak may be up to 25-35 mIU / ml. The expression “inducing in a female human subject an increase in endogenous luteinizing hormone levels” means increasing the plasma LH concentration above the baseline concentration. Preferably plasma LH concentration is increased at least 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or 2000% above baseline concentra lion .

[0090] The dosage and mode of administration of HCG or LH may vary depending on the patient and the circumstances and may be determined by those of skill in the art. Subcutaneous injection is a preferred mode of administration although other modes of administration may be used. A single injection of about 5000 to about 20,000 IU of HCG is generally sufficient. In one embodiment, a single dose of about 10, 000 IU of HCG is carried out.

[0091] In the case of a subject having a regular menstrual cycle, the time for inducement may be determined relative to the commencement of menstrual bleeding, which is considered “day 0”. In a subject having an irregular or absent menstrual cycle, commencement of menstrual bleeding (i.e, the ini tiation of “day 0”) can be achieved by procedures known in the art, e.g. by the administration of progesterone (available from e.g. Prometrium; Schering, Pointe-Claire, Quebec, Canada), e.g. intravaginally, in an amount of approximately 300 mg / day for 10 days.

[0092] Typically, on day three and day eight, the ovaries are examined by ultrasound to assess the size and the number of follicles. Usually, the dominant follicle reaches a diameter of at least about 16 mm by day 10-14 priming.

[0093] In instances where a dominant follicle is present (e.g. as in normal ovarian function, as in the case of a healthy subject or a subject suffering from e.g. endometriosis or tubal blockage) the increase in endogenous LH activity is induced (e.g. by administration of HCG) when the dominant follicle reaches a diameter of about 15- 17 mm, preferably at least about 16 mm. If a dominant follicle is not present, as in cases of e.g. PCOS, inducement of an increase in endogenous LH levels preferably occurs when the follicles are less than about 12 mm in diameter.

[0094] Oocytes are generally retrieved from about 32 to about 40 hours after priming, more preferably about 34 to 38 hours after priming and even more preferably about 36 hours after priming.

[0095] Means for retrieving oocytes are known in the art. In one embodiment, trans vaginal ultrasonographically-guided oocyte collection is done using a 17-20 gauge, preferably 19 gauge single-lumen aspiration needle at an aspiration pressure of from about 5 to about 10 kPa, preferably about 7.5 kPa.

[0096] As used herein, the term “immature human oocyte” refers to a human oocyte that has not yet reached metaphase- II (M-II). As discussed previously, metaphase-II is characterized by exclusion of one polar body from the cytoplasm. Immature human oocytes used in the invention are typically at the germinal vesicle (GV) stage.

[0097] Oocytes may be cultured with their cumulus intact, in a form known as a “cumulus-oocyte- complex” (COC), or oocytes may be partially or entirely denuded from cumulus cells.

[0098] COC can be stripped with hyaluronidase in an appropriate medium (e.g. HEPES buffered medium) and mechanically pipetted until oocytes are denuded. An oocyte that is “essentially free of cumulus cells” is an oocyte that is associated with sufficiently few cumulus cells that the cumulus cells have no detectable physiological effect on the oocyte.

[0099] As mentioned, the oocytes are contacted with an agent which increases an amount and / or activity of a down-stream effector of the receptor activator of NF-KB (RANK) receptor on the oocyte.

[0100] The term “RAN K” refers to the receptor on the oocyte having the identifier TNFRSF11 A.

[0101] In one embodiment, the agent is an agonist of the receptor, thereby increasing a downstream effector thereof (e.g. tumor necrosis factor receptor-associated factor 6 (TRAF6). In one embodiment, the amount of TRAF6 increases by at least 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold or even at least 5 fold (e.g. as measured by Western blot analysis or RT-PCR analysis), as compared in the absence of the agent.

[0102] In another embodiment, the agent increases an amount of at least one, at least two, at least three, at least four or each of the transcription factors selected from the group consisting of NFATcl, CREB, NFKB, AP-1, c-fos, and MITF (e.g. as measured by Western blot analysis or RT- PCR analysis).

[0103] Agents capable of upregulating the activity of RANK include activating antibodies, (e.g. activating antibody). The term "antibody" as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, Fv or single domain molecules such as VH and VL to an epitope of an antigen. These functional antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; (5) Single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule; and (6) Single domain antibodies are composed of a single VH or VL domains which exhibit sufficient affinity to the antigen.

[0104] In a particular embodiment, the antibody is a monoclonal antibody.

[0105] Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference and the Examples section which follows).

[0106] Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

[0107] Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nafl Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio / Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.

[0108] Another form of an antibody fragment is a peptide coding for a single complementaritydetermining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].

[0109] Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323- 329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].

[0110] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0111] Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boemer et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boemer et al., J. Immunol., 147(l):86-95 (1991)]. Similarly, human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio / Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856- 859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845- 51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).

[0112] One exemplary polypeptide activator is the protein RANKL.

[0113] As used herein the term “RANKL” refers to the peptide which comprises an amino acid sequence at least 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % identical or similar to the amino acid sequence as set forth in SEQ ID NO: 17 or SEQ ID NO: 18. Human sRANK Ligand (CHO derived), Accession Number: 014788 is available for example at Peprotech (Cat. # 310-01C). It is a 22.1 kDa polypeptide comprised of the TNF- homologous region of R.ANKL and a N-terminal His-tag.

