Compositions, kits and methods for restoring and enhancing fertility

Abstract

The present disclosure relates to methods for producing mature oocytes comprising transplanting ovarian tissue or any fragment thereof of a human female into at least one recipient site of an immunodeficient rabbit, and collecting mature oocytes from said transplanted ovarian tissue. In addition, the methods comprises providing conditions for follicles development and oocytes maturation in said rabbit.

Description

[0001] COMPOSITIONS, KITS AND METHODS FOR RESTORING AND ENHANCING FERTILITY

[0002] TECHNOLOGICAL FIELD

[0003] The present disclosure relates to methods for restoring fertility and for enhancing fertility.

[0004] BACKGROUND ART

[0005] References considered to be relevant as background to the presently disclosed subject matter are listed below:

[0006] Man, L., et al., Xenograft model of heterotopic transplantation of human ovarian cortical tissue and its clinical relevance; Reproduction, 2022 165(1):31-47.

[0007] Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

[0008] GENERAL DESCRIPTION

[0009] In accordance with some aspects, the present disclosure provides a method for producing mature oocytes, the method comprises (a) transplanting an ovarian tissue or any fragment thereof of a human female into at least one recipient site of an immunodeficient rabbit, and (b) collecting mature oocytes from the transplanted ovarian tissue.

[0010] In accordance with some other aspects, the present disclosure provides a method for producing mature oocytes, the method comprises (a) transplanting an ovarian tissue or any fragment thereof of a human female into at least one recipient site of an immunodeficient rabbit, and (b) providing conditions for follicles development and oocytes maturation in said rabbit.

[0011] In accordance with some further aspects, the present disclosure provides a method for producing mature oocytes, the method comprises (a) transplanting an ovarian tissue or any fragment thereof of a human female into at least one recipient site of an immunodeficient rabbit, (b) providing conditions for follicles development and oocytes maturation in said rabbit and (c) collecting mature oocytes from the transplanted ovarian tissue.

[0012] In accordance with yet some aspects, the present disclosure provides mature oocytes obtained by the method described herein.

[0013] In accordance with yet some other aspects, the present disclosure provides mature oocytes obtainable by the method described herein.

[0014] In accordance with yet some further aspects, the present disclosure provides a method for fertility preservation, fertility restoration and treating infertility or infertility- related conditions in a human female, the method comprising: producing mature oocytes as described herein and performing IVF procedure.

[0015] In accordance with yet some further aspects, the present disclosure provides a method for fertility preservation, fertility restoration and treating infertility or infertility- related conditions in a human female, the method comprising: performing IVF procedure using the mature oocytes obtained or obtainable by the method described herein.

[0016] In accordance with yet some further aspects, the present disclosure provides a method for of gametogenesis, the method comprises: transplanting a tissue or any fragment thereof of a one animal into at least first recipient site of an immunodeficient second animal, and providing conditions for gametes maturation in the second animal.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0019] Figures 1A-1C shows serum FSH levels following weekly administration of different doses of Gonal F, Figure 1A shows serum levels of FSH at the first week after injection of different dose of Gonal F, Figure IB shows serum levels of FSH at the second week of injection (the second injection) of different dose of Gonal F, Figure 1C shows serum levels of FSH at the third week of injection (the third injection) of different dose of Gonal F. Figures 2A-2C is a time-Dependent changes in serum FSH following weekly dosing of Gonal F at different concentrations, Figure 2A shows serum levels of FSH after administration of 25IU Gonal F at different times after first week (Wl), second week (W2), third week (W3), Figure 2B shows serum levels of FSH after administration of 37.5IU Gonal F at different times after first week (Wl), second week (W2), third week (W3) of administration, Figure 2C shows serum levels of FSH after administration of 50IU Gonal F at different times after second week (W2), third week (W3) of administration.

[0020] Figures 3A-3B shows a comparison of serum FSH levels between male and female subjects following Gonal F administration, Figure 3A shows serum levels of FSH at the first week after injection of Gonal F (25IU) in male compared to female, Figure 3B shows serum levels of FSH at the second week after injection of Gonal F (37.5 IU) in male compared to female.

[0021] Figures 4A-4C shows serum FSH and LH levels following weekly administration of different doses of Gonal F and Luveris, Figure 4A shows serum levels of FSH after injection of Gonal F (25IU) and different doses of Luveris in male rabbits, Figure 4B shows serum levels of LH after injection of different concentrations of Gonal F and different concentrations of Luveris in male, on week 2, Figure 4C shows serum levels of LH in males, at week 3 after injection of different concentrations of Gonal F and Luveris.

[0022] Figure 5 shows serum levels of LH in female rabbits, after injection of Luveris at different concentrations, on weeks 1-3.

[0023] Figure 6 shows serum levels of FSH in males, after injection of Elonva at different concentrations.

[0024] Figures 7A-7B shows serum progesterone levels following administration of Gonal F or Elonva, Figure 7A shows serum levels of progesterone after injection of Gonal F at different doses at week 1, 2 and 3, Figure 7B shows serum levels of progesterone after injection of Elonva after up to 12 days.

[0025] Figures 8A-8C shows serum human FSH profiles in male and female rabbits after Gonal F and Luveris administration, Figure 8A shows human FSH levels in male rabbits following administration of human gonadotropin Gonal F and Luveris, Figure 8B shows human FSH levels in female rabbits following administration of human gonadotropin Gonal F and Luveris, Figure 8C is a schematic representation of the sequential administration of Gonal F and Gonal + Luveris.

[0026] Figures 9A-9C shows serum human FSH and PHCG levels in male rabbits after Menopur administration, Figure 9A shows human FSH levels in male rabbits following administration of human menopausal gonadotropin Menopur, Figure 9B shows PHCG levels in male rabbits following administration of human menopausal gonadotropin Menopur, Figure 9C is a schematic representation of the sequential administration of Menopur.

[0027] Figures 10A-10C shows FSH and LH levels following several dilutions in different media of Gonal F, LH and Elonva, Figure 10A shows levels of FSH at different Gonal F doses, in PBS or DMEM media, Figure 10B shows levels of LH of Luveris at different doses, in PBS or DMEM media, Figure IOC shows levels of FSH of at different concentrations of Elonva.

[0028] Figures 11A-11B shows FSH bioactivity of serum of castrated rabbits versus serum from women undergoing IVF, Figure HA shows cAMP levels of Rabbit Physiological baseline and growth media following different times of incubation with human granulosa cells, Figure 11B shows cAMP levels of serum from women undergoing IVF following different times of incubation.

[0029] Figures 12A-12B shows FSH bioactivity of serum of castrated rabbits following injection with Elonva or Menopur, Figure 12A shows cAMP levels of rabbit serum 2 days and 8 days after injection of Elonva following different times of incubation, Figure 12B shows cAMP levels of rabbit serum 12 hours and 48 hours after injection of Menopur following different times of incubation.

[0030] Figure 13 shows cAMP levels (serum FSH bioactivity) of castrated Male Rabbits following repeated injections of human gonadotropins Gonal F and Luveris.

[0031] Figures 14A-14C shows Exemplary images showing preparations of ovarian tissue from Pl-VL and transplantation.

[0032] Figures 15A-15C are images showing hematoxylin and eosin (H&E)-stained histological sections exhibiting necrotic tissue with residual ovarian follicles, and early fibrosis and eosinophilic infiltrate. Figures 16A-16C are images showing H&E-stained histological sections exhibiting prominent chronic inflammation associated with numerous eosinophils, focal microabscess formation, and focal necrotizing granuloma and focal fibrosis

[0033] Figures 17A-17B are images showing H&E-stained histological sections.

[0034] Figures 18A-18B are images showing H&E-stained histological sections.

[0035] Figures 19A-19D are images showing H&E-stained histological sections exhibiting hyper stimulated Ovaries.

[0036] Figures 20A-20C are images showing H&E-stained histological sections showing WT rejection of transplant.

[0037] Figures 21A-21B are images showing H&E-stained histological sections showing survival and growth of transplant in IL rabbit strains.

[0038] Figure 22: is a graph showing follicles counting in transplants of IL2 and RAG rabbit strains.

[0039] Figures 23A-23L are images showing H&E-stained histological sections and immunofluorescent staining of Human and Rabbit Blood Vessels detection in Ovarian Xenotransplantation 2W post transplantation into WT, IL2 and RAG2 rabbit strains. Magnification: 20x, Zoom 1.9. H&E stain (Figures 23A-23C); aSMA+ nuclei stain (DAPI), (Figures 23D- Figures 23F); CD31+CD34 (Figures 23G-23I), Merged (Figures 23J- 23L).

[0040] Figures 24A-24L are images showing H&E-stained histological sections and immunofluorescent staining of Human and Rabbit Blood Vessels detection in Ovarian Xenotransplantation 2W post transplantation into WT, IL2 and RAG2 rabbit strains. Magnification: 20x, Zoom 6.6. H&E stain (Figures 24A-24C); aSMA+ nuclei stain (DAPI), (Figures 24D- 24F); CD31+CD34 (Figures 24G- Figures 241), Merged (Figures 24J- Figures 24L).

[0041] Figures 25A-25L are images showing H&E-stained histological sections and immunofluorescent staining of Human and Rabbit Blood Vessels detection in Ovarian Xenotransplantation 3M post transplantation into WT, IL2 and RAG2 rabbit strains. Magnification: 20x, Zoom 1.9. H&E stain (Figures 25A-25C); aSMA+ nuclei stain (DAPI), (Figures 25D-25F); CD31+CD34 (Figures 25G-25I), Merged (Figures 25J- 25L).

[0042] Figures 26A-26L are images showing H&E-stained histological sections and immunofluorescent staining of Human and Rabbit Blood Vessels detection in Ovarian Xenotransplantation 3M post transplantation into WT, IL2 and RAG2 rabbit strains. Magnification: 20x, Zoom 6.6, H&E stain (Figures 26A-26C); aSMA+ nuclei stain (DAPI), (Figures 26D-26F); CD31+CD34 (Figures 26G-26I), Merged (Figures 26J- 26L).

[0043] Figures 27A-27D shows Follicle counting in Ovarian Xenotransplantation 2W post transplantation into WT, IL2 and RAG2 rabbit strains. Patients: EA (Figures 27A-27B); LV (Figures 27C-27D). Counting: Primordial (PMF) and Growing Follicle / ovary (Figures 27A, 27C); Total follicle s / o vary (Figures 27B, 27D).

[0044] Figures 28A-28D shows Follicle counting in Ovarian Xenotransplantation 3M post transplantation into WT, IL2 and RAG2 rabbit strains. Patients: EA (Figures 28A and 28B); LV (C, D). Counting: Primordial (PMF) and Growing Follicle / ovary (Figures 28A and 28C); Total follicles / ovary (Figures 28B and 28D).

[0045] Figures 29A-29D are images showing H&E staining in human ovary transplanted into WT Rabbits (Figures 29A-29B) and into immunodeficient rabbits two weeks after subcutaneous transplantation and three months after intra peritoneal transplantation (Figures 29C-29D, respectively), arrows in Figures 29C-29D point to exemplary follicles.

[0046] Figures 30A-30D are graphs showing follicle count two weeks after intraperitoneal transplantation, Figures 30A and 30C show follicle count per ovary in extracellular- matrix-assisted protocol (EA) and lentiviral-vector-mediated vascularization (LV) protocol, respectively and Figures 30B and 30D show total follicle count per ovary in extracellular-matrix-assisted protocol (EA) and lentiviral-vector-mediated vascularization (LV) protocol, respectively.

[0047] Figures 31A-31D are graphs showing follicle count thee weeks after peritoneal transplantation, Figures 31A and 31C show follicle count per ovary in extracellular- matrix-assisted protocol (EA) and lentiviral-vector-mediated vascularization (LV) protocol, respectively and Figures 31B and 31D show total follicle count per ovary in extracellular-matrix-assisted protocol (EA) and lentiviral-vector-mediated vascularization (LV) protocol, respectively.

[0048] Figures 32A-32D are images showing human blood vessels outside transplant, Figure 32A shows the transplant -dashed lines and the arrows indicate staining for human blood vessels.

[0049] Figures 33A-33D are images showing rabbit blood vessels with the transplant, Figure 32A shows the transplant -dashed lines and the arrows indicate staining for rabbit blood vessels.

[0050] DETAILED DESCRIPTION OF EMBODIMENTS

[0051] The present disclosure relates to fertility preservation and fertility restoration and is based on development of compositions, kits and methods that are suitable for female subjects that refrained from conception or fertility and provide an improvement in fertility preservation and implantation techniques, increasing the success rate of these processes.

[0052] The compositions, kits and methods described herein are applicable for different female subject populations having different health condition or undergoing (past and / or present) medical treatments, including for example, female subjects diagnosed with cancer and / or treated with an anti-cancer treatment. Thus, the composition, kits and methods described herein may be advantageous for example, in cancer female patients.

[0053] The novel technology described herein is based on ovarian xenotransplantation, optionally ovarian tissue cryopreservation and xenotransplantation (OTCX) as a source of purified mature oocytes suitable for in-vitro fertilization (IVF). Accordingly, the ovarian tissue obtained (collected / harvested / recovered / procured) from a female subject, optionally after cryopreservation and thaw, is transplanted to an immunodeficient host animal. The transplanted ovarian tissue (grafted tissue) is engrafted in the host and follicle development initiates, optionally under exogenous hormonal stimulation. Mature oocytes may be isolated (harvested) from the graft (engrafted ovarian tissues), for use, for example in IVF.

[0054] It was suggested that the technology described herein may advance the fertility preservation research field and potentially develop a clinically applicable OTCX protocol that could assist patients who have fertility preservation needs. Specifically, this technology may enable fertility and live births in female subjects who were chronically treated for many years with immunologic, biologic therapies or even chemotherapies, during which they refrained from conception and IVF. In addition, it was suggested that this technology may potentially circumvent the problem of cancer cell transmission via the graft.

[0055] Studies have shown that xenotransplantation of ovarian tissue from different animal species in immunodeficient mice works resulting in follicle growth and metaphase II oocytes maturation, however, the relatively small size and short lifespan of such animals limit their application.

[0056] As shown in the Examples below and specifically in Example 1, a human-like hormonal milieu (environment) was established in non-human host, e.g. wild type or transgenic immunodeficient rabbits, thereby permitting survival and maturation of human ovarian graft follicles following xenotransplantation of cryopreserved or freshly excised human ovarian tissue.

