Apparatus, kit and method for delivering a fertilized ovum / embryo to an implantation site in a uterus

Abstract

An apparatus configured to facilitate delivery of an embryo to an implantation site on an endometrium of a patient is provided. The apparatus comprises one or more walls defining a cavity, the walls comprising a rim configured for adhering to the endometrium and defining an opening to the cavity. The walls comprise one or more throughgoing apertures, each sized to allow passage therethrough of intrauterine environmental molecules, and to prevent passage therethrough of a blastocyst. The material of the walls is configured to degrade in the intrauterine environment within a predetermined range of time.

Description

APPARATUS, KIT AND METHOD FOR DELIVERING A FERTILIZED OVUM / EMBRYO TO AN IMPLANTATION SITE INA UTERUSFIELD OF THE INVENTION

[0001] The presently disclosed subject matter relates in general to in vitro fertilization (IVF) systems and methods, and, in particular, to novel system and method for delivering a fertilized ovum / embryo to a preferred implantation site within a uterus of a patient to facilitate implantation and increase successful pregnancy rate.BACKGROUND

[0002] In vitro fertilization (IVF) is a process of fertilization where an egg is combined with a sperm outside the body. Theoretically, IVF can be performed by collecting an ovum, mixing it with sperm, and reinserting the fertilized ovum into the uterus. However, the chances of pregnancy are extremely small, unless other techniques are also combined, such as ovarian hyper-stimulation to generate multiple eggs, ultrasound-guided transvaginal oocyte retrieval from the ovaries, injecting sperm into the oocyte, incubating the fertilized eggs (zygotes) for 2-6 days for embryo culture and selection of resultant embryos before introducing them into the uterus for establishing pregnancy.

[0003] Current IVF procedures involve two major flaws: low live birth rate primarily due to low implantation rate; and high risk for unsafe / unwanted implantation sites.

[0004] Current implantation rates are averaged in around 30%, and are highly variable and depend on patients’ biographic and clinical parameters, as well as clinic to clinic differences. Due to this low rate there is a substantial wastage of embryo and a need for repeated IVF cycles, resulting in parallel increase in medical risks and morbidity as well as substantial emotional and financial costs.

[0005] Another byproduct of the low implantation rates is the tendency to transfer / retum multiple embryos into the uterus, resulting in concomitant increase in multiple fetal pregnancies (twins, triplets, etc.). Such pregnancies are considered as “high risk pregnancies” and carry a dramatic increased risk of pregnancy complications, such as preterm labor, gestational diabetes, hypertension, etc. A multiple fetal pregnancy is regarded as an unwanted result in most IVF treatments, and the medical community is advocating single embryo transfer. Despite such trends, as long as implantation rates remain low this will still be a challenge.

[0006] Unsafe / unwanted implantation sites: Implantation does not necessarily lead to successful pregnancy. The transferred embryo lacks movement