[0114] Murine sRANK Ligand (CHO derived). Accession Number: 035235 is available for example at Peprotech (Cat. # 315-11C). It is a 19.8 kDa polypeptide comprising the TNF- homologous region of RANKL (178 amino acid residues).

[0115] The polypeptide agents described herein may be expressed (using an expression construct), directly in the subject (i.e. in vivo gene therapy) or in a cell system and then administered to the subject.

[0116] Other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the polypeptide), the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed peptide.

[0117] Various methods can be used to introduce the expression vector of the present invention into the host cell system. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0118] Transformed cells are cultured under effective conditions, which allow for the expression of high amounts of recombinant polypeptide. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. An effective medium refers to any medium in which a cell is cultured to produce the recombinant polypeptide of the present invention. Such a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art. Depending on the vector and host system used for production, resultant polypeptides of the present invention may either remain within the recombinant cell, secreted into the fermentation medium, secreted into a space between two cellular membranes, such as the periplasmic space in E. coll: or retained on the outer surface of a cell or viral membrane.

[0119] Following a predetermined time in culture, recovery of the recombinant polypeptide is affected.

[0120] The phrase "recovering the recombinant polypeptide" used herein refers to collecting the whole fermentation medium containing the polypeptide and need not imply additional steps of separation or purification.

[0121] Recovering is also covered by the term “isolating” or “purifying”, which can also be from the host cells.

[0122] Thus, polypeptide RAN K activators can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.

[0123] To facilitate recovery, the expressed coding sequence can be engineered to encode the RANK activator and fused cleavable moiety. Such a fusion protein can be designed so that the polypeptide can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the cleavable moiety. Where a cleavage site is engineered between the polypeptide and the cleavable moiety, the polypeptide can be released from the chromatographic column by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site [e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988); and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)].

[0124] Exemplary purification tags for purposes of the invention include but are not limited to polyhistidine, V5, myc, protein A, gluthatione-S-fransferase, maltose binding protein (MBP) and cellulose-binding domain (CBD) [Sassenfeld, 1990, TIBTECH, 8, 88-9].

[0125] The polypeptides of the present invention are preferably retrieved in "substantially pure" form.

[0126] As used herein, the phrase "substantially pure" refers to a purity that allows for the effective use of the protein in the applications described herein.

[0127] As used herein, “percent homology”, “percent identity”, "sequence identity" or "identity" or grammatical equivalents as used herein in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are considered to have "sequence similarity" or "similarity". Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Henikoff S and Henikoff JG. [Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. U.S.A. 1992, 89(22): 10915- 9].

[0128] Percent identity can be determined using any homology comparison software, including for example, the BLASTN software of the National Center of Biotechnology Information (NCBI) such as by using default parameters.

[0129] In one embodiment, the agent which increases the amount and / or activity of a down-stream effector of the RANK receptor is not a growth factor (e.g. does not belong to the epidermal growth factor (EGF) family; platelet derived growth factor (PDGF) family; insulin-like growth factor (IGF) family; nerve growth factor (NGF) family; transforming growth factor (TGF) family; fibroblast growth factor (FGF) family; hepatocyte growth factor (HGF) family). In another embodiment, the agent which increases the amount and / or activity of a down- stream effector of the RANK receptor is not a hemapoietic growth factor or a cytokine.

[0130] Suitable culture conditions include e.g. culturing the oocytes at 37° C. in an atmosphere of 95% air and 5% CO? at high humidity, e.g. 100% humidity. A “triple gas” atmosphere of 5% O?, 5% CCh and 90% Njmay also be used. Mineral oil may be overlaid on the medium to control evaporation and / or temperature. Oocytes are typically cultured in a well containing 1 ml of culture medium or more, or may be cultured in 10 ul of culture medium or less in a droplet in a culture dish.

[0131] In one embodiment, the oocytes are not denuded - i.e. for the entire culturing phase, the oocytes are comprised in a cumulus-oocyte complex.

[0132] In another embodiment, the oocytes are denuded (e.g. at about 24 hours after initial culturing). The base medium in which the cells are cultured comprises inorganic salts, essential and non-essential amino acids, and energy source. Suitable salts, amino acids and energy sources are known in the art.

[0133] Inorganic salts may be provided to buffer the pH of the medium within a range preferably of about 7.2-7.4 and to maintain correct osmolarity of the medium with the oocytes. Suitable inorganic salts and concentrations thereof as used in culture media are known in die art. Typical inorganic salts include CaCl2, KC1, MgSO4, NaCl, NaHCOs, NaH2PO4.H2O, FE(NO3)3.9H2O, KH2PO4, Na acetate, Na2H2PO4, etc.