[0057] Example 1 further shows that conditioning the host animal to maintain a serum follicle-stimulating-hormone (FSH) concentration comparable to that observed during controlled ovarian stimulation in human IVF cycles (about 20 IU L1± 5 IU L ') supports follicular development and oocyte maturation. The conditioning regimen may comprise exogenous FSH administration, modulation of endogenous gonadotropin secretion (e.g., via a GnRH agonist or antagonist), or a combination thereof, delivered in a dosing schedule sufficient to maintain the target endocrine profile throughout the engraftment period.

[0058] Example 3 shows that human FSH is active Granulosa Cells in the presence of rabbit serum. In addition and as shown in this Example, serum from castrated rabbits exhibited no detectable FSH bioactivity on human granulosa cells in vitro, whereas serum from women undergoing IVF demonstrated clear FSH bioactivity. Interestingly, GnRH antagonist treatment had no inhibitory effect on the efficacy of FSH to induce aromatase expression in human granulosa cells.

[0059] Hence, in accordance with some aspects, the present disclosure provides a composition comprising an effective amount of at least one reproductive hormone, wherein the composition induces in a host animal an endocrine profile that is comparable to that observed during ovarian stimulation in human IVF cycles in human females.

[0060] As shown in Figures 29C and 29D, appearance of PMF and growing follicles was observed in human ovarian tissue transplant into immunodeficient rabbits. Figures 30A- 30D and Figures 31A-31D demonstrate that immunodeficient rabbits supported growth and maturation of follicles originating from the transplanted human ovary.

[0061] Moreover, Figures 32A demonstrates growth of human blood vessels in the rabbit outside the transplanted human ovary and Figures 33 A demonstrates growth of rabbit blood vessels in the rabbit inside the transplanted human ovary. These results demonstrate that human ovarian cortical xenografts in immunodeficient rabbits survive and activate both resting (PMF) and growing follicle populations and undergo progressive maturation, progressing through primary to antral stages over time. In addition, these results demonstrate that human ovarian cortical xenografts in immunodeficient rabbits establish bidirectional, functional vasculature, with human-derived vessels sprouting outward into the host stroma and host vessels infiltrating inward throughout the graft.

[0062] Taken together, these findings confirm that immunodeficient rabbits provide a permissive in vivo microenvironment that (i) prevents xenograft rejection, (ii) supports folliculogenesis to the metaphase II stage, and (iii) promotes robust host-graft vascular integration. This dual vascularization not only sustains graft viability but also enables reliable harvesting of mature human oocytes for downstream applications such as in vitro fertilization or fertility restoration.

[0063] Hence, in accordance with some aspects, the present disclosure provides a method for xenotransplanting an ovarian tissue or any fragment thereof into a non-human mammalian animal, the method comprising transplanting an ovarian tissue of a first mammalian animal into at least one recipient site of a second mammalian animal, wherein said first mammalian animal and said second mammalian animal are different animal species.

[0064] In some examples, the first mammalian animal is a human female.

[0065] In some examples the second mammalian animal is a rabbit. In some examples the second mammalian animal is an immunodeficient rabbit. In accordance with some aspects, the present disclosure provides a method for producing mature oocytes, the method comprising transplanting an ovarian tissue or any fragment thereof of a human female into at least one recipient site of an immunodeficient rabbit, and collecting mature oocytes from the rabbit.

[0066] In accordance with some aspects, the present disclosure provides a method for producing mature oocytes, the method comprising transplanting an ovarian tissue or any fragment thereof of a human female into at least one recipient site of an immunodeficient rabbit, and collecting mature oocytes from the transplanted ovarian tissue.

[0067] In some examples which may be considered as aspects of the invention, the method comprises the steps of transplanting an ovarian tissue or any fragment thereof of a human female into at least one recipient site an immunodeficient rabbit, and providing conditions for follicles development and oocytes maturation in the transplanted ovarian tissue in the rabbit. In some examples, the method comprises collecting mature oocytes from the transplanted ovarian tissue.

[0068] In accordance with some aspects, the present disclosure provides a method for transplanting an ovarian tissue of a human female into at least one recipient site of an immunodeficient rabbit.

[0069] In some embodiments, the method comprising transplanting an ovarian tissue or any fragment (section thereof).

[0070] The ovarian tissue or any fragment thereof may be transplanted into one or more sites in the immunodeficient rabbit that would support transplantation, follicles development and oocytes maturation (denoted herein recipient site).

[0071] As used herein, the term "recipient site” refers to any anatomical location within the rabbit where transplantation of ovarian tissue is performed to support graft survival, follicular development, and oocyte maturation.

[0072] In some embodiments, the recipient site in the rabbit is one or more of back region, abdominal area, intra peritoneal, a kidney capsule or a combination thereof.

[0073] In some embodiments, the recipient site in the rabbit is one or more of back region, abdominal area (subcutaneous, intramuscular), intra peritoneal or a combination thereof. In some embodiments, the recipient site in the rabbit is one or more of a subcutaneous site (e.g., back region, abdominal area), an intramuscular site, an intraperitoneal site, a renal capsule site, an omentum site, a subrenal adipose tissue site, a mammary fat pad site, or a combination thereof.

[0074] The method comprises monitoring transplant acceptance of the ovarian tissue in the immunodeficient rabbit.

[0075] Transplant acceptance (or “acceptance of the transplanted tissue”), as used herein, refers to a state in which the xenografted ovarian tissue is tolerated by the host (rabbit) without immunological rejection and achieves functional integration.

[0076] In the context of the claimed methods, and in some examples, transplant acceptance is evidenced by one or more of the following criteria: (1) absence of host immune- mediated damage as may be determined by lack of inflammatory cell infiltrates (e.g., no peri-vascular lymphocyte cuffs or macrophage accumulation) in and around the graft and / or no histological signs of tissue necrosis or endothelial apoptosis within the transplanted tissue; (2) structural integrity of the graft as may be determined retention of normal ovarian cortical architecture and follicular morphology (i.e., intact primordial, primary, and growing follicles) and / or maintenance of stromal and extracellular-matrix frameworks without fibrotic scarring; (3) bidirectional vascular integration as may be determined host-to-graft: Infiltration of host (rabbit) blood vessels throughout the human ovarian tissue, demonstrated by staining for a rabbit-specific endothelial marker within the graft (e.g., rabbit CD31+lumens) and / or graft-to-host: Sprouting of human-derived vessels outward into host stroma, shown by human-specific endothelial markers (e.g., hCD31+vessels) extending beyond the graft margin; (4) functional viability as may be determined sustained oocyte viability and progression to metaphase II (Mil), with measurable retrieval of mature oocytes and / or hormonal or paracrine activity indicative of folliculogenesis (e.g., detectable estradiol or AMH levels in host serum consistent with follicle growth).

[0077] In some examples, monitoring transplant acceptance may by assessing one of more of (i) oocyte viability, (ii) ovarian follicle count, (iii) presence of human-derived blood vessels, (iv) presence of rabbit-derived blood vessels within the transplant, or (v) any combination thereof. In some examples, the method comprises a step of providing conditions for follicles development and oocytes maturation.

[0078] In some examples, the method comprises a step of providing conditions for follicles development and oocytes maturation after the step of transplanting an ovarian tissue of a human female into at least one recipient site of an immunodeficient rabbit.

[0079] In accordance with some other aspects, the present disclosure provides a method for producing mature oocytes comprising:

[0080] (a) transplanting an ovarian tissue of a first mammalian animal into at least one recipient site of a second mammalian animal, wherein said first mammalian animal and said second mammalian animal are different animal species,

[0081] (b) providing conditions for follicles development and oocytes maturation in said transplanted ovarian tissue, and

[0082] (c) collecting said mature oocytes from said transplant ovarian tissue.

[0083] In some examples, the step of providing conditions for follicles development and oocytes maturation in said transplanted ovarian tissue comprise administration to the rabbit of at least one reproductive hormone.

[0084] In accordance with some aspects, the present disclosure provides a method for inducing a human endocrine environment in a non-human mammal, the method comprising administering to the immunodeficient rabbit an effective amount of at least one reproductive hormone, to thereby induce a serum concentration of the at least one reproductive hormone in the immunodeficient rabbit of at least about 8 IU. As described herein, the method comprises a step of providing conditions for follicles development and oocytes maturation in a transplanted ovarian tissue.

[0085] In some embodiments, the conditions comprise administering a hormonal treatment to the immunodeficient rabbit.

[0086] As used herein, “hormonal treatment” or “hormonal conditioning” refers to the administration of one or more exogenous hormones to a recipient animal to promote, enhance, or regulate the growth, maturation, and / or survival of ovarian follicles and oocytes within transplanted ovarian tissue. Hormonal conditioning may include the administration of gonadotropins such as Follicle-Stimulating Hormone (FSH), Luteinizing Hormone (LH), or human menopausal gonadotropin (hMG), as well as steroid hormones such as estradiol or progesterone, alone or in combination, at one or more time points. Hormonal conditioning may further involve the use of long -acting hormone analogs (e.g., corifollitropin alfa) or recombinant hormone preparations (e.g., recombinant FSH or recombinant LH).

[0087] It should be noted that hormonal treatment may refer to the composition and kits of the present disclosure.

[0088] In some examples, the reproductive hormone induces in the immunodeficient rabbit an endocrine profile that is comparable to a hormonal profile during IVF stimulation in human females.

[0089] As used herein, the term "reproductive hormone” or "hormone" or "hormonal agent” refers to an active molecule capable of modulating one or more reproductive functions. The modulation may be a direct effect or an indirect effect.

[0090] In some examples, the reproductive hormone is one or more of a natural hormone, a recombinant hormone, a synthetic hormone or any combination thereof.

[0091] In some examples, the reproductive hormone is a natural hormone. In some examples, the reproductive hormone is of urinary origin, a placental origin or a pituitary origin. In some examples, the reproductive hormone is of urinary origin.

[0092] In some examples, the reproductive hormone is one or more of a peptide, a protein, a steroid, a glycoprotein, a small molecule, a small molecule analogue or any combination thereof.

[0093] Non-limiting examples include one or more gonadotropins (e.g., FSH, LH, hMG) and / or one or more steroid hormones (e.g., estradiol, progesterone).

[0094] As used herein, "gonadotropin hormone” (or “gonadotrophin”) refers to a biologically active glycoprotein heterodimer composed of a common a-subunit 92 amino acids) and a hormone-specific P-subunit, wherein the -subunit confers selective binding to a follicle-stimulating hormone receptor (FSHR) or luteinising hormone / choriogonadotropin receptor (LHCGR). Gonadotropins are hormones secreted by gonadotropic cells of the anterior pituitary of vertebrates and are considered central to the complex endocrine system regulating normal growth, sexual development, and reproductive function.

[0095] This hormone family includes the mammalian hormones follicle-stimulating hormone (FSH) and luteinizing hormone (LH), human chorionic gonadotropin (hCG) and equine chorionic gonadotropin (eCG), as well as at least two forms of fish gonadotropins.

[0096] In some embodiments, the gonadotropin hormone is selected from the group consisting of follicle-stimulating hormone (FSH), luteinising hormone (LH), human menopausal gonadotropin (hMG), chorionic gonadotropin, piscine gonadotropin or any combination thereof.

[0097] In some examples, the chorionic gonadotropins is one or more of human chorionic gonadotropin (hCG), equine chorionic gonadotropin (eCG, pregnant-mare-serum gonadotropin (PMSG)) or any combination thereof.

[0098] In some examples, the fiscine gonadotropin is one or more of fish FSH, fish LH; or any combination thereof.

[0099] In some embodiments, the gonadotropin hormone is at least one of follicle- stimulating hormone (FSH), luteinizing hormone (LH), human menopausal gonadotropin (hMG), placental / chorionic gonadotropin, human chorionic gonadotropin (hCG), equine chorionic gonadotropin (eCG), as well as at least two forms of fish gonadotropins or any combination thereof.

[0100] LH and FSH are secreted by the anterior pituitary gland, while hCG and eCG are secreted by the placenta in pregnant women and mares, respectively. The gonadotropins act on the gonads, controlling gamete and sex hormone production.

[0101] In some examples, the at least one reproductive hormone is at least one of human follicle-stimulating hormone (FSH), human luteinising hormone (LH) and / or human menopausal gonadotrophin (hMG).

[0102] In some embodiments, the reproductive hormone may be at least one of follicle- stimulating hormone (FSH), luteinizing hormone (LH), or human menopausal gonadotropin (hMG), as well as steroid hormones such as estradiol or progesterone.

[0103] In some examples, the reproductive hormone is or comprises FSH. FSH is one of the two pituitary and is produced by gonadotroph cells in the anterior pituitary and released into the bloodstream under the control of hypothalamic GnRH and gonadal feedback (inhibin, estradiol, testosterone). Functionally it drives growth and maturation of ovarian follicles in females and supports Sertoli-cell-mediated spermatogenesis in males.

[0104] In some examples which may be considered as aspects of the invention, the method comprises administration of FSH at a concentration of between about 10 IU and about 100 IU, at times between about 10 IU and about 90IU, at times between about 10 IU and about 80IU, at times between about 10 IU and about 60IU, at times between about 12.5 IU and about 90IU, at times between about 20 IU and about 75 IU, at times between about 30 IU and about 60 IU.

[0105] In some examples which may be considered as aspects of the invention, the method comprises administration of FSH at a concentration of between about 10 IU to about 60 IU. In some examples which may be considered as aspects of the invention, the method comprises administration of FSH at a concentration of between about 12.5 IU to about 50 IU. In some examples which may be considered as aspects of the invention, the method comprises administration FSH of between about 25 IU and about 75 IU. In some examples which may be considered as aspects of the invention, the method comprises administration of FSH at a concentration of between about 30 IU and about 60 IU. In some examples which may be considered as aspects of the invention, the method comprises administration of FSH at a concentration of between 35 IU and 50 IU. In some examples which may be considered as aspects of the invention, the method comprises administration of FSH at a concentration of between 40 IU and 50 IU.

[0106] In some examples which may be considered as aspects of the invention, the method comprises administration of FSH at a concentration of 12.5 IU. In some examples which may be considered as aspects of the invention, the method comprises administration of FSH at a concentration of 25 IU. In some examples which may be considered as aspects of the invention, the method comprises administration of FSH at a concentration of 37.5 IU. In some examples which may be considered as aspects of the invention, the method comprises administration of FSH at a concentration of 50 IU. As used herein, "FSH" encompasses naturally derived or recombinant follicle- stimulating hormone and functional analogues thereof. It should be noted that the FSH as used herein binds and activates the FSH receptor.

[0107] In some examples, FSH is follitropin alfa, follitropin beta, urofollitropin, corifollitropin alfa, follitropin delta or any combination thereof.

[0108] In some examples, FSH is a recombinant FSH. In some examples, FSH is a recombinant FSH from Chinese-hamster-ovary (CHO) cells.

[0109] In some examples, FSH is a FSH preparation.

[0110] In some examples, FSH is follitropin alfa. Follitropin alfa refers to a recombinant human FSH (rhFSH) expressed in serum-free CHO cells, enriched in a2,3- and a2,6- linked sialylated N-glycans. Follitropin alfa may be supplied as pre-filled syringes or pens (75-450 IU, e.g., and is commercially available under the trade name Gonal-F®, Bemfola®, Ovaleap®).

[0111] In some examples, FSH is follitropin beta. Follitropin beta is an rhFSH produced in CHO cells and bearing a lower sialylation. Follitropin beta may be commercially available under the trade name Puregon® / Follistim® AQ.

[0112] In some examples, FSH is urofollitropin. Urofollitropin is a highly purified urinary FSH obtained from the urine of post-menopausal women. Urofollitropin contain < 1 IU UH or hCG per 75 IU FSH and may be formulated as lyophilised vials (75 or 150 IU). Urofollitropin Bravelle®, Fostimon®).

[0113] In some examples, FSH is corifollitropin alfa. Corifollitropin alfa is a rhFSH analogue in which the FSH -subunit is fused to the 28-residue C-terminal peptide of hCG, conferring a markedly increased sialic -acid content. Corifollitropin alfa may be commercially available under the trade name Elonva®.

[0114] Unless otherwise specified, the term “FSH preparation” further includes pharmaceutically acceptable salts, solvents, sustained-release depots, biosimilar or glycoengineered variants of the foregoing preparations, and any combination thereof.

[0115] As noted above, the various FSH preparations differ principally in their glycosylation patterns and structural modifications, and those differences translate directly into distinct pharmacokinetic behaviors. Preparations with a higher degree of sialylation or with C-terminal peptide fusions (e.g., corifollitropin alfa) exhibit reduced renal and hepatic clearance, giving an extended elimination half-life and a functionally sustained-release profde that permits once-weekly — or even less frequent — administration. By contrast, less sialylated forms such as follitropin beta are cleared slightly faster, necessitating standard daily dosing during controlled ovarian stimulation, while highly purified urinary FSH (urofollitropin) closely parallels the pharmacokinetics of recombinant follitropin alfa. Accordingly, when the present disclosure refers to an “FSH preparation,” it is intended to encompass this spectrum of molecules — ranging from conventional daily-dosing formulations to long-acting, extended-half-life analogues — together with any pharmaceutically acceptable salts, depot compositions, or glycoengineered variants that achieve a comparable or prolonged systemic exposure.

[0116] As used herein, a "sustained-release hormone" refers to a hormone or hormone formulation capable of maintaining elevated serum hormone levels for an extended period following a single administration, typically sustaining a biological effect. The sustained biological effect may be observed for at least one day, at least two days, at least three days, at least four days, at least five days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 15 days, at least 20 days, at least 25 days.

[0117] In some embodiments, the sustained-release hormone is a long-acting recombinant FSH analog. A "long -acting recombinant FSH analog" refers to a modified FSH molecule engineered to exhibit an extended serum half-life compared to native or recombinant FSH, such that a single administration results in prolonged stimulation of follicular development without the need for daily injections. Long-acting recombinant FSH analogs typically include structural modifications, such as the addition of the C-terminal peptide (CTP) of human chorionic gonadotropin (hCG), which prolongs their pharmacokinetic profile by reducing clearance.

[0118] In some specific embodiments, the sustained-release hormone is corifollitropin alfa.

[0119] In some specific embodiments which may be considered as aspects of the invention, In some examples which may be considered as aspects of the invention, the method comprises administration of a sustained-release hormone. In some specific embodiments which may be considered as aspects of the invention, In some examples which may be considered as aspects of the invention, the method comprises administration of corifollitropin alfa. corifollitropin alfa may be commercially available under the trade name Elonva®. Corifollitropin alfa is a recombinant fusion protein comprising the alpha-subunit of human FSH and a beta-subunit modified by fusion with the C-terminal peptide of hCG.

[0120] As shown in Examples IE and IF, administration of Elonva to rabbits resulted in sustained elevation of serum FSH levels for at least 8 to 12 days following a single injection, confirming its sustained-release properties and supporting its use in compositions designed to mimic the endocrine profile observed during human IVF stimulation.

[0121] In some examples which may be considered as aspects of the invention, the method comprises administration of a long-acting recombinant follicle-stimulating hormone (FSH) analog at a concentration of about 40 IU.

[0122] In some examples which may be considered as aspects of the invention, the method comprises administration of a long-acting recombinant FSH analog at a concentration of between about 30 IU and about 100 IU. In some examples which may be considered as aspects of the invention, the method comprises administration of a long-acting recombinant FSH analog at a concentration of between about 30 IU and about 80 IU. In some examples which may be considered as aspects of the invention, the method comprises administration of a long-acting recombinant FSH analog at a concentration of between about 35 IU and about 75 IU. In some examples which may be considered as aspects of the invention, the method comprises administration of a long-acting recombinant FSH analog at a concentration of between about 40 IU and about 70 IU. In some examples which may be considered as aspects of the invention, the method comprises administration of a long-acting recombinant FSH analog at a concentration of between about 40 IU and about 60 IU.

[0123] In some examples which may be considered as aspects of the invention, the method comprises administration of FSH as defined herein which provides a sustained elevation of serum FSH levels for at least 8 to 12 days following a single injection. In some examples which may be considered as aspects of the invention, the method comprises administration of a sustained release FSH providing a sustained elevation of serum FSH levels for at least 8 to 12 days following a single injection.

[0124] In some examples, the method comprises administration of LH.

[0125] LH is a heterodimeric glycoprotein consisting of two non-covalently linked peptide chains: a-subunit 92 aa) - identical in sequence to the a-chain of the other glycoprotein hormones (FSH, TSH, hCG) and -subunit 121 aa) - unique to LH and determines its selective binding to the LH / chorionic -gonadotropin receptor. Each subunit carries two N- linked glycans; the total carbohydrate makes up roughly one-third of the ~30 kDa molecular mass and is essential for bioactivity and serum half-life.

[0126] In some examples which may be considered as aspects of the invention, the method comprises administration of LH at a concentration of between about 10 IU and about 100 IU, at times between about 12.5 IU and about 90IU, at times between about 20 IU and about 75 IU, at times between about 30 IU and about 60 IU.

[0127] In some examples which may be considered as aspects of the invention, the method comprises administration of LH at a concentration of between about 25 IU and about 50 IU. In some examples which may be considered as aspects of the invention, the method comprises administration of LH at a concentration of between about 30 IU and about 70 IU. In some embodiments, the method comprises administration of LH at a concentration of between about 40 IU and about 75 IU. In some embodiments, the method comprises administration of LH at a concentration of between about 50 IU and about 75 IU. In some examples, the method comprises administration of LH at a concentration of between about 25 IU and about 75 IU.

[0128] In some embodiments which may be considered as aspects of the invention, the method comprises administration of LH at a concentration of about 25 IU. In some embodiments which may be considered as aspects of the invention the method comprises administration of LH at a concentration of about 50 IU. In some embodiments the method comprises administration of LH at a concentration of about 75 IU.

[0129] In some embodiments, the LH is lutropin alfa. Lutropin alfa is a recombinant human LH produced in Chinese-hamster-ovary (CHO) cells that contains the native 92-residue a-subunit and the LH-specific 121 -residue P-subunit.

[0130] In some embodiments, the LH is lutropin alfa commercially available under the trade name Luveris®.

[0131] In some examples, the method comprises administration of a combination of FSH and LH.

[0132] In some embodiments, the method comprises administration of FSH at a concentration of between about 10 IU and about 100 IU, and LH at a concentration of between about 10 IU and 100 about IU.

[0133] In some embodiments, the method comprises administration of FSH at a concentration of between about 25 IU and about 37.5 IU, and LH at a concentration of between about 25 IU and about 75 IU. In some embodiments, the method comprises administration of FSH at a concentration of between about 20 IU and about 50 IU, and LH at a concentration of between about 20 IU and about 100 IU. In some embodiments, the method comprises administration of FSH at a concentration of between about 25 IU and about 40 IU, and LH at a concentration of between about 40 IU and about 80 IU. In some embodiments, the method comprises administration of FSH at a concentration of between about 30 IU and about 50 IU, and LH at a concentration of between about 50 IU and about 100 IU.

[0134] In some embodiments, the method comprises administration of a combination of FSH at a concentration of 25 IU and LH at a concentration of 75 IU. In some embodiments, the method comprises administration of a combination of FSH at a concentration of 25 IU and LH at a concentration of 25 IU. In some embodiments, the method comprises administration of a combination of FSH at a concentration of 25 IU and LH at a concentration of 50 IU. In some embodiments, the method comprises administration of a combination of FSH at a concentration of 25 IU and LH at a concentration of 75 IU. In some embodiments, the method comprises administration of a combination of FSH at a concentration of 37.5 IU and LH at a concentration of 50 IU. In some embodiments, the method comprises administration of a combination of FSH at a concentration of 37.5 IU and LH at a concentration of 75 IU. In some examples, the method comprises administration of a gonadotropin preparation comprising FSH and LH biological activity.

[0135] As used herein, a "gonadotropin preparation comprising FSH and LH biological activity" refers to a hormone preparation derived from natural, urinary, or recombinant sources, containing biologically active forms of both FSH and LH in a single formulation. Such preparations are used to promote follicular development and maturation by simultaneously providing exogenous FSH and LH support, mimicking the natural hormonal environment necessary for reproductive function.

[0136] In some embodiments, the gonadotropin preparation is a human menopausal gonadotropin (hMG) preparation. Human menopausal gonadotropins are typically purified from the urine of postmenopausal women and contain both FSH and LH bioactivity.

[0137] In some embodiments, the gonadotropin preparation is Menopur®, a commercially available hMG preparation that provides both FSH and LH activity in a balanced ratio. Menopur is commonly used in assisted reproductive technologies (ART) for controlled ovarian stimulation protocols.

[0138] As shown in Example II, administration of Menopur to rabbits at concentrations of 37.5 IU and 50 IU resulted in sustained serum FSH levels above 10 IU / L for up to 48 hours following injection, confirming its effective endocrine activity and supporting its use in compositions designed to simulate the hormonal environment observed during human IVF stimulation.

[0139] In some embodiments, the method comprises administration of a gonadotropin preparation comprising FSH and LH at a concentration of about 37.5 IU. In some embodiments, the method comprises administration of gonadotropin preparation comprising FSH and LH activity at a concentration of about 50 IU. In some embodiments, the method comprises administration of gonadotropin preparation comprising FSH and LH activity at a concentration of between about 30 IU and about 100 IU. In some embodiments, the method comprises administration of a gonadotropin preparation comprising FSH and LH activity at a concentration of between about 30 IU and about 80 IU. In some embodiments, the method comprises administration of a gonadotropin preparation comprising FSH and LH activity at a concentration of between about 35 IU and about 75 IU. In some embodiments, the method comprises administration of a gonadotropin preparation comprising FSH and LH activity at a concentration of between about 40 IU and about 70 IU. In some embodiments, the method comprises administration of a gonadotropin preparation comprising FSH and UH activity at a concentration of between about 40 IU and about 60 IU.

[0140] In some embodiments, the reproductive hormone may be at least one as steroid hormone.

[0141] As used herein, steroid hormones are biologically active organic compounds derived from cholesterol that regulate numerous physiological processes, particularly those related to reproduction. Relevant examples include estradiol, the primary estrogen involved in the regulation of the menstrual cycle and endometrial preparation for implantation, and progesterone, which is essential for endometrial receptivity, maintenance of early pregnancy, and inhibition of uterine contractions.

[0142] In some embodiments, the reproductive hormone may be at least one steroid hormone.

[0143] In some examples, the at least one steroid hormone is one or more of 17p-estradiol, estrone, estriol, progesterone, 17a-hydroxyprogesterone, testosterone, pharmaceutically acceptable esters or salts thereof or any combination thereof.

[0144] In some examples, the at least one steroid hormone is one or more of 17p-estradiol, progesterone or any combination thereof.

[0145] As shown in Example IF, administration of FSH analogs (Gonal-F® and Elonva®) resulted in an endogenous rise in serum progesterone levels in female rabbits, with progesterone levels reaching approximately 9.9 nmol / E after administration of Elonva® and remaining elevated for several days. These observations suggest that the inclusion of progesterone supplementation or modulation in compositions aiming to mimic the endocrine milieu associated with human IVF stimulation.

[0146] In some embodiments, the steroid hormone is progesterone.

[0147] Progesterone is a natural progestogen that binds to the progesterone receptor to exert effects essential for endometrial receptivity and maintenance of pregnancy. In some embodiments, the progesterone is administered in a form such as micronized progesterone (e.g., Utrogestan®, Endometrin®), injectable progesterone, or vaginal progesterone formulations.

[0148] It should be understood that any form of these hormones may be suitable, including natural, recombinant, synthetic, or urinary-derived preparations. For example, recombinant human FSH (r-hFSH, e.g., GONAL-f®), recombinant human LH (r-hLH, e.g., Luveris®), or recombinant human chorionic gonadotropin (r-hCG, e.g., Ovidrel®) may be used. Likewise, urinary-derived preparations such as Menopur® (hMG containing both FSH and LH activity) or purified urinary FSH preparations such as Bravelle® may be employed. With regard to steroid hormones, estradiol may be provided as estradiol valerate (e.g., Progynova®) or micronized estradiol (e.g., Estrace®), and progesterone may be administered as micronized progesterone (e.g., Utrogestan® or Endometrin®) or as progesterone-containing vaginal inserts, gels, or intramuscular injections.

[0149] Hence, in accordance with some aspects, the present disclosure provides a kit comprising an effective amount of at least one reproductive hormone as described herein and instructions for use. The instructions for use of kit include that the kit is configured for use in inducing an endocrine profile in an animal that is comparable to a hormonal profile during human IVF stimulation, optionally wherein the animal is intended for receiving a reproductive tissue xenotransplant, such as a xenotransplant of ovarian tissue.