[0134] The IVM medium may contain at least one amino acid or source thereof. Preferably, at least the essential amino acids, or sources thereof, are included in IVM medium. “Essential” amino acids are those amino acids not synthesized in the oocyte and that are essential for protein synthesis. The essential amino acids are generally considered to be isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. A commercial amino acid mixture, containing the eight essential amino acids as well as some or all of the remaining non-essential amino acids may conveniently be used. Non-naturally occurring amino acids or amino acid derivatives as are known in the art may also be included in the IVM medium. In a particularly preferred embodiment, the IVM medium of the invention comprises alanine, arginine, asparagine, aspartic acid, cystine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0135] The IVM medium may further contain vitamins as are known in the art for inclusion in culture media. Vitamins that may be included in the media include, without limitation, vitamins Al (retinol), A2 (an alternative form of retinol), Bl (thiamine), B2 (riboflavin), B6 (pyridoxine), B9 (folic acid), B12 (cyanocobalamin), B17, C (ascorbic acid), D, D2 (calciferol), D3 (cholecalciferol), E (tocopherol), H (biotin), K, KI (phylloquinone), K2, K3 (menadione), P. etc. A particularly preferred combination of vitamins comprises biotin, D-Ca pantothenate, choline chloride, folic acid, i-inositoL nicotinamide, pyroxidal-HCl, riboflavin, and thi amine- HC1.

[0136] The IVM medium may also contain at. least one hormone. Preferred hormones include insulin, estradiol, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Human menopausal gonadotropin (HMG) can generally be substituted for FSH, and human chorionic gonadotropin (HCG) can generally be substituted for LH. In a preferred embodiment, the IVM medium comprises insulin. Normally, insulin is involved in energy metabolism and amino acid transportation into cells. The IVM medium preferably contains from 0.5 mg / L to 50 mg / L insulin, more preferably 0.25 mg / L to 10 mg / L insulin, and even more preferably about 5 mg / L insulin. In a particularly preferred embodiment, the IVM medium further comprises estradiol, LH and FSH. The amount of estradiol is preferably about 1 pg / ml±10%,±20%, ±30%, ±40% or ±50%. The amount of each of FSH and LH is preferably about 0.08 IU / ml±10%, ±20%. ±30%, ±40% or ±50%.

[0137] In one particular embodiment, the IVM medium also contains human transferrin (TF). TF is a 75 kDa glycoprotein containing 679 amino acids and two glycan chains. For each, the last residue is a sialic (N-acetyl neuraminic) acid, which can be hydrolyzed by neuraminidase. TF not only transports iron in all extracellular fluid but also exerts many additional properties, including ceil growth stimulation (Gross- Weege et al., 1986). The IVM medium preferably contains from 5 mg / L to 500 mg / L TF, more preferably from 25 mg / L to 100 mg / L TF, and even more preferably about 50 mg / L TF.

[0138] Exemplary energy sources which may be added to the medium include, but are not limited to glucose, sodium pyruvate, lactate, or a mixture of some or all of these energy sources.

[0139] The IVM medium may additional contain additional components such as selenite or selenium and hydrocortisone. Antibiotics such as penicillin G and streptomycin may be added to the IVM medium to prevent contamination. Synthetic Serum Supplement (SSS) may be included in the IVM medium as a source of protein for the oocyte and to prevent cells from adhering to glassware during in vitro culture.

[0140] An exemplary medium which can be used for culturing the oocytes is TCM-199.

[0141] Techniques for fertilizing oocytes and culturing embryos are known in the art. In one embodiment, fertilization is accomplished by intracytoplasmic sperm injection (ICS I). Generally, spermatozoa can be prepared by gradient separation by centrifugation or by “swim-up” followed by washing with a suitable medium such as HTF supplemented with 10% SSS. A single spermatozoon may be injected into an M-H oocyte. Following ICS I, the oocyte may be transferred to e.g. fertilization medium supplemented with 10% SSS in a tissue culture dish. Fertilization can be detected by the appearance of two distinct pronuclei and two polar bodies approximately 16-18 hours after ICSI.

[0142] In one embodiment, fertilized oocytes may be cultured in fertilization media until about 72 hours after ICSI and then transferred to e.g. embryo developmental medium (Vitrolife, Goteborg, Sweden) in a tissue culture dish under mineral oil for an additional 48 hours.

[0143] Also contemplated is in vivo treatment, whereby the agent which activates RANK can be provided to the subject per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

[0144] The agent may be administered as a peptide or as a nucleic acid agent which encodes the peptide / protein (as further described herein above). In another aspect, the present inventors contemplate agents that reduce the amount of a down- stream effector from a RANK receptor of an oocyte as contraceptive agents.

[0145] Such agents include inhibitory antibodies and agents that sequester RANKL (e.g. osteoprotegerin).

[0146] Thus, according to another aspect of the present invention there is provided a non-hormonal contraceptive composition comprising an agent that reduces the amount of a down-stream effector from a RANK receptor of an oocyte and a pharmaceutically acceptable carrier, with the proviso that when the agent is osteoprotegerin, said osteoprotegerin is present in an amount which reduces the risk of pregnancy and / or the composition is formulated for local delivery.

[0147] Exemplary down-stream effectors include for example TRAF6, NFATcl, CREB, NFKB, AP-1, c-fos, and MITF, as further described herein above. In one embodiment, the agent reduces the down-stream effector by at least 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or even 100 %, as compared in the absence of the agent (e.g. as measured by Western blot analysis or RT-PCR analysis).