[0150] As used herein, the term "kit" refers to a packaged set of one or more therapeutic agents, optionally together with one or more pharmaceutically acceptable carriers, diluents, administration devices, or instructions for use, wherein the components are physically or functionally associated and intended for combined use in fertility support, preservation, or restoration procedures. In some embodiments, the kit comprises at least one gonadotropin hormone, at least one steroid hormone, or any combination thereof, as described above. In some embodiments, the kit comprises at least one gonadotropin hormone. In some embodiments, the kit comprises FSH (e.g., Gonal-F®, follitropin alfa). In some embodiments, the kit comprises FSH at a concentration of between about 10 IU and about 100 IU. In some embodiments, the kit comprises FSH at a concentration of about 12.5 IU. In some embodiments, the kit comprises FSH at a concentration of about 37.5 IU. In some embodiments, the kit comprises FSH at a concentration of about 50 IU. In some embodiments, the kit may further comprise:

[0151] - one or more unit dose containers (e.g., vials, pre-fdled syringes, pens, ampoules) containing the hormone (s);

[0152] - one or more administration devices suitable for delivering the hormone(s) (e.g., syringes, injection pens, infusion systems);

[0153] - instructions for use providing guidance on dosing regimens, routes of administration, and timing relative to fertility treatments or xenotransplantation protocols.

[0154] In some embodiments, the hormones comprised in the kit are provided in separate reservoirs or containers, such as separate syringes, vials, cartridges, divided bottles, or divided foil packets, allowing separate storage and administration of the individual hormonal components. In some embodiments, the reproductive hormones are provided in different dosage forms, including solid, liquid, or solution forms. In certain embodiments, the hormones are provided as solid pharmaceutical compositions that may be reconstituted with a suitable solvent prior to administration. In such embodiments, the kit may optionally include a reconstituting solvent. In some embodiments, the components of the kit are intended for simultaneous or sequential administration. When sequential administration is employed, the hormones may be administered in any suitable order according to a prescribed dosing schedule.

[0155] In some embodiments, the instructions for use provided in the kit include guidance on simultaneous or sequential administration, and on any required reconstitution procedures when applicable.

[0156] As described herein, the composition or kit may be used to induce an endocrine profile induced in a host animal that is comparable to a hormonal profile during IVF stimulation in human females. When referring to an endocrine profile induced in a host animal that is comparable to a hormonal profile during IVF stimulation in human females it should be understood as systemic hormonal environment, quantified by circulating concentrations of one or more gonadotropins and / or downstream steroid hormones, as measured in serum or plasma of the subject.

[0157] Unless otherwise specified, an “endocrine profile comparable to that observed during human IVF stimulation” is achieved when one or more of the following is achieved: the serum FSH concentration in the host animal is at least 8IU, at times > 10 IU maintained for a continuous period of at least 1 hour after administration, serum LH concentration in the host animal is > 10 IU L1maintained for a continuous period of at least 1 hour after administration.; and / or progesterone, estradiol, or p-hCG levels in the host animal is within ±30 % of those typically recorded in controlled ovarian stimulation of human IVF patients.

[0158] Concentrations of hormones (for example serum hormone) may be determined by any method known in the art. For example, Hormone levels are determined with a validated immunoassay (e.g., electrochemiluminescence on a COBAS e801 analyzer) calibrated against WHO International Standards. In some examples, the concentrations of the hormones may be determined as described herein in the Examples below.

[0159] It was suggested that inducing an endocrine profile in a host animal that is comparable to a hormonal profile during IVF stimulation in human females is a step in the methods described herein for xenotransplantation of ovarian cortex tissue, optionally frozen-thawed ovarian cortex tissue.

[0160] Hence, the methods described herein comprises a step of providing conditions for follicles development and oocytes maturation in a transplanted ovarian tissue.

[0161] As used herein, providing conditions includes, in some embodiments, inducing one or more hormonal conditions in a non-human mammalian recipient into which ovarian tissue (or a fragment thereof) has been, or is intended to be, transplanted.

[0162] As mentioned above, it should be understood that any form of these hormones may be employed, including natural, recombinant, synthetic, or urinary-derived preparations. For example, recombinant human FSH (r-hFSH, e.g., GONAL-f®), recombinant human LH (r-hLH, e.g., Luveris®), or recombinant human chorionic gonadotropin (r-hCG, e.g., Ovidrel®) may be used. Likewise, urinary-derived preparations such as Menopur® (hMG containing both FSH and LH activity) or purified urinary FSH preparations such as Bravelle® may be employed. With regard to steroid hormones, estradiol may be provided as estradiol valerate (e.g., Progynova®) or micronized estradiol (e.g., Estrace®), and progesterone may be administered as micronized progesterone (e.g., Utrogestan® or Endometrin®) or as progesterone-containing vaginal inserts, gels, or intramuscular injections. In some embodiments, the hormonal treatment comprises administering at least one of a gonadotropin preparation (e.g., human menopausal gonadotropin such as Menopur), a follicle-stimulating hormone preparation (e.g., Gonal-F or long-acting corifollitropin alfa such as Elonva), a luteinizing hormone preparation (e.g., recombinant LH such as Luveris), estradiol, progesterone, or any combination thereof.

[0163] In some embodiments, the hormonal conditioning comprises administration of at least one hormonal agent at a dose effective to promote follicular development, oocyte maturation, or endocrine support in the recipient animal. The dose of the hormonal agent may vary widely depending on the species, size, physiological status of the recipient, and the specific hormonal agent administered.

[0164] In some embodiments, the dose of a gonadotropin (e.g., FSH, LH, hMG) or steroid hormone (e.g., estradiol, progesterone) may range from about 1 IU to about 500 IU per administration. In some embodiments, the dose may range from about 1 IU to about 400 IU, from about 1 IU to about 300 IU, from about 1 IU to about 250 IU, from about 1 IU to about 200 IU, from about 1 IU to about 150 IU, or from about 1 IU to about 100 IU. In some embodiments, the dose may range from about 5 IU to about 250 IU, from about 5 IU to about 200 IU, from about 5 IU to about 150 IU, or from about 5 IU to about 100 IU. In some embodiments, the dose may range from about 10 IU to about 200 IU, from about 10 IU to about 150 IU, from about 10 IU to about 100 IU, or from about 10 IU to about 75 IU. In some embodiments, the dose may range from about 25 IU to about 150 IU, or from about 25 IU to about 100 IU. In some embodiments, the dose may range from about 30 IU to about 80 IU. In some embodiments, the dose may range from about 35 IU to about 70 IU. In some embodiments, the dose may range from about 40 IU to about 60 IU. In some embodiments, the dose is about 25 IU, about 30 IU, about 35 IU, about 37.5 IU, about 40 IU, about 45 IU, about 50 IU, about 55 IU, about 60 IU, about 65 IU, about 70 IU, about 75 IU, about 80 IU, or about 100 IU.

[0165] In some embodiments, the doses are selected to achieve transient or sustained elevations of serum hormone levels above a predefined threshold (e.g., above 10 IU / L for FSH).

[0166] In some embodiments, the method comprises maintaining a serum concentration of the at least one reproductive hormone in the non -human mammal of at least 10 IU. In some embodiments, the method comprises maintaining a serum concentration of the at least one reproductive hormone in the non-human mammal of at least 8 IU for at least 1 hour after administration. In some embodiments, the method comprises maintaining a serum concentration of the at least one reproductive hormone in the non-human mammal of at least 10 IU for at least 1 hour after administration. In some embodiments, the method comprises maintaining a serum concentration of the at least one reproductive hormone in the non-human mammal of at least 10 IU for at least 4 hours after administration, at times at least 8 hours, at times at least 12 hours.

[0167] In accordance with the experimental data described herein, specific doses administered to rabbits include 25 IU, 37.5 IU, and 50 IU of recombinant FSH (Gonal- F®); 25 IU, 50 IU, and 75 IU of recombinant UH (Uuveris®); and 37.5 IU or 50 IU of human menopausal gonadotropin (Menopur®).

[0168] In some specific embodiments, recombinant human FSH (Gonal-F®) is administered at a dose of 25 IU, 37.5 IU, or 50 IU per injection. In some embodiments, recombinant human UH (Uuveris®) is administered at a dose of 25 IU, 50 IU, or 75 IU per injection. In some embodiments, human menopausal gonadotropin (Menopur®) is administered at a dose of 37.5 IU or 50 IU per injection. In some embodiments, long- acting recombinant FSH (Elonva®) is administered at doses corresponding to 40 IU per injection. In some embodiments, the hormones are administered once weekly, or multiple times per week (e.g., on days 1, 2, 3, 4, and 6), as exemplified in the sequential administration protocols detailed in Examples 1H and II.

[0169] In some embodiments, the hormonal treatment may be administered to the recipient in sequential steps. Sequential hormonal administration protocols, including administration on multiple days within a week or combinations of different gonadotropins and / or steroid hormones over successive weeks, are supported by the examples described herein, e.g. in the sequential administration of Gonal-F and Luveris (Example 1H, Figure 8) and Menopur

[0170] In some embodiments, the hormonal treatment comprises sequential administration of one or more hormonal agents to the recipient animal over an extended period ranging from at least two weeks to about one month, or even up to two months. In some embodiments, the sequential administration includes repeated administration of the same hormonal agent, or alternating administration of different hormonal agents, such as gonadotropins (e.g., FSH, LH, hMG), steroid hormones (e.g., estradiol, progesterone), or combinations thereof.

[0171] In some embodiments, the administration may involve periodic injections on selected days within each week, continuous administration over multiple days, or intermittent administration with defined rest periods between treatment cycles.

[0172] In some embodiments, different hormonal agents may be administered simultaneously (co-administration) or sequentially (staggered administration) across different time points (e.g. to mimic physiological endocrine patterns or to optimize follicular development and oocyte maturation).

[0173] In some embodiments, the frequency, dose, and type of hormonal agent administered may be adjusted dynamically based on the stage of follicular development, the hormonal levels measured in the recipient, or other physiological parameters.

[0174] In some embodiments, sequential administration schemes include one or more phases of stimulation followed by rest phases (e.g. with the goal of sustaining optimal follicular growth, promoting oocyte maturation, or supporting endocrine function of the grafted ovarian tissue).

[0175] In some specific embodiments, the sequential administration comprises administering a first gonadotropin during a first week, optionally followed by a rest period, and subsequently administering a combination of gonadotropins during one or more additional weeks. For example, in some embodiments, a first administration of recombinant follicle-stimulating hormone (r-hFSH; Gonal-F®) is performed during the first week (e.g., on day 1), followed by a rest period of about 10 days, and then coadministration of r-hFSH (Gonal-F®) and recombinant luteinizing hormone (r-hLH; Luveris®) during a second week, optionally followed by a rest period of about 4 days, and further co-administration of r-hFSH and r-hLH during a third week.

[0176] In some other specific embodiments, the sequential administration comprises repeated administration of a gonadotropin preparation containing both FSH and LH activity (e.g., human menopausal gonadotropin, Menopur®) across at least four weeks, such as administration on days 1, 2, 3, 4, and 6 of each week. In some further embodiments, the hormonal treatment is continued beyond three weeks, optionally including additional weekly cycles of administration with or without rest periods.

[0177] In some embodiments, the doses, frequency, and combinations of administered hormones may be adjusted for maintaining serum human FSH levels, human serum P- hCG levels or progesterone levels above a certain relevant level supporting follicular development, oocyte maturation, endocrine activity and / or survival of grafted ovarian tissue. In some embodiments, the doses, frequency, and combinations of administered hormones may be adjusted for maintaining serum human FSH levels, human serum P- hCG levels or progesterone levels to human IVF-equi valent levels.

[0178] In some embodiments, the hormonal treatment is administered in an amount effective to maintain human serum FSH levels above about 8IUm, at times above about 10 IU / L and / or human serum p-hCG levels above about 2 IU / L in the recipient animal.

[0179] The hormonal agent may be administered in an effective amount. The effective amount as used herein is sufficient to achieve a desired biological effect in the recipient animal. In some embodiments, an effective amount is an amount sufficient to maintain human serum FSH levels above about 10 IU / L and / or human serum P-hCG levels above about 2 IU / L following administration. In some embodiments, an effective amount is sufficient to promote follicular development, support oocyte maturation, stimulate endocrine activity of grafted ovarian tissue, or sustain hormonal profiles conducive to reproductive tissue function and survival.

[0180] As described herein, xenotransplanting an ovarian tissue or any fragment thereof from a human subject into an immunodeficient rabbit is suggested to provide mature oocytes.

[0181] In accordance with some aspects, the present disclosure provides a method for producing mature oocytes.

[0182] In some examples, the method comprises removing an ovarian tissue or a fragment thereof from a human female.

[0183] In some embodiments, the method comprises a step of obtaining an ovarian tissue from the human female. In some embodiments, the method comprises obtaining the ovarian tissue from the human female. In some embodiments the method comprising obtaining an ovarian tissue from the human female.

[0184] When referring to “obtained” or “obtaining” or "removing" and the like an ovarian tissue it should be understood as a procedure in which an ovarian tissue has been taken / excised / removed from a female. The ovarian tissue may be taken / excised / removed from the first mammalian subject by any method known in the art. For example, an ovarian tissue or any part thereof can be taken / excised / removed during a surgery.

[0185] The ovarian tissue taken / excised / removed from the human female, as described herein may be manipulated / treated before transplanted into the second mammalian animal.

[0186] In some embodiments, the ovarian tissue removed from the human female may be sectioned (cut into sections / fragments) prior to transplantation. In some embodiments, the method comprises cutting the ovarian tissue into fragments having a size suitable to allow transplantation. In some embodiments, the method comprises cutting ovarian tissue to be transplanted to dimensions of between about 0.1cm and about 2cm in a first dimension and about 0.1cm and about 2cm in a second dimension, at times between about 0.3cm and about 1.5cm in a first dimension and about 0.3cm and about 1.5cm in a second dimension, at times between about 0.5cm and about 1cm in a first dimension and about 0.5cm and about 1cm in a second dimension.

[0187] In some embodiments, the method comprises cutting ovarian tissue to be transplanted to dimensions of about 0.5cm in a first dimension and about 0.5cm in a second dimension. In some embodiments, the method comprises cutting ovarian tissue to be transplanted to dimensions of about 0.5cm in a first dimension and about 1cm in a second dimension.

[0188] In some embodiments, the ovarian tissue has a size suitable to allow transplantation. In some embodiments, the ovarian tissue to be transplanted has dimensions of between about 0.1cm and about 2cm in a first dimension and about 0.1cm and about 2cm in a second dimension, at times between about 0.3cm and about 1.5cm in a first dimension and about 0.3cm and about 1.5cm in a second dimension, at times between about 0.5cm and about 1cm in a first dimension and about 0.5cm and about 1cm in a second dimension. In some embodiments, the ovarian tissue to be transplanted has dimensions of about 0.5cm in a first dimension and about 0.5cm in a second dimension. In some embodiments, the ovarian tissue to be transplanted has dimensions of about 0.5cm in a first dimension and about 1cm in a second dimension.

[0189] In some embodiments, the method comprising cryopreservation the ovarian tissue prior to transplantation. In some embodiments, the method comprising cryopreservation and thawing the ovarian tissue prior to transplantation.

[0190] The term ovarian tissue encompasses specific types of cells and structures including germ cells, follicles such as primordial follicles, and primary and secondary follicles. In some embodiments, the ovarian tissue is an ovarian cortex tissue. The ovarian cortex as used herein refers to the outermost layer of the ovary, predominantly composed of dense connective tissue and is where the ovarian follicles are located.

[0191] In some embodiments, the ovarian tissue is an ovarian cortex tissue.

[0192] The term ovarian tissue as used herein encompasses an ovarian tissue and any fragment (section) thereof.

[0193] In some embodiments, the ovarian tissue is frozen-thawed ovarian cortex (i.e. ovarian cortex that has been cryopreserved and thawed prior to transplantation).

[0194] In some embodiments, the ovarian tissue is fresh ovarian cortex.

[0195] In some embodiments, the ovarian tissue is full ovarian tissue slices (i.e. including medullary and cortical regions, not only cortex).

[0196] In some embodiments, the human female is in the reproductive years.

[0197] In some embodiments, the human female subject may be at any age ranging between 17-50 years, 17-25, 25-30. 30-35, 35-40, 40-45, 45-50 or older than 50-years.

[0198] In some embodiments, the human female subject is in the reproductive years. The term reproductive years refers in general to the age prior to menopause.

[0199] In some embodiments, the human female subject is suffering from a genetic abnormality or a non-genetic abnormality. In some embodiments, the non-genetic abnormality is at least one of an autoimmune disorder, a metabolic disorder, an infection, an environmental factor, or any combinations thereof. Fertility preservation has become an important component in the management of young cancer patients. Among the currently available clinical procedures, ovarian tissue cry opreservation and transplantation (OTCT) may be effective, yet, it is often not applicable for some specific groups of patients such as caner patients, including, leukemia patients, due to the potential risk malignant cells in the tissue (optionally cryopreserved) that could lead to recurrence of the primary disease after reimplantation of malignancy reintroduction on transplantation.

[0200] In some embodiments, the human female subject is diagnosed or classified with premature ovarian failure.

[0201] In some embodiments, the human female subj ect is diagnosed or classified as a poor ovarian responder (POR).

[0202] Poor ovarian responder (POR) as used herein refers to an individual female subject whose ovaries respond insufficiently to controlled ovarian hyperstimulation.

[0203] In some embodiments, the human female subject is diagnosed or classified as a diminished ovarian reserve (DOR).

[0204] Diminished ovarian reserve (DOR) as used herein refers to a condition where the ability of the ovary to produce eggs is reduced, optionally due to lower quantity and / or quality of remaining oocytes (egg cells).

[0205] In some embodiment, the human female subject is or was diagnosed with cancer.

[0206] As used herein to describe the present invention, “proliferative disorder”, “cancer”, “tumor” and “malignancy” all relate equivalently to a hyperplasia of a tissue or organ. If the tissue is a part of the lymphatic or immune systems, malignant cells may include non-solid tumors of circulating cells. Malignancies of other tissues or organs may produce solid tumors. In general, the methods, compositions and kits of the present invention may be applicable for a patient suffering from any one of non-solid and solid tumors. Specifically Cancer as defined herein is a disorder displaying cell division and growth that is not part of normal cellular turnover, metabolism, growth, or propagation of the whole organism. Unwanted proliferation of cells is seen in tumors and other pathological proliferation of cells, does not serve normal function, and for the most part will continue unbridled at a growth rate exceeding that of cells of a normal tissue in the absence of outside intervention. In accordance with some examples, the caner is at least one of hematological malignancies (including lymphoma, leukemia, myeloproliferative disorders, Acute lymphoblastic leukemia; Acute myeloid leukemia), hypoplastic and aplastic anemia (both virally induced and idiopathic), myelodysplastic syndromes, all types of paraneoplastic syndromes (both immune mediated and idiopathic) and solid tumors (including GI tract, colon, lung, liver, breast, prostate, pancreas and Kaposi's sarcoma. The invention may be applicable as well for the treatment or inhibition of solid tumors such as tumors in lip and oral cavity, pharynx, larynx, paranasal sinuses, major salivary glands, thyroid gland, esophagus, stomach, small intestine, colon, colorectum, anal canal, liver, gallbladder, extrahepatic bile ducts, ampulla of Vater, exocrine pancreas, lung, pleural mesothelioma, bone, soft tissue sarcoma, carcinoma and malignant melanoma of the skin, breast, vulva, vagina, cervix uteri, corpus uteri, ovary, fallopian tube, gestational trophoblastic tumors, penis, prostate, testis, kidney, renal pelvis, ureter, urinary bladder, urethra, carcinoma of the eyelid, carcinoma of the conjunctiva, malignant melanoma of the conjunctiva, malignant melanoma of the uvea, retinoblastoma, carcinoma of the lacrimal gland, sarcoma of the orbit, brain, spinal cord, vascular system, hemangiosarcoma, Adrenocortical carcinoma; AIDS-related cancers; AIDS-related lymphoma; Anal cancer; Appendix cancer; Astrocytoma, childhood cerebellar or cerebral; Basal cell carcinoma; Bile duct cancer, extrahepatic; Bladder cancer; Bone cancer, Osteosarcoma / Malignant fibrous histiocytoma; Brainstem glioma; Brain tumor; Brain tumor, cerebellar astrocytoma; Brain tumor, cerebral astrocytoma / malignant glioma; Brain tumor, ependymoma; Brain tumor, medulloblastoma; Brain tumor, supratentorial primitive neuroectodermal tumors; Brain tumor, visual pathway and hypothalamic glioma; Breast cancer; Bronchial adenomas / carcinoids; Burkitt lymphoma; Carcinoid tumor, childhood; Carcinoid tumor, gastrointestinal; Carcinoma of unknown primary; Central nervous system lymphoma, primary; Cerebellar astrocytoma, childhood; Cerebral astrocytoma / Malignant glioma, childhood; Cervical cancer; Childhood cancers; Chronic lymphocytic leukemia; Chronic myelogenous leukemia; Chronic myeloproliferative disorders; Colon Cancer; Cutaneous T-cell lymphoma; Desmoplastic small round cell tumor; Endometrial cancer; Ependymoma; Esophageal cancer; Ewing's sarcoma in the Ewing family of tumors; Extracranial germ cell tumor, Childhood; Extragonadal Germ cell tumor; Extrahepatic bile duct cancer; Eye Cancer, Intraocular melanoma; Eye Cancer, Retinoblastoma; Gallbladder cancer; Gastric (Stomach) cancer; Gastrointestinal Carcinoid Tumor; Gastrointestinal stromal tumor (GIST); Germ cell tumor: extracranial, extragonadal, or ovarian; Gestational trophoblastic tumor; Glioma of the brain stem; Glioma, Childhood Cerebral Astrocytoma; Glioma, Childhood Visual Pathway and Hypothalamic; Gastric carcinoid; Hairy cell leukemia; Head and neck cancer; Heart cancer; Hepatocellular (liver) cancer; Hodgkin lymphoma; Hypopharyngeal cancer; Hypothalamic and visual pathway glioma, childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi sarcoma; Kidney cancer (renal cell cancer); Laryngeal Cancer; Leukemias; Leukemia, acute lymphoblastic (also called acute lymphocytic leukemia); Leukemia, acute myeloid (also called acute myelogenous leukemia); Leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia); Leukemia, chronic myelogenous (also called chronic myeloid leukemia); Leukemia, hairy cell; Lip and Oral Cavity Cancer; Liver Cancer (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphomas; Lymphoma, AIDS-related; Lymphoma, Burkitt; Lymphoma, cutaneous T-Cell; Lymphoma, Hodgkin; Lymphomas, Non- Hodgkin (an old classification of all lymphomas except Hodgkin's); Lymphoma, Primary Central Nervous System; Marcus Whittle, Deadly Disease; Macroglobulinemia, Waldenstrom; Malignant Librous Histiocytoma of Bone / Osteosarcoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular (Eye); Merkel Cell Carcinoma; Mesothelioma, Adult Malignant; Mesothelioma, Childhood; Metastatic Squamous Neck Cancer with Occult Primary; Mouth Cancer; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma / Plasma Cell Neoplasm; Mycosis Lungoides; Myelodysplastic Syndromes; Myelodysplastic / Myeloproliferative Diseases; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Adult Acute; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple (Cancer of the Bone-Marrow); Myeloproliferative Disorders, Chronic; Nasal cavity and paranasal sinus cancer; Nasopharyngeal carcinoma; Neuroblastoma; NonHodgkin lymphoma; Non-small cell lung cancer; Oral Cancer; Oropharyngeal cancer; Osteosarcoma / malignant fibrous histiocytoma of bone; Ovarian cancer; Ovarian epithelial cancer (Surface epithelial-stromal tumor); Ovarian germ cell tumor; Ovarian low malignant potential tumor; Pancreatic cancer; Pancreatic cancer, islet cell; Paranasal sinus and nasal cavity cancer; Parathyroid cancer; Penile cancer; Pharyngeal cancer; Pheochromocytoma; Pineal astrocytoma; Pineal germinoma; Pineoblastoma and supratentorial primitive neuroectodermal tumors, childhood; Pituitary adenoma; Plasma cell neoplasia / Multiple myeloma; Pleuropulmonary blastoma; Primary central nervous system lymphoma; Prostate cancer; Rectal cancer; Renal cell carcinoma (kidney cancer); Renal pelvis and ureter, transitional cell cancer; Retinoblastoma; Rhabdomyosarcoma, childhood; Salivary gland cancer; Sarcoma, Ewing family of tumors; Sarcoma, Kaposi; Sarcoma, soft tissue; Sarcoma, uterine; Sezary syndrome; Skin cancer (nonmelanoma); Skin cancer (melanoma); Skin carcinoma, Merkel cell; Small cell lung cancer; Small intestine cancer; Soft tissue sarcoma; Squamous cell carcinoma - see Skin cancer (nonmelanoma); Squamous neck cancer with occult primary, metastatic; Stomach cancer; Supratentorial primitive neuroectodermal tumor, childhood; T-Cell lymphoma, cutaneous (Mycosis Fungoides and Sezary syndrome); Testicular cancer; Throat cancer; Thymoma, childhood; Thymoma and Thymic carcinoma; Thyroid cancer; Thyroid cancer, childhood; Transitional cell cancer of the renal pelvis and ureter; Trophoblastic tumor, gestational; Unknown primary site, carcinoma of, adult; Unknown primary site, cancer of, childhood; Ureter and renal pelvis, transitional cell cancer; Urethral cancer; Uterine cancer, endometrial; Uterine sarcoma; Vaginal cancer; Visual pathway and hypothalamic glioma, childhood; Vulvar cancer; Waldenstrom macroglobulinemia and Wilms tumor (kidney cancer).

[0207] It should be noted that the term cancer when used herein encompasses non-invasive cancer, invasive cancer and metastatic cancer.

[0208] "Non-invasive" cancer is to be understood as a cancer that do not grow into or invade normal tissues within or beyond the primary location, for example the ovary.

[0209] "Invasive cancers" is to be understood as cancer that invades and grows in normal, healthy tissues to form metastasis.

[0210] "Metastatic cancer" or "metastatic status" is to be understood as a cancer that has spread from the place where it first started to another place in the body. Such a tumor formed by metastatic cancer cells is called a metastatic tumor or a metastasis.

[0211] Malignancy encompasses any one of carcinomas, melanomas, lymphomas, leukemias, myeloma and sarcomas.

[0212] Malignancies of tissues or organs may produce solid tumors. If the tissue is a part of the lymphatic or immune systems, malignant cells may include non-solid tumors of circulating cells. In general, the methods of the present invention may be applicable for patients suffering of non-solid tumors as well as of solid tumors. The cancer may be selected from the group consisting of breast cancer, renal cancer, melanoma, lung cancer, glioblastoma, head and neck cancer, prostate cancer, ovarian carcinoma, bladder carcinoma, primary peritoneal carcinomatosis, genitourinary cancer, metastatic peritoneal carcinomatosis, and lymphoma.

[0213] The cancer may be any one of leukemias and lymphoma.

[0214] The cancer may be any one of carcinoma, melanoma, sarcoma, glioma and blastoma.

[0215] The cancer may be a carcinoma. The carcinoma may be an adenocarcinoma, a basal cell carcinoma, or squamous cell carcinoma.

[0216] The cancer may be selected from the group consisting of brain cancer, breast cancer, and lung cancer.

[0217] In some embodiment, the human female subject is or was treated with an anti -cancer treatment.

[0218] In some embodiments, the anti-cancer treatment is at least one of chemotherapy, radiotherapy or a combination thereof. In some embodiments, the anti-cancer treatment is chemotherapy. The side effects of chemotherapy may include short-term and long-term effects and are often associated with reduced fertility.

[0219] In some embodiments, the invention is applicable to human female subject being treated or was treated with an anti-cancer treatment.

[0220] In some embodiments, the invention is applicable to human female subject undergoing treatment with chemotherapy or was treated with chemotherapy.

[0221] The present invention is not related to a specific chemotherapeutic agent and is applicable to various chemotherapeutic agents. In some embodiments, the chemotherapy is at least one of at least one alkylating agent, at least one anti metabolite, at least one anti-tumor antibiotics, at least one Topoisomerase inhibitor, at least one mitotic inhibitor, at least one plant alkaloid or a combination thereof.

[0222] In some embodiments, the anti-cancer treatment is radiotherapy. In other embodiments, the subject is a human female subject undergoing treatment with radiotherapy or was treated with radiotherapy. In some embodiments, the treatment is a gonadotoxic treatment. As used herein the term gonadotoxic treatment refers chemotherapy, radiation, or surgical resection (for treatment of disease or gender affirmation surgery).

[0223] As used herein, an “immunodeficient animal” specifically immunodeficient rabbit refers to a host rabbit that possesses an impaired or absent immune response, thereby reducing or eliminating immune-mediated rejection of transplanted tissues. In some embodiments, the immunodeficient animal includes animals carrying genetic mutations that compromise T-cell function, B-cell function, or both.

[0224] Specific examples of immunodeficient animals used herein include:

[0225] (i) NUDE rabbits, which possess a genetic mutation leading to a lack of thymus and deficient T-cell production;

[0226] (ii) RAG-mutant rabbits, which carry mutations in the Recombination Activating Genes (RAG1 or RAG2), resulting in the absence of functional B-cells and T-cells; and

[0227] (iii) IL2 receptor gamma chain-deficient (IL2RG-deficient) rabbits, characterized by severe combined immunodeficiency (SCID) due to impaired signaling necessary for the development of multiple immune lineages.