[0148] In some embodiments, the agent is a RANK or RANKL antagonist.. The RANK antagonist can be any chemical species having the activity of R ANK / RANKL antagonists as commonly understood in the ail, or can be an agent that partially or fully blocks, inhibits, or neutralizes one or more biological activities of RANKL or RANK, such as binding of RANKL to RANK, in vitro, in situ, or in vivo. .An antagonist may function in a direct or indirect manner. For instance, the antagonist may directly bind to RANKL or RANK, thus partially or fully blocking, inhibiting or neutralizing one or more biological activities of RAN KL or RANK, in vitro, in situ, or in vivo. The antagonist may also function indirectly to partially or fully block, inhibit or neutralize one or more biological activities of RANKL or RANK, in vitro, in situ, or in vivo as a result of interacting with, e.g., activating, inducing, blocking or inhibiting, another compound that can bind to RANK or RANKL. The antagonist may also function indirectly to partially or fully block, inhibit or neutralize one or more biological activities of RANKL or RANK, in vitro, in situ, or in vivo as a result of modulating or affecting the production of RANKL or RANK.

[0149] In some embodiments, the RANK antagonist is a soluble RANK protein that is capable of binding RANKL that can comprise all or a fragment of the extracellular domain of a RANK protein.

[0150] In some embodiments, the RANK / RANKL antagonist can be OPG (osteoprotegerin) a variant thereof or an anti-RANKL antibody. In some embodiments, the RANK / RANKL antagonist can be a monoclonal anti-RANKL antibody (e.g. Denosumab). In some embodiments, the RANK / RANKL antagonist can be a small interfering RNA, a microRNA, a precursor molecule, a ribozyme, an antisense nucleic acid sequence, or an aptamer targeting RANKL. In some embodiments, the RANK / RANKL antagonist can be a humanized monoclonal anti-RANKL antibody.

[0151] Further examples of RANK or RANKL antagonists are provided in US 9,493,571, the contents of which are incorporated herein by reference.

[0152] The agents may be administered per se or as part of a pharmaceutical composition.

[0153] As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0154] Herein the term "active ingredient" refers to the agent that reduces the amount of a downstream effector from a RANK receptor of an oocyte accountable for the biological effect.

[0155] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

[0156] Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0157] Techniques for formulation and administration of drugs may be found in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

[0158] Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.

[0159] Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via an intrauterine device.

[0160] Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0161] For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0162] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0163] Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

[0164] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0165] Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (e.g. agents that reduces the amount of a down-stream effector from a RANK receptor of an oocyte) effective to prevent pregnancy.

[0166] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0167] For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0168] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 P-l).

[0169] Dosage amount and interval may be adjusted individually to provide tissue levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0170] In one embodiment, the agent is formulated in a daily dosage unit and comprised in a packaging unit. Each packaging unit contains 21-28 daily active dose units. Each daily active dosage unit. In one embodiment, the daily active dosage unit does not contain any hormones (e.g. estrogen or estrogen derivative such as ethinyl estradiol, ethinyl estradiol methyl ether or 8- prenylnaringenin). In other words, the present agents are preferably present in daily active dosage units that do not contain estrogen.

[0171] In a specific embodiment, the agent that reduces the amount of a down-stream effector from a RANK receptor of an oocyte is the only contraceptive ingredient contained in the daily active dosage unit.

[0172] Each packaging unit may optionally contain 1 -7 daily dosage units of a pharmaceutically acceptable placebo. In some embodiments, the contraceptive kit is characterized in that each packaging unit comprises 28 daily dosage units and does not contain a pharmaceutically acceptable placebo daily dosage unit. Such contraceptive kits are particularly suitable for performing the contraceptive methods of the present invention, which consist in "continuous" administration of the active agent without a period of non-avoidaiice.

[0173] In other embodiments, each packaging unit comprises 21 to 27 daily dosage units of a contraceptive composition substantially as described herein, and -optionally, 1 to 7 pharmaceutically acceptable placebo daily dosage units. Such a contraceptive kit is particularly suitable for carrying out the contraceptive method of the invention, which comprises:

[0174] -a first phase wherein an active daily dosage unit of the invention is administered to a female patient over a period of 21 to 27 consecutive days, followed by:

[0175] -a second phase wherein the female patient is not administered a contraceptive composition for a period of 1 to 7 consecutive days.

[0176] In some other embodiments, each packaging unit of the kit comprises 4 daily dosage units comprising an effective amount of 24 daily dosage units of the contraceptive composition described herein and optionally a pharmaceutically acceptable placebo.

[0177] The above-described packaging unit may have a conventional form commonly used for oral contraceptives.

[0178] For example, the packaging unit may be a conventional blister pack comprising an appropriate number of dosage units in a blister pack sealed with a cardboard, paperboard, foil or plastic backing and enclosed in a suitable cover. Each blister container may be conveniently numbered or marked to promote compliance.

[0179] The packaging units may comprise daily dosage units in the order in which they are to be taken, i.e. starting with the first of at least 21 dosage units comprising the active agent (i.e. the agent that reduces the amount of a down-stream effector from a RANK receptor of an oocyte) composition, optionally followed by 7 or less empty blisters, or 7 or less dosage units comprising a pharmaceutically acceptable placebo,

[0180] The kits of the invention may comprise other suitable components, such as instructions for use.

[0181] As used herein the term “about” refers to ± 10 %.

[0182] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0183] The term “consisting of’ means “including and limited to”.