[0228] In some embodiments, the receipt site may be prepared / treated / manipulated prior to transplantation, to support transplantation, follicles development and oocytes maturation. In some embodiments, the method comprising preparing the recipient site to enhance follicles development and oocytes maturation. In some embodiments, preparing the recipient site comprises enriching the recipient site with at least one material that promotes follicles development and oocytes maturation. In some embodiments, the material is at least one hormone. In some other embodiments, the material suppresses cellular and humoral immune responses in the second mammalian animal.

[0229] In some embodiments, the immunodeficient rabbit should undergo oophorectomy or castration.

[0230] Oophorectomy refers to the surgical excision of one or both ovaries in a female mammalian animal, while castration denotes the surgical removal of the testes in a male mammalian animal. These procedures are employed to eliminate endogenous gonadal hormone production, thereby facilitating controlled experimental conditions, particularly in studies involving reproductive physiology, transplantation, or hormone-related research.

[0231] In certain embodiments, a pharmacological hypophysectomy was induced in the recipient animal by administration of a Gonadotropin-Releasing Hormone (GnRH) antagonist (such as Degarelix). Pharmacological hypophysectomy refers to the functional suppression of pituitary gonadotropin secretion without surgical intervention. Degarelix acts by competitively inhibiting the binding of endogenous GnRH to its receptor in the anterior pituitary, thereby preventing the release of luteinizing hormone (LH) and follicle- stimulating hormone (FSH). This approach effectively suppresses endogenous gonadal function, creating a hormonally controlled environment suitable for evaluating the transplantation, survival, and functional integration of reproductive tissues or cells.

[0232] In accordance with some further aspects, the present disclosure provides a method for producing mature oocytes comprising:

[0233] (a) obtaining an ovarian tissue from a human female,

[0234] (b) transplanting the ovarian tissue into at least one recipient site of an immunodeficient rabbit, and

[0235] (c) providing conditions for follicles development and oocytes maturation in the transplanted ovarian tissue.

[0236] The term “mature oocytes” relates an oocyte that has progressed to metaphase II (Mil) of meiosis, characterized by the extrusion of the first polar body, and is capable of being fertilized.

[0237] As used herein, the term “maturated oocytes” relate to oocytes exhibiting a more advanced progression of maturation in comparison with their initial state of maturation before transplantation into the recipient.

[0238] In some embodiments, the maturated oocyte may be at least one of the following maturation stages: Primordial follicle Primary follicle, Secondary follicle (Preantral follicle), early Antral follicle, Antral follicle (late), preovulatory (Graafian) follicle, germinal vesicle (GV) stage oocyte, metaphase I (MI) oocyte and / or metaphase II (Mil) oocyte (mature). Oocyte maturation stages and their markers are detailed below:

[0239] Primordial follicle: Small oocyte surrounded by a single layer of flattened (squamous) granulosa cells. Key molecular markers of primordial follicle are Oocyte markers: VASA (DDX4), OCT4 (POU5F1) and Granulosa cell markers: FOXL2.

[0240] Primary follicle: The oocyte enlarges and is surrounded by a single layer of cuboidal granulosa cells. Granulosa cells become cuboidal; zona pellucida formation starts. Key molecular markers of primary follicles are ZP1, ZP2, ZP3 (zona pellucida proteins) and GDF9 (Growth Differentiation Factor 9).

[0241] Secondary follicle (Preantral follicle): Multiple layers of granulosa cells form, and a theca cell layer appears. Key molecular markers are GDF9, BMP 15 (important for oocyte and granulosa interaction) and AMH (Anti-Mullerian Hormone) in granulosa cells.

[0242] Early Antral follicle: Appearance of small fluid-filled spaces (antrum formation). Key molecular markers are FSH receptor (FSHR) expression increases in granulosa cells and CYP19A1 (aromatase).

[0243] Antral follicle (late): Large central antrum; cumulus granulosa cells surround oocyte; mural granulosa cells line follicular wall. Key molecular markers are LH receptor (LHR) begins to appear in mural granulosa cells and PTGS2 (prostaglandin synthase) in cumulus cells.

[0244] Preovulatory (Graafian) follicle: Fully mature follicle; oocyte resumes meiosis upon LH surge. Key molecular markers are high expression of PTX3 (Pentraxin 3), HAS2 (Hyaluronan Synthase 2), and TNFAIP6 in cumulus cells.

[0245] Germinal Vesicle (GV) stage oocyte: Nucleus (germinal vesicle) visible inside oocyte. Key molecular markers are high expression of PTX3 (Pentraxin 3), HAS2 (Hyaluronan Synthase 2), and TNFAIP6 in cumulus cells.

[0246] Metaphase I (MI) oocyte: Germinal vesicle breaks down (GVBD); no polar body yet. Key molecular markers are reduction of cAMP and activation of MPF (Maturation- Promoting Factor; CDKl / cyclin B complex). Metaphase II (Mil) oocyte (mature): First polar body extruded; oocyte arrested at Mil. Key molecular markers are high activity of MPF, proper spindle formation (detected by tubulin staining) and normal cortical granule distribution (detected by lectin staining).

[0247] In some examples, the method comprises collecting mature oocytes at the metaphase II (Mil) stage.

[0248] In some embodiments, the mature oocytes are collected from the transplant ovarian tissue by any method known in the art.

[0249] In accordance with some further aspects, the present disclosure provides a method for producing mature oocytes comprising:

[0250] (a) obtaining an ovarian tissue from a human female,

[0251] (b) transplanting the ovarian tissue into at least one recipient site of an immunodeficient rabbit,

[0252] (c) providing conditions for follicles development and oocytes maturation in the transplanted ovarian tissue, and

[0253] (d) collecting said mature oocytes from the transplant ovarian tissue.

[0254] In some embodiments, the method comprising purifying the collected mature oocytes.

[0255] In some embodiments, the method comprising applying (using) the collected mature oocytes in IVF procedure.

[0256] In some embodiments, the method is for fertility preservation and fertility restoration.

[0257] As noted herein, the method includes transplantation of ovarian tissue or any fragment thereof in intermediate and / or large animals. It was suggested that this would allow repeated collecting of mature oocytes at multiple time points. In other words, the transplanted ovarian tissue may be suitable for more than a single, one time, oocyte collecting and hence the second mammalian animal may be considered as a repeated source of mature oocytes.

[0258] In some embodiments, the method comprises repeating the step of collecting mature oocytes. In some embodiments, the method comprises repeating the steps of providing conditions for follicles development and oocytes maturation in said transplanted ovarian tissue, and collecting said mature oocytes from said transplant ovarian tissue.

[0259] As described herein, the collected mature oocytes may be used for IVF procedure. Hence, in accordance with some other aspects, the present disclosure provides a method for fertility preservation, fertility restoration and treating infertility or infertility-related conditions in a mammalian subject, the method comprising producing mature oocytes as described herein and performing an IVF procedure.

[0260] In some other aspects, the present disclosure provides a method for fertility preservation, fertility restoration and treating infertility or infertility-related conditions in a mammalian subject, the method comprising performing IVF procedure using the mature oocytes obtained or obtainable by the method described herein.

[0261] In some other aspects, the present disclosure provides mature oocytes obtained or obtainable by the method described herein.

[0262] Follicles development as used herein refers to the process by which ovarian follicles, grow and mature, oocytes maturation as used herein involves the process in which the oocyte progresses from a primordial state to a fully mature state, capable of being fertilized.

[0263] As used herein, the forms "a", "an" and "the" include singular as well as plural references unless the context clearly dictates otherwise.

[0264] Further, as used herein, the term "comprising" is intended to mean that the composition include the recited components, e.g. at least one modulator. "Consisting of shall thus mean excluding more than trace amounts of other components. Embodiments defined by each of these transition terms are within the scope of this invention.

[0265] Further, all numerical values, e.g. when referring the amounts or ranges of the components, are approximations which are varied (+) or (-) by up to 20%, at times by up to 10%, at times up to 5% of from the stated values. It is to be understood, even if not always explicitly stated that all numerical designations are preceded by the term "about".

[0266] It should be noted that various embodiments of this invention may be presented in a range format. The description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 or between 1 and 6 should be considered to have specifically disclosed sub ranges 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.

[0267] It should be further noted that the various embodiments and examples detailed herein in connection with various aspects of the invention may be applicable to one or more aspects disclosed herein. It should be further noted that any embodiment described herein, for example, related to methods of the invention, may be applied separately or in various combinations as well as may be applied separately or in various combinations to oocytes. 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. The phrases “in another embodiment” or any refence made to embodiment as used herein do not necessarily refer to different embodiment, although it may. Thus, various embodiments of the invention can be combined (from the same or from different aspects) without departing from the scope of the invention.

[0268] The invention will now be exemplified in the following description of experiments that were carried out in accordance with the invention. It is to be understood that these examples are intended to be in the nature of illustration rather than of limitation. Obviously, many modifications and variations of these examples are possible in light of the above teaching. It is therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise, in a myriad of possible ways, than as specifically described hereinbelow.

[0269] NON-LIMITING EXAMPLES

[0270] Example 1: Evaluation and establishment of a human endocrine environment

[0271] Example 1A: Gonal-F (follitropin alfa) FSH in male rabbits

[0272] Three wild type (WT) New Zealand (NZ) rabbit creed castrated males, age at start - 20 weeks, weight at start: 3260g; 3100g and 2950g were used in this experiment. Blood was withdrawn from the ear and vein. The male rabbits were administered with Gonal-F (follitropin alfa, FSH, 22 pg / 0.5ml, Serono) with a single dose once a week for 3 weeks. Serum blood hormones were repeatedly measured. Human serum FSH was measured using an Elecsys FSH electrochemiluminescence immunoassay kit (ref. 8932387190, COBAS e801, Roche Diagnostics GmbH, Mannheim, Germany), at the Endocrine Laboratory, Division of Endocrinology, Diabetes and Metabolism, Sheba medical center.

[0273] Results

[0274] Serum FSH levels after administration of Gonal-F are shown in Tables 1A-1C and Figures 1A-1C.

[0275] Table 1A shows FSH levels at different times after administration.

[0276] Table 1A: Serum FSH levels at different hours after administration

[0277] The data is provided in Figure 1A showing serum levels of FSH at the first week after injection of different doses of Gonal F. As can be seen, FSH levels in male rabbits after Gonal-F injection were low with 12.5 IU and 25 IU doses but increased significantly after administration of 37.5 IU — indicating a dose-dependent response. As can be seen, a single dose of 37.5 IU Gonal-F induced IVF-equivalent human. As can be seen from Figure 1A and Table 1A, low FSH serum levels were observed after injection of 12.5IU and 25IU, whereas high FSH serum levels was observed after injection with 37.5IU.

[0278] Table IB shows FSH levels at different times after the second administration, at the second week.

[0279] Table IB: FSH levels at different times after the second administration Figure IB shows the serum levels of FSH at the second week of injection (the second injection) of different doses of Gonal F. As can be seen from Figure IB, at the second week, serum FSH levels decline below 10 IU faster with lower doses. The 50 IU dose maintained higher levels up to 36h.

[0280] As can be seen from Figure IB and Table IB, there was a reduction in FSH serum levels below 10IU FSH after several hours.

[0281] Table 1C shows FSH levels at different times after the third administration (third week).

[0282] Table 1C: FSH levels at different times after the third administration

[0283] Figure 1C shows serum levels of FSH at the third week of injection (the third injection) of different dose of Gonal F. As can be seen from Figure 1C and Table 1C, administration of 37.5IU and 50IU resulted in FSH serum blood levels above 10IU which decayed below 10IU 36h post injection.

[0284] Serum FSH levels after administration of different doses of Gonal-F at different week of administration are shown in Tables 2A-2C and Figures 2A-2C.

[0285] Table 2A shows FSH levels at different times after administration of 25IU Gonal. Figure 2A shows serum levels of FSH after administration of 25IU Gonal F at different times after first week (Wl), second week (W2), third week (W3).

[0286] Table 2A: FSH levels at different times after first week (Wl), second week (W2), third week (W3) of administration

[0287] As can be seen in Figure 2A and Table 2A, after repeated injections of FSH, higher FSH levels were observed in the serum. Week-over-week injections of 25 IU led to increased serum FSH levels overtime.

[0288] Table 2B shows FSH levels at different times after the administration of 37.5IU Gonal F. Figure 2B shows serum levels of FSH after administration of 37.5IU Gonal F at different times after first week (Wl), second week (W2), third week (W3) of administration.

[0289] Table 2B: FSH levels at different times after first week (Wl), second week (W2), third week (W3) of administration.

[0290] As can be seen in Figure 2B and Table 2B, after repeated injections of 37.5IU FSH, serum levels of FSH increased.

[0291] Table 2C shows FSH levels at different times after administration of 50IU Gonal F. Figure 2C shows serum levels of FSH after administration of 50IU Gonal F at different times after second week (W2), third week (W3) of administration. Table 2C: FSH levels at different times after second week (W2), third week (W3) of administration.

[0292] As can be seen in Figure 2C and Table 2C, after repeated injections of 50IU FSH, serum levels of FSH increased and were reduced to below 10IU after 36h.

[0293] Example IB: Gonal-F (follitropin alfa) FSH in male rabbits and female rabbits

[0294] Castrated male rabbit and female rabbit, age at start 20 weeks, were used in this experiment. Blood samples were withdrawn from the ear and vein.

[0295] Rabbits were administered with Gonal F (follitropin alfa 22 ug / 0.5ml). A single dose at different Gonal F (FSH) doses were administered once a week for 3 weeks in male rabbits. Blood Hormones were repeatedly measured. Human Serum FSH was measured as detailed above.

[0296] Results

[0297] Table 3A and Figure 3A show serum levels of FSH at the first week after injection of Gonal F (25IU) in male rabbit and in female rabbit.

[0298] Table 3A: FSH levels at different times after administration

[0299] As can be seen from Figure 3A and Table 3A, higher FSH serum levels were observed after injection in female rabbit. Figure 3B and Table 3B show serum levels of FSH at the second week after injection of

[0300] Gonal F (37.5 IU) in male rabbit and in female rabbit.

[0301] Table 3B: FSH levels at different times after administration

[0302] As can be seen from Figure 3B and Table 3B, higher FSH serum levels were observed after injection in female rabbit.

[0303] When given the same Gonal-F dose, female rabbits exhibited higher serum FSH levels than castrated males.

[0304] Example 1C: combination of LH Luveris and Gonal F

[0305] Three castrated males, age at start: 20 weeks, weight at start: 3500g; 3500g; 3300g were used in this experiment. Blood was withdrawn from the ear and vein.

[0306] Rabits were administered with Luveris (Lutropin alfa 75 IU, Merck) and Gonal F (Follitropin alfa 22 ug / 0.5ml, Serono). Blood Hormones were repeatedly measured as follows: human Serum LH was measured using an Elecsys LH electrochemiluminescence immunoassay Human kit (ref. 7027575190) and human Serum FSH was measured as described above.

[0307] Results

[0308] Table 4A and Figure 4A show serum levels of FSH after injection of Gonal F (25IU) and different doses of Luveris in male rabbits.

[0309] Table 4A: FSH levels at different times after administration Table 4B and Figure 4B show serum levels of LH after injection of different concentrations of Gonal F and different concentrations of Luveris in male, on week 2.

[0310] Table 4B: LH levels at different times after administration

[0311] Table 4C and Figure 4C show serum levels of LH in males, at week 3 after injection of different concentrations of Gonal F and Luveris.

[0312] Table 4C: LH levels at different times after administration

[0313] As can be seen from Figures 4A-4C and Tables 4A-4C, increasing Luveris doses (25IU- 75IU) resulted in increased LH serum levels. LH serum levels declined below 10 IU after 12h in some of the combinations. In addition, co administration of Luveris with FSH increased FSH serum levels in line with the increase in Luveris doses. Luveris (lutropin alfa) increased serum LH rapidly, peaking around 3 hours and dropping below 10 IU after 12-24h. When co-administered with Gonal-F, it enhanced FSH levels.

[0314] Example ID: Luveris in female rabbit

[0315] A female rabbit, age at start: 20 weeks, weight 3500g was used in this experiment.

[0316] Blood was withdrawn from the ear and vein. The rabbit was administered with Luveris (LH) (Lutropin alfa 75). Female rabbit received Luveris (LH) injections in different doses. Serum Blood Hormones were repeatedly measured. Human serum LH and human serum FSH were measured as described above.

[0317] Table 5 and Figure 5 show serum levels of LH in female rabbits, after injection of

[0318] Luveris at different concentrations. Table 5: Levels of LH at different times after administration.

[0319] As can be seen, LH serum levels following injection were similar in male rabbits and in female rabbit.

[0320] Example IE: Elonva FSH (Human)

[0321] Three castrated males age at start: 20 weeks, weight at start: 3500g; 3500g; 3300g were used in this experiment. Blood withdrawal from: ear and vein. Elonva (FSH) (Corifolltropin alfa, Organon) was injected at different doses to the rabbits. Blood Hormones were repeatedly measured. Human serum FSH was measured as described above.

[0322] Table 6 and Figure 6 show serum levels of FSH in males, after injection of Elonva at different concentrations.

[0323] Table 6: FSH levels at different times after administration

[0324] As can be seen in Table 6 and Figure 6, serum LH levels after Elonva administration to male rabbits were high and remained above 10IU between 8-12 days after a single injections in all doses. This suggested that Elonva induced a sustained and potent FSH response in male rabbits as this level was high and stable.

[0325] Example IF: Progeterone levels after Elonva Gonal F injection

[0326] One female WT NZ rabbit, age at start: 20 weeks, weight at start: 3500g was administered with Gonal F or with Elonva (FSH) in different doses. Blood withdrawal from: ear and vein. Blood Hormones were repeatedly measured. Human Serum FSH was measured as described above. Table 7A and Figure 7A show serum levels of progesterone after injection of Gonal F at different doses at week 1, 2 and 3.

[0327] Table 7A: progesterone levels at different times after administration

[0328] Table 7B and Figure 7B shows serum levels of progesterone after injection of Elonva after up to 12 days.

[0329] Table 7B: progesterone levels at different times after administration

[0330] 3. 1siweek time 0 below the detection

[0331] 4. 3t<!week na time 0 and 48h

[0332] As can be seen., in female rabbit, progesterone levels increased after Gonal F administration, whereas estrogen levels remained undetectable after injection of Gonal F (data not shown).

[0333] Progesterone levels were higher and stable when the rabbit was administered with Elonva.

[0334] Example 1H Gonal F & Luveris Administration Induces IVF-Equivalent Human FSH Levels in Rabbits

[0335] Male and female rabbits were administered with a combination of Gonal F and and Luveris at indicated time points over 3 weeks. Human FSH levels were then examined up to 48 hours following administration.

[0336] Figure 8A and 8B show human FSH levels in male and female rabbits following administration of human gonadotropin Gonal F and Luveris as detailed in Figure 8C. As can be seen in these figures, combination of Gonal F at 37.5 IU and Luveris 50IU at W2 as well as Gonal F 37.5 IU and Luveris 75IU at W3, resulted in FSH levels that were above 10IU / L up to 36 hours.

[0337] Example II: Menopur Administration Induces IVF-Equivalent Human FSH Levels in Rabbits

[0338] Male rabbits were administered with Menopur (a post human menopausal gonadotropin) at different concentrations. Human FSH and PHCG levels were then examined up to 48 hours following administration.

[0339] Figure 9A and 9B shows human FSH levels and PHCG levels in male rabbits following administration of human menopausal gonadotropin Menopur as detailed in Figure 9C. As can be seen from these figures, serum levels of FSH were above 10IU / L up to 48 hours for both concentration of 37.5 IU and 50 IU. The levels of human PHCG were above 2 IU / L only for concentration of 50 IU of Menopur.

[0340] Example 2: Hormone Stability and Dilution in vitro Assays

[0341] Human FSH and LH levels and their stability was tested after administration of Gonal F, Luveris or Elonva at different solvents and dilutions. Human FSH and LH were measured as detailed above.

[0342] Table 10A and Figure 10A show levels of FSH at different Gonal F doses, in PBS or DMEM media.

[0343] Table 10A: FSH levels of Gonal F dilutions in different solvents at different times.

[0344] As can be seen, FSH levels increased linearly with Gonal F doses of in both solvents.

[0345] Table 10B and Figure 10B show levels of LH of Luveris at different doses, in PBS or DMEM media. Table 10B: LH levels at different times

[0346] As can be seen, in in-vitro, LH levels increased linearly with increasing doses of Luveris.

[0347] Table IOC and Figure IOC show levels of FSH of at different concentrations of Elonva.

[0348] Table IOC: FSH levels at different dilutions

[0349] As can be seen, FSH levels after Elonva were very high (>200).

[0350] EXAMPLE 3: in vitro bioactivitv assay

[0351] Human FSH Bioactivity in Rabbit Serum - Assay following Gonadotropins Treatment

[0352] Rabbit serum may potentially interfere with human gonadotropin administration through FSH antibodies and / or competitive malfunction rabbit FSH. FSH Bioactivity was then examined in rabbit serum.

[0353] In vitro assessment of FSH Bioactivity was performed on human granulosa cells. Castrated Rabbits’ Serum was compared with serum from women undergoing hormonal stimulation for IVF.

[0354] As shown in Figure 11A andllB, serum from castrated rabbits exhibited no detectable FSH bioactivity on human granulosa cells in vitro, whereas serum from women undergoing IVF demonstrated clear FSH bioactivity. Figure 12A and 12B shows in vitro FSH Bioactivity in castrated male rabbit serum following human gonadotropins administration. As can be seen in these figures, following a single administration of long -acting FSH (Elonva) to castrated male rabbits, serum FSH bioactivity remained stable for 8 days post-injection.

[0355] Figure 13 shows repeated injections of human gonadotropins in castrated male rabbits did not change human FSH bioactivity in serum. As can be seen in this figure, potential anti-human FSH antibodies in rabbit serum, if present, did not diminish the in vitro biological efficacy of administered human FSH.

[0356] Based on these results, it was concluded that potential anti-human FSH antibodies in rabbit serum, if present, did not diminish the in vitro biological efficacy of administered human FSH.

[0357] Example 4: Transplantation of frozen / thawed human ovarian tissue to WT rabbits

[0358] The purpose of this example was to optimize the surgical approach for xenografting human ovarian cortical tissue into WT rabbits, comparing several graft sites and to assess whether the grafted human tissue shows immune rejection after a defined post-operative interval, by removing the grafts and analyzing them histologically.

[0359] Ovarian tissue donor details

[0360] Patient 1, (Pl VL)” has a clinical background of cervical carcinoma with no prior chemotherapy. Fragment size for transplantation after thawing 5-6 cm2cortical strip. Follicles seen post-thaw 10 primordial follicles and 4 primary follicles. No malignancy is identified and no fibrosis or inflammatory infiltrate are identified.

[0361] Patient 2, here after (P2 HT)” has a clinical background of Non-Hodgkin lymphoma; with no prior chemotherapy. Pathology pre freezing: Ovary, medulla: Fragments of ovarian tissue rich in blood vessels and containing corpus albicans. -One primordial follicle is identified, no malignancy is identified. - Multiple deeper sections were performed. Fragments of ovarian tissue cortical type with acorpus albicans. CD20 and CD30 immunostaining were negative, confirming the diagnosis. MNF116 immunostaining highlighted the follicle. Ovary, cortex: Fragments of ovarian tissue rich in blood vessels and containing corpus albicans. 3 primordial follicles, 1 primary follicle and 1 secondary follicle were identified. No malignancy is identified. - Multiple deeper sections were performed. - CD20 CD30 immunostaining are negative, confirming the diagnosis. MNF116 immunostaining highlights the follicles. No follicles were identified. No malignancy was identified. Multiple deeper sections were performed. Fragment size for transplantation after thaw 2-3 cm2cortical + medullary fragments. Follicles seen post-thaw Cortex: 3 primordial, 1 primary, 1 secondary Medulla: 1 primordial. No malignancy is identified and CD20 / CD30 (confirms lymphoma origin absent); MNF116 highlights follicles

[0362] Surgical procedure

[0363] Rabbits were used for transplantation of ovarian fragments denoted herein as C- 7808 (male rabbit weight 3830 g) and D-7801 (female rabbit weight 4585 g). different translation sites were tested.

[0364] Ovary fragments were transplanted to C-7808 at the back right side and left side by SC (4 pockets) and abdomen left side and right side by IP (4 pockets). Figure 14C shows an exemplary image of transplantation.

[0365] Ovary fragments were transplanted to D-7801 at abdomen above fascia right side and left side by SC (4) and by IP bottom and top (4).

[0366] Figure 14A and 14B are an exemplary image showing preparations of ovarian tissue from Pl-VL.

[0367] Pathology at 14 days post-transplant of male rabbit

[0368] Several pathology samples were obtained from the rabbit back, details of which are as follows:

[0369] #1008 right side right incision from patient Pl VL.

[0370] #1007 right left incision from patient P2 HT.

[0371] # 1006 left side right incision from patient Pl VL.

[0372] # 1005 left side left incision from patient P2 HT.

[0373] Several pathology samples were obtained from the rabbit abdomen, details of which are as follows:

[0374] # 1002 left side upper incision from patient P2 HT.

[0375] # 1001 left side bottom incision from patient Pl VL. #1004 right side upper incision from patient P2 HT.

[0376] #1003 right side bottom incision from patient Pl VL.

[0377] Results

[0378] # 1001 : Pl VL - included skeletal muscle with central graft. The graft was replaced by fibroblastic fibrosis with prominent chronic inflammatory infiltrate with numerous eosinophils and occasional neutrophils. Two suppurative granulomata were also present. No follicles were identified. # 1002: P2 HT, IP, AB - Skeletal muscle with central graft. The graft showed marked fibroblastic fibrosis with numerous neutrophils and eosinophils. Peripheral lymphoid rimming as well as patchy lymphoid infiltrate were also seen. Three epithelioid granulomata were present, one with central suppuration and other with central necrosis. No obvious oocytes were identified. # 1003: Pl VL, IP, AB - Skeletal muscle with central graft. The graft is replaced by fibroblastic fibrosis with prominent chronic inflammatory infiltrate with lymphoid aggregates, numerous eosinophils and rare neutrophils. Two confluent suppurative granulomata were also present. No follicles were identified. # 1004: P2 HT, IP, AB Skeletal muscle with adjacent graft. The skeletal muscle showed focal interstitial chronic inflammation rich in neutrophils and eosinophils. Adjacent fibrous tissue was also rich in eosinophils with patchy lymphoid infiltrate. The graft showed focal fibroblastic fibrosis with numerous eosinophils with eosinophilic micro abscess formation. Extensive but patchy mononuclear cell infiltrate was seen replacing most of the graft and irregularly extending between the skeletal muscle bundles. One epithelioid granuloma was present with central suppuration. No obvious oocytes were identified. # 1005: P2 HT, SC, Back - Small fragment of skeletal muscle and adipose tissue with adjacent graft. The graft was replaced by prominent fibroblastic fibrosis with numerous eosinophils and neutrophils. Focal small lymphoid infiltrate was also seen. There was a large incompletely formed epithelioid granuloma with central necrosis and signs of early organization with fibrosis and eosinophilic infiltrate. No obvious oocytes were identified. # 1006:Pl VL, SC, back - Skeletal muscle and adipose tissue with adjacent graft. The graft showed extensive fibrosis and mild eosinophilic infiltrate. Poorly formed granulomata were seen in the periphery of the graft. There are eighty one (81) necrotic primordial follicles, thirty nine (39) necrotic primary follicles and two (2) necrotic secondary follicles. # 1007: P2 HT, SC, Back -Skeletal muscle, tendon and fibroadipose tissue with adjacent graft. The graft showed fibroblastic fibrosis with prominent chronic mononuclear infiltrate with numerous eosinophils and some neutrophils. A nodular fibrotic area was present with more collagenous stroma. One small epithelioid granulomata was present. No obvious oocytes were identified. # 1008: Pl VL, SC, Back - Adipose tissue with adjacent graft. Skeletal muscle was not identified. The graft was mostly replaced by a large irregular granuloma with suppurative and fibrotic center. Fibroblastic fibrosis and chronic inflammatory infiltrate rich in eosinophils were seen around the granuloma. No follicles were identified.

[0379] Pathology at 14 days post-transplant of female rabbit

[0380] Two samples of blood taken: one was frozen and the second was transferred to hormone’s analysis. Several pathology samples were obtained from the rabbit abdomen.