[0184] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0185] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0186] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0187] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0188] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0189] When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

[0190] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0191] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0192] EXAMPLES

[0193] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0194] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

[0195] MATERIALS AND METHODS

[0196] IVF Protocol’. Mural Granulosa Cells (MGCs) were obtained from large follicles at the time of oocyte retrieval from women at the age of < 38 years that undergoing IVF treatment due to mechanical problems, pre-implantation genetic disorders (PGD), or malefactors. In brief, the patients underwent suppression using a GnRH (Gonadotropin-Releasing Hormone) antagonist protocol (0.25 mg / day, Cetrorelix, Cetrotide; Merck-Serono, Darmstadt, Germany). Ovarian stimulation was performed with a daily subcutaneous dose of recombinant FSH (either Gonal-F; Merck Serono, Darmstadt, Germany, or Puregon Pen; Schering Plough, North Wales PA, USA), which was commenced on the third day of the menstrual cycle for five days. This was followed by a daily dose of human menopausal gonadotrophin (hMG; Menogon, Ferring, Switzerland). The initial dose used was dependent upon age, body mass index, and prior IVF treatment history. When three leading follicles had reached 18 mm in diameter, patients received 250 pg human chorionic gonadotrophin (hCG) (Ovitrelle, Merck-Serono, Darmstadt, Germany).

[0197] Oocyte retrieval was arranged 36 hours after hCG injection and performed by transvaginal ultrasound-guided needle aspiration. Follicular fluids (FFs) were collected in culture tubes containing a flushing medium with heparin (MediCult).

[0198] MGC Collection and Grouping’.

[0199] MGCs were collected from the FFs and re-suspended in a phosphate-buffered solution (PBS, Sigma St Louis MO, USA). After allowing the cells to settle by gravity for a few minutes, the top medium was aspirated, and this step was repeated 2-3 times until the medium became clear. The cells were centrifuged at 1000 rpm for 5 min at room temperature. Purified MGCs of three to four women undergoing IVF procedures were pooled and represented a biological replicate. Pooled cells were subjected to total RNA purification. The resulting pellets were stored at -80°C until RNA isolation.

[0200] Cumulus Cell ( CC) Collection and Grouping:

[0201] CCs were obtained during oocyte denudation for the intracytoplasmic sperm injection (ICSI) procedure. After oocyte retrieval, the CCs of each oocyte were removed by hyaluronidase (SAGE, Trumbull, CT) and a glass denudation pipette (Swemed, Sweden). The cells were washed in PBS and centrifuged at 3000 x g for 5 min at room temperature. The resulting pellets were stored at -80°C until RNA isolation.

[0202] Total CCs mass derived from three women was pooled together to create the cumulus group that represented a biological replicate.

[0203] Cumulus cells isolated from individual oocytes were divided into different groups according to the corresponding oocytes maturation stages: CCGV, CCMI, CCMIL

[0204] Mural Granulosa Cell Culture:

[0205] MGCs were collected from FF and washed in PBS, as described above. The resulting pellets were re-suspended in basic medium (Medium 199) (Sigma Aldrich St Louis MO, USA) supplemented with 5% fetal calf serum (FCS) (Invitrogen Grand Island, NY, USA) and 1% penicillin / streptomycin (Sigma Aldrich St Louis, MO, USA). MGCs were plated in 24-well plates at a density of 100,000 cells / well and incubated at 37°C in a humidified atmosphere with 5% CO2 in the air. MGCs were cultured for 4 days with a daily medium exchange ("early non luteinized like MGCs") (Ophir et al. 2014). MGCs were then treated with hCG in a time course: 0, 3, 6, 9, 12, 24, and 36 hours. In addition, MGCs were plated in 6-well plates for Western blot at a density of 500,000 cells / well and incubated at 37°C in a humidified atmosphere with 5% CO2, they were cultured for 4 days with a daily medium exchange. MGCs were then stimulated with hCG alone or in combination with PGE2 for an additional 8h.

[0206] RNA Extraction and qPCR:

[0207] For mural granulosa cells, total RNA was extracted using a Mini / Micro RNA Isolation I kit (Zymo Research, CA, USA), according to the manufacturer’s instructions.

[0208] RNA purity and concentration were assessed using nanodrop (NanoDrop 2000C, Thermo Scientific Waltham, MA). Total RNA from each sample was used for cDNA synthesis using a High-Capacity Reverse Transcription kit (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer’s instructions in a 10 pl total volume reaction. mRNA levels were analyzed by real-time PCR using the StepOnePlus real-time PCR system (Applied Biosystems). The real-time PCR mix contained one pl of cDNA, fast SYBR Green Master Mix (Applied Biosystems), and specific primers for OPG, RANK, RANKL, and P- actin (housekeeping gene) in a total volume of 10 pl. Cycling parameters were: 1 cycle at 95°C for 20 s, and 40 cycles each at 95°C for 3 s and 60°C for 30 s. A melting curve analysis was performed at the end of each run to ensure a single amplicon. All samples were run in duplicates.

[0209] Analysis of the qPCR results was carried out using Step One software. Relative gene expression was calculated using the delta-delta Ct method. Details of the primers used are shown in Table 1.