[0381] # 1009: from patient Pl VL, IP, AB - Scant adipose tissue and adjacent graft. Skeletal muscle tissue was not identified. The graft was replaced by fibroblastic fibrosis with prominent chronic inflammatory infiltrate with numerous eosinophils and occasional neutrophils. One large suppurative granuloma and small well circumscribed granulomata were also present. No follicles were identified. # 1010: from patient HT, IP, AB - Skeletal muscle and adipose tissue with adjacent graft. The graft was completely replaced by fibroblastic fibrosis with numerous eosinophils, small lymphocytes and occasional neutrophils. Patchy peripheral lymphoid aggregates are also seen, focally extending in the skeletal muscle interstitial tissue. Numerous small epithelioid granulomata were present, one with central suppuration. No obvious oocytes were identified. # 1011: from patient Pl VL, SC, AB - Skeletal muscle with adjacent graft. The graft showed prominent fibrosis and chronic inflammation The inflammatory infiltrate focally extended into the endomysium. Residual but partly necrotic follicles were present: nine (9) primordial, three (3) primary and one (1) suggested secondary. Other areas showed neutrophilic infiltrate with abscess formation. # 1012: from patient P2 HT, SC, AB - Skeletal muscle and adipose tissue with prominent vasculature with central graft. The graft showed central fibrosis with suppuration. Peripheral mononuclear rimming rich in eosinophils and occasional neutrophils was present. Two epithelioid granulomata were present. No obvious oocytes were identified. # 1013: from patient P2 HT, SC, AB - Fibroadipose tissue with prominent vasculature with central graft. Skeletal muscle was not identified. The graft was replaced by sheets of mononuclear cells rich in eosinophils and occasional neutrophils. Focal fibroblastic fibrosis was suggested. Several epithelioid granulomata were present, part of them with central suppuration, one of them irregular. No obvious oocytes were identified. # 1014: from patient Pl VL, SC, AB - Fibroadipose tissue and adjacent graft. Most of the graft was replaced by fibroblastic fibrosis with prominent chronic inflammatory infiltrate with numerous eosinophils. Residual viable ovarian tissue was present with seven (7) primordial follicles, five (5) primary follicles) and one (1) secondary follicle. Several suppurative granulomata were also present. # 1015: from patient P2 HT, IP, AB - Skeletal muscle and adipose tissue with central graft. The graft showed prominent fibroblastic fibrosis with a striking mononuclear cell infiltrate rich in eosinophils and occasional neutrophils. The mononuclear infiltrate focally extended into the skeletal muscle interstitium. One small epithelioid granuloma was present. No obvious oocytes were identified. # 1016: from patient Pl VL, IP, AB - Scant skeletal muscle and adjacent fibroblastic fibrotic areas with myxoid changes. No follicles were identified.

[0382] Histological analysis

[0383] Histological analysis by H&E of sample 1006 is shown in Figures 15A-15C. As can be seen, most of the tissue is necrotic tissue with residual ovarian follicles, and early fibrosis and eosinophilic infiltrate. Histological analysis by H&E of sample 1003 is shown in Figures 16A-16C. As can be seen in Figures 16A and 16B, the tissue was characterized by prominent chronic inflammation associated with numerous eosinophils, focal microabscess formation, and focal necrotizing granuloma (with eosinophilic debris). As can be seen in Figure 16C, the tissue was characterized by fibrosis associated with chronic inflammation rich in eosinophils. Small epithelioid granuloma. Histological analysis by H&E of sample 1002 is shown in Figures 17A-17B. Histological analysis by H&E of sample 1010 is shown in Figures 18A-18B.

[0384] Hormonal conditioning with Elonva on Rabbit D-7801

[0385] 0.2ml (40IU) of Elonva was injected to 7801 female rabbit. Six days later the day of Extraction operation, various hormones were measured as shown in Table 11. Table 11: hormone measurements

[0386] Both Ovaries were removed 6-day after Elonva administration.

[0387] Figure 19A(zoom xl.8) and Figurel9B (zoom 8x), show H&E stating of right ovary. Figure 19C-(zoom xl.8) and Figurel9D (zoom 8x), show H&E stating of left ovary.

[0388] As can be seen, H&E clearly shows hyper stimulated Ovaries. More than 12 antral follicles in section. Cumulus granulosa cells and oocytes in an antral Follicles containing many granulosa layers. As shown in this example, extraperitoneal location in the abdomen was more convenient than back area.

[0389] Recovery post operation:

[0390] The 2 rabbits recovered well, gained weight, wounds recovered well, back sites- reached by rabbits and bites.

[0391] Tissue recovery

[0392] In all locations transplanted tissue was well recognized and removed. Both large and smaller tissue.

[0393] Conclusions for Pathology

[0394] The pathological criteria fortissue rejection were tested and established. Two weeks after implantation, in both rabbits (male and female) and in both sites of implantation (SC and IP), the implanted ovary fragments exhibited strong signs of early rejection, including parameters of inflammation, fibrosis, and in some fragments also granuloma formation. These results were typical for heterologous transplantation of human tissue into immunocompetent rabbits.

[0395] Ovarian stimulation endocrine + pathology (rabbit D-7801):

[0396] After 1 week of Elonva 40IU administration, E2 was not detected, Progesterone rose to 9.9 nmole / L. EXAMPLE 5:

[0397] Xenograft transplantation of cryopreserved / thawed human ovarian tissue in immune deficient rabbits- Detection and analysis of Follicle survival and vascularization in transplants.

[0398] Immune deficient rabbits were tested as a Xenotransplantation animal model. The use of rabbits (an animal with a longer lifespan than a mouse or rat) is necessary since the implant is required to remain in the animal for a long period of time, which can extend beyond the lifetime of mice or rats. Another advantage of using rabbits is due to their size - which allows a larger number of pieces to be implanted in each animal.

[0399] Male and female rabbits are suitable for ovarian tissue transplantation following oophorectomy / castration and hormonal manipulation.

[0400] Wild type rabbits, NUDE rabbits, RAG mutation immunodeficient rabbits, IL2 immune deficient rabbits and double knockout immunodeficient rabbits were used.

[0401] Human ovarian tissue was transplanted in subcutaneous locations, abdominal supra-facial locations and intraabdominal peritoneal pockets, in subcutaneous locations, abdominal supra-facial locations and intraabdominal peritoneal pockets.

[0402] In Wild type rabbits, follicles were destroyed within few weeks.

[0403] In RAG and IL mutation rabbits:

[0404] - Tissue survives for a long period,

[0405] - human blood vessels grew into rabbit tissue,

[0406] - rabbit blood vessels were present in human ovarian tissue,

[0407] - Human ovarian follicles morphology and counting post transplantation showed nice survival.

[0408] As shown in Figure 20A-20C, rejection of the transplant was observed in Wild type rabbit. However, survival and growth of the transplant was observed in IL2 rabbit strain, as shown in Figure 21A-21B. It was possible to observe numerous follicles in RAG and IL2 rabbit strains, indicating survival in subcutaneous grafts, as shown in Figure 22. As shown in Figures 23, 24, 25 and 26, human and rabbit blood vessels and follicles were observed post transplantation after two weeks and after 3 months. Human Blood vessels were present in rabbit tissue and rabbit blood vessels were present in human ovarian graft. Primordial (PMF) and Growing Follicle / ovary were also observed and counted.

[0409] Follicle counting in Ovarian Xenotransplantation was performed for 2 weeks and 3 months post transplantation into WT, IL2 and RAG2 rabbit strains and are shown in Figures 27 and 28, respectively.

[0410] Figures 29A-29D show human ovaries in WT rabbit (Figures 29A and 29B) and in immune deficient rabbits (Figures 29Cand 29D). Figures 29A and 29B show that human ovaries were rejected in the WT rabbit. In contrast, Figures 29C and 29D show that in immune deficient rabbits after ovarian transplantation, follicles were observed after 2 weeks (Figure 29C) and after 2 months (Figure 29D).

[0411] Figure 30A-30D shows follicle count in peritoneal grafts after two weeks. As can be seen in Figures 30A and 30B, showing extracellular-matrix assisted (“EA”) protocol, WT rabbits exhibited essentially no detectable follicles, whereas RAG1 / RAG2 knockout (RAG) supported survival of Primordial follicles (PMF) and growing follicles and interleukin-2 knockout (IL2) rabbits showed further enhancement in PMF and in growing follicles. As can be seen in Figures 30C and 30D, showing lentiviral-vector-mediated vascularization (“LV”) protocol, WT rabbits showed minimal survival, whereas RAG and IL2 rabbits supported survival of PMF and growing follicles. These data demonstrate that immunodeficient rabbits supports in vivo maturation of human oocytes.

[0412] Figure 31A-31D shows follicle count in peritoneal grafts after three months. As can be seen in Figures 31A and 31B, showing EA protocol, WT rabbits exhibited essentially no surviving follicles, whereas the immunodeficient rabbits (IL2) supported both follicle preservation and activation, with an average of 3 PMF and 7 primary follicles. As can be seen in Figures 30C and 30D, showing LV protocol, WT rabbits exhibited negligible follicle survival, whereas immunodeficient rabbits and specifically RAG immunodeficient rabbits exhibited robust long-term engraftment, with ~23 PMF and ~18 primary follicles. Figures 32A-32D show human blood vessels outside the transplant (i.e. the rabbit tissue). Figure 32A shows the transplant ovary by the dashed line. The arrows in Figure 32A to blood vessels stained positively for a human-specific endothelial marker. These vessels extend well beyond the graft margin into the surrounding rabbit stroma. These data demonstrate that human ovarian tissue transplantation into immunodeficient mice not only preserves follicular structures but also actively drives formation of functional, human-derived microvasculature into the host organ, a critical step for long-term graft survival and oocyte maturation.

[0413] Figures 33A-33D show rabbit microvascular ingrowth into transplanted human ovarian tissue in immunodeficient rabbit recipients, blood within the transplant (i.e. the human tissue). Figure 33A shows the transplant ovary by the dashed line and a higher- magnification view of the boxed region. The arrows in Figure 33A point to vessels within the human tissue that stain positively for a rabbit-specific endothelial marker. These results demonstrate that immunodeficient rabbits mount extensive host vascularization into human ovarian xenografts without evidence of rejection, thereby establishing a fully perfused microenvironment conducive to human follicle maintenance and maturation.

Claims

CLAIMS:

1. A method for producing mature oocytes comprising:(a) transplanting an ovarian tissue or any fragment thereof of a human female into at least one recipient site of an immunodeficient rabbit, and(b) collecting mature oocytes from said transplanted ovarian tissue.

2. The method of claim 1, comprising monitoring transplant acceptance of the ovarian tissue by assessing one of more of (i) oocyte viability, (ii) ovarian follicle count, (iii) presence of human-derived blood vessels, (iv) presence of rabbit-derived blood vessels within the transplant, or (v) any combination thereof.

3. The method of claim 1 or 2, comprising providing conditions for follicles development and oocytes maturation in said rabbit.

4. The method of claim 3, wherein said conditions comprise administration to the rabbit of at least one reproductive hormone.

5. The method of claim 4, wherein said reproductive hormone is or comprises at least one gonadotropin hormone, at least one steroid hormone or any combination thereof.

6. The method of claim 5 , wherein said at least one gonadotropin hormone is selected from the group consisting of follicle-stimulating hormone (FSH), luteinising hormone (LH), human menopausal gonadotropin (hMG), chorionic gonadotropin, piscine gonadotropin or any combination thereof.

7. The method of claim 5, wherein said at least one gonadotropin hormone is FSH.

8. The method of claim 5, wherein said FSH is at least one of follitropin alfa, follitropin beta, urofollitropin, corifollitropin alfa, follitropin delta or any combination thereof.

9. The method of claim 5, wherein said at least one gonadotropin hormone is LH.

10. The method of claim 5, wherein said LH is lutropin alfa.

11. The method of any one of claims 1 to 6, wherein said human female is in the reproductive years.

12. The method of any one of claims 1 to 6, wherein said human female is suffering from a genetic abnormality or a non-genetic abnormality.

13. The method of claim 8, wherein said non-genetic cause is at least one of an autoimmune disorder, a metabolic disorder, an infection, an environmental factor, or any combinations thereof.

14. The method of any one of claims 1 to 9, wherein said human female is or was diagnosed with cancer.

15. The method of any one of claims 1 to 10, wherein said human female is or was treated with an anti -cancer treatment.

16. The method of claim 11, wherein said anti -cancer treatment is at least one of chemotherapy, radiotherapy or a combination thereof.

17. The method of claim 11 or 12, wherein said anti -cancer treatment is chemotherapy.

18. The method of any one of claims 1 to 14, where said human female is diagnosed or classified with premature ovarian failure.

19. The method of any one of claims 1 to 15, wherein said human female is diagnosed or classified as a poor ovarian responder (POR).

20. The method of any one of claims 1 to 15, wherein said human female is diagnosed or classified as a diminished ovarian reserve (DOR).

21. The method of any one of claims 1 to 16, comprising removing an ovarian tissue or any fragment thereof from said human female prior to said transplantation.

22. The method of claim 17, wherein said ovarian tissue or any fragment thereof is sectioned into fragments prior to transplantation.

23. The method of any one of claims 1 to 18, wherein said ovarian fragment thereof has a thickness of about 0.5 and about 2 mm.

24. The method of any one of claims 1 to 19, wherein said ovarian tissue or any fragment thereof is cryopreserved and thawed prior to transplantation.

25. The method of any one of claims 1 to 20, wherein said immunodeficient rabbit is a nude rabbit, a SCID rabbit, or a Ragl / Rag2 knockout rabbit.

26. The method of any one of claims 1 to 21 , wherein said recipient site is one or more of back region, abdominal area, intra peritoneal, a kidney capsule or a combination thereof.

27. The method of any one of claims 1 to 22, comprising collecting mature oocytes at the metaphase II (Mil) stage.

28. The method of any one of claims 1 to 23, comprising purifying said collected mature oocytes.

29. The method of any one of claims 1 to 24, comprising using said collected mature oocytes in in vitro fertilization (IVF).

30. The method of any one of claims 1 to 25, for fertility preservation and fertility restoration.

31. Mature oocytes obtained by the method of any one of claims 1 to 26.

32. Mature oocytes obtainable by the method of any one of claims 1 to 26.

33. A method for fertility preservation, fertility restoration and treating infertility or infertility-related conditions in a human female, the method comprising: producing mature oocytes as defined in any one of claims 1 to 26 and performing IVF procedure.

34. A method for fertility preservation, fertility restoration and treating infertility or infertility-related conditions in a human female, the method comprising: performing IVF procedure using the mature oocytes obtained or obtainable by the method of any one of claims 1 to 26.

35. A method of gametogenesis, the method comprises:(a) transplanting a tissue or any fragment thereof of a human subject into at least first recipient site of an immunodeficient second rabbit, and(b) providing conditions for gametes maturation in said rabbit.

36. The method of claim 35, wherein said conditions comprises hormonal treatment.

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