[0210] Table 1

[0211] RANK expression in mouse oocytes characterized using Single-cell RT-PCR Kit (Cell to CT, Thermo Fisher Scientific) comprising 4 functional steps: cell lysis, reverse transcription, cDNA pre-amplification, and Real-Time PCR according to the manufacturer’s instructions. RANK Primers (TaqMan Gene Expression assays) were ordered from Thermo Fisher Scientific.

[0212] Western blot:

[0213] Cells were harvested using 0.1 mL NP-40 lysis buffer with protease inhibitor cocktail and incubated for 30 min on ice before removal of nuclei and debris by centrifugation. Protein concentration was determined by the Bradford method (Protein Assay Dye Reagent, Bio-Rad, Hercules, CA, USA). Equal amounts (50 pg) of protein were loaded and separated on SDS- Polyacrylamide gel (10% acrylamide). Proteins were then transferred onto nitrocellulose membranes. Membranes were blocked in 5% milk in TBST for one hour and afterward incubated with a primary antibody against RANKL (Santa Cruz biotechnology, 1:400) or a-tubulin (housekeeping gene, 1: 1,000) overnight at 4°C. The membranes were then treated with a horseradish peroxidase-conjugated secondary antibody and developed using Image Lab™ software (Bio-Rad, version 5.2.1).

[0214] Animals and hormonal treatment

[0215] All mice used were of the C57BL / 6 (Envigo Inc., Israel) genetic background, and were maintained under controlled lighting (12 hours light / 12 hours dark) with continuous access to food and water. For Mil oocytes, 25- 31 day old female mice were injected with lOU / mice of PMSG (Sigma, St. Louis, MO, USA) to stimulate follicle growth and 48 h later with lOU / mice of hCG to induce ovulation. For COCs and immature oocytes, female mice were injected with lOU / mice of PMSG and sacrificed after 48h.

[0216] For the RANKL inhibitor experiment mice were injected with lOU / mice PMSG for 48h and then injected with lOU / mice hCG, 12mg / mice murine OPG, and 40ng / mice RANKL or hCG with OPG.

[0217] Oocyte collection:

[0218] Mice: Mil ovulated mouse oocytes were obtained by dissecting cumulus-oocyte masses from the ampulla of hCG treated mice and then transferred into PBS. Oocyte maturation was assessed by denudation with hyaluronidase enzyme and mechanical pipetting. Immature oocytes were obtained by puncturing the ovary of PMSG treated mice and denuded by a needle. 15 MII / GV oocytes were collected into groups and transferred immediately to liquid nitrogen and stored at - 80°c until used for RT-PCR.

[0219] Human: GV oocyte were obtained from patients undergoing a protocol of oocyte freezing. Immature oocytes were nor frozen and instead used in the present study. GV oocytes were collected only when two or more GV oocyte were available.

[0220] IVM:

[0221] COCs were obtained by puncturing the ovary of PMSG treated mice and washed in PBS, then transferred into IVM maturation medium (Oocyte maturation medium (Sage) with 75 mIU / mL FSH and 75 mIU / mL LH) with 150 mM dibutyryl cAMP to prevent GV oocyte spontaneous maturation. COCs were treated with or without (control group) murine RANKL peptide. Following 16h of incubation, oocytes were denuded and Mil oocytes were assessed by double-blind PB identification.

[0222] Statistical analysis:

[0223] Data were expressed as mean ± Standard error of the mean (SEM) and evaluated with students t-test with a two-tailed distribution, with two samples equaling variance, or with ANOVA for more than two variances using the post hoc Tukey test, assuming equal variances, or the Games-Howell test for unequal variances. For all statistical analyses, SPSS 25 software (IBM, Armonk, NY, USA) was used.

[0224] P values < 0.05 were considered statistically significant.

[0225] RESULTS

[0226] RANK expression in mouse oocytes

[0227] RANK ligand was shown to be upregulated in cumulus cells (CCs) during the ovulatory process [7]. To examine the expression of RANK in the oocyte, RANK expression was examined in the mouse oocyte at various stages of maturation, GV or Mil. RANK mRNA was purified using a mRNA purification kit for limited cells number (Cell to CT, Thermo Fisher Scientific). Expression levels of RANK mRNA were assessed by qPCR. RANK was detected in mouse oocytes, in both GV and Mil oocytes, with no difference in expression (Figure 1).

[0228] RANK, RANKL, and OPG mRNA expression in cumulus and mural granulosa cells

[0229] To study RANK, RANKL, and OPG expression in the ovary, the present inventors compared mRNA levels in MGCs, purified from follicular fluid obtained during IVF, and CCs, obtained during oocyte denudation before an intracytoplasmic sperm injection procedure, by qPCR. The level of RANKL mRNA was significantly higher in mural granulosa cells (MGCs) than in CC (Figure 2A). OPG mRNA level was also higher in MGCs than CCs but did not reach significance (Figure 2B). Figure 2C illustrates that RANK is present in oocytes, but not in granulosa cells.

[0230] RANK, RANKL, and OPG mRNA expression in CCs according to maturation stage.

[0231] Next, RANK, RANKL, and OPG expression patterns were evaluated in CCs according to the COCs maturation stage. CCs were isolated from COCs according to their maturation, GV, or Mil stages. Expression levels of RANK, RANKL, and OPG mRNA was assessed by qPCR. RANKL level increased significantly with the maturation stage, from GV to Mil (Figure 3A). No difference was found in the expression levels of OPG between CCs isolated from GV and Mil COCs (Figure 3B). RANK was not detected.

[0232] RANK, RANKL, and OPG expression during the ovulatory interval in vitro.

[0233] MGCs from 2-5 women were mixed and cultured for 4 days with a daily medium exchange before being stimulated with human chorionic gonadotropin (hCG). Cells were harvested at different time points as indicated, and mRNA was quantified by qPCR. RANKL increased and reached the maximal level at 9h after hCG administration, and at 12h RANKL was significantly higher compared to time zero control (Figure 4A). OPG was at a higher level 6h after hCG administration but with no significance (Figure 4B). RANK was not detected. Induction ofRANKL protein in MGCs by hCG

[0234] To confirm the effect of hCG on RANKL expression, RANKL protein induction was examined in in vitro MGCs. MGCs aspirated during IVF procedures were cultured for 4 days with a daily medium exchange followed by stimulation by hCG (lU / ml) for 16h. Protein levels of RANKL expression were determined by Western blotting. RANKL protein level was upregulated by hCG compared to control (Figure 5). Quantification of the protein levels was made using the “Image J” software.

[0235] RANK, RANKL, and OPG expression in mural granulosa cells treated with different concentrations of PGE2

[0236] To examine whether RANK, RANKL, and OPG are regulated by PGE2 in the ovary, MGCs were cultured and treated with different concentrations of PGE2. Cells were administrated with hCG (lU / ml) as a positive control, or PGE2 at different concentrations or both, and harvested after 8h. RANKL (Figure 6A) and OPG (Figure 6B) were quantified by qPCR. RANK was not detected. PGE2 with hCG or alone upregulated RANKL expression significantly (P=0.047 and P=0.003, respectively). The maximal level of RANKL was evident at a concentration of Ing / ml PGE2. hCG downregulated OPG levels (P<0.001), while PGE2 with hCG elevated it to the control level. PGE2 alone increased OPG expression, but not significantly. The maximal level of OPG was evident after treatment with 10 ng / ml PGE2. A higher concentration of 100 ng / ml PGE2 reduced both RANKL and OPG levels.

[0237] RANKL expression in mural granulosa cells treated with hCG, PGE2, and Indomethacin

[0238] To confirm that RANKL is upregulated by PGE2 in MGCs, the effect of the PGE2 synthesis inhibitor, indomethacin, on RANKL mRNA was examined. Cells were treated with hCG, PGE2 with or without indomethacin. hCG and PGE2 alone upregulated RANKL while indomethacin blocked the elevated levels of RANKL by hCG and PGE2 (Figure 7). RANKL expression levels in cells treated with hCG, PGE2, and indomethacin remained low.

[0239] The effect ofRANKL on mouse GV oocyte undergo IVM treatment.

[0240] Female mice were primed with lOU / mice Pregnant mare's serum gonadotropin (PMSG) for 48 hours. Cumulus-oocyte complexes (COCs) obtained from the ovary were transferred into IVM maturation medium with 150 mM dibutyryl cAMP to prevent spontaneous maturation.

[0241] It was found that COCs obtained from PMSG treated mice’s ovaries presented in two different appearances and therefore separated into two distinct groups. A group named "complete compact COCs", includes COCs with a rough and tight cumulus, while the other group named "partial compact COCs" is characterized by less tight connections between the cumulus cells layers (Figures 8A-B).

[0242] All COCs were cultured in an IVM medium, half the COCs in each group were treated with the addition of murine RANKL peptide and the other half remained without RANKL as a control. Following 16h of incubation, oocytes were denuded and oocytes maturation were assessed by polar body identification.

[0243] Following IVM, the complete compact COCs remain tight compact without oocyte maturation. The partial compact COCs showed an increase in cumulus fraction but not cumulus expansion in both, RANKL treated and control. Oocyte maturation was assessed by oocyte denudation with hyaluronidase enzyme and mechanical pipetting. Results show oocyte maturation in the control group was 31% while RANKL treatment significantly elevated the maturation rate to 64 %.

[0244] Effect of OPG on ovulation and oocytes maturation

[0245] To investigate RANKL function on ovulation and oocyte maturation, RANKL function was inhibited in a superovulation mouse model. RANKL was inhibited by the injection of endogenous antagonist OPG. To further support these results, RANKL was added in rescue experiments. Oocytes were obtained by dissecting cumulus-oocyte masses from the ampulla, the number of ovulated COCs were counted and oocytes were denuded to elucidate their maturation stage. Mice after hCG treatment ovulated 47 oocytes on average, while OPG treatment reduced ovulation to 34 oocytes per mouse, and OPG with RANKL increased to 40 oocytes per mouse (Figure 9 A). The differences between groups were not significant.

[0246] Denuded oocytes were assessed for maturation by polar body identification. The maturation rate after hCG was 79%, whilst OPG treatment reduced to 10% (P<0.001) maturation rate. RANKL addition partially rescued oocytes maturation rate to 41% (P<0.001) (Figure 9B). The effect of RANKL on human GV oocyte

[0247] Oocytes from the same patient were separated and icubated in two mediums, one step medium (Sage) and one step medium with RANKL peptide (100 ng / ml) for 16h. After 16h oocyte were examined for polar-body apperance. The results (Figure 10) show that RANKL peptide increased the maturation rate to 80% while the spontaneous maturation was 27% only.

[0248] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0249] All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

Claims

WHAT IS CLAIMED IS:

1. A method of maturing an immature oocyte comprising contacting the immature oocyte with an agent which increases an amount and / or activity of a down-stream effector of a RANK receptor on the oocyte, thereby maturing the immature oocyte.

2. An agent which increases an amount and or activity of a down- stream effector of a RANK receptor on an oocyte for use in assisted fertilization.

3. The method or agent of claims 1 or 2, wherein said agent binds to said RANK receptor.

4. The method or agent of 2, wherein said agent is an activating antibody.

5. The method or agent of any one of claims 1-4, wherein said down-stream effector comprises tumor necrosis factor receptor-associated factor 6.

6. The method or agent of any one of claims 1-5, wherein said agent increases an amount of at least one transcription factor selected from the group consisting of NFATcl, CREB, NFKB, AP-1, c-fos, and MITF.

7. The method or agent of any one of claims 1-6, wherein said agent is a peptide agent.

8. The method or agent of claim 7, wherein said peptide has an amino acid sequence at least 99 % identical to SEQ ID NOs: 17 or 18.

9. The method or agent of claims 1 or 2, wherein said immature oocyte is comprised in a cumulus-oocyte complex.

10. The method of claim 1, wherein said contacting comprises culturing said immature oocyte in a medium comprising said agent.

11. The method or agent of any one of claims 1-9, wherein said oocyte is a human oocyte.

12. The method of claim 1, wherein said oocyte is retrieved from a female subject having a polycystic ovarian syndrome.

13. The method of claim 1, being an in vitro method.

14. The method of claim 1, being an in vivo method.

15. A method of preparing oocytes from a human subject for in-vitro fertilization comprising:(a) retrieving an immature oocyte from the human subject; and(b) maturing the immature oocyte according to the method of claim 1 , thereby preparing oocytes from the human subject for in-vitro fertilization.

16. An oocyte culture medium comprising an agent which increases an amount and or activity of a down-stream effector of a RANK receptor on the oocyte.

17. The oocyte culture medium of claim 16, wherein said agent binds to said RANK receptor.

18. The oocyte culture medium of claims 16 or 17, wherein said down-stream effector comprises tumor necrosis factor receptor-associated factor 6.

19. The oocyte culture medium of claims 16 or 17, wherein said agent increases an amount of at least one transcription factor selected from the group consisting of NFATcl, CREB, NFKB, AP-1, c-fos, and MITF.

20. The oocyte culture medium of claim 16, wherein said agent is a peptide agent.

21. The oocyte culture medium of claim 20, wherein said peptide has an amino acid sequence at least 90 % identical to SEQ ID NO: 17 or 18.

22. The oocyte culture medium of any one of claims 16-21, further comprising at least one immature oocyte.

23. A non-hormonal contraceptive composition comprising an agent that reduces the amount of a down- stream effector from a RANK receptor of an oocyte and a pharmaceutically acceptable carrier, with the proviso that when said agent is osteoprotegerin, said osteoprotegerin is present in an amount which reduces the risk of pregnancy and / or the composition is formulated for local delivery.

24. The contraceptive composition of claim 23, formulated for oral delivery.

25. The contraceptive composition of claim 23, wherein said local delivery comprises intra-uterine delivery.

26. The contraceptive composition of claim 23, wherein said agent binds to and sequesters RANK.

27. The contraceptive composition of claim 23, wherein said agent is a neutralizing antibody which binds to RANKL or said RANK receptor.

28. The contraceptive composition of claim 27, wherein said agent binds to the extracellular domain of said RANKL.

29. The contraceptive composition of claim 27, wherein said neutralizing antibody is Denosumab.

30. An article of manufacture having a plurality of separate dosage units of an agent that reduces the amount of a down-stream effector from a RANK receptor of an oocyte, each of said dosage units, formulated for oral administration.

31. The article of manufacture of claim 30, wherein said plurality of separate dosage units comprises at least 20.

32. The article of manufacture of claim 30, wherein said agent binds to and sequestersRANK.

33. The article of manufacture of claim 30, wherein said agent is a neutralizing antibody which binds to RANKL or said RANK receptor.

34. The article of manufacture of claim 33, wherein said agent binds to the extracellular domain of said RANKL.

35. The article of manufacture of claim 34, wherein said neutralizing antibody is Denosumab.

36. A method of reducing the risk of pregnancy in a female subject comprising administering to the subject a preventatively effective amount of an agent that reduces the amount of a down- stream effector from a RANK receptor of an oocyte, thereby reducing the risk of pregnancy in the subject.

37. The method of claim 36, wherein said agent binds to and sequesters RANK.

38. The method of claim 37, wherein said agent is osteoprotegerin.

39. The method of claim 36, wherein said agent is a neutralizing antibody which binds to RANKL or said RANK receptor.

40. The method of claim 39, wherein said agent binds to the extracellular domain of said RANKL.

41. The method of claim 40, wherein said neutralizing antibody is Denosumab.

42. The method of any one of claims 36-41, wherein said administering comprises daily administering.

43. The method of any one of claims 36-42, wherein a dosage of said agent alternates according to the menstrual cycle of the subject.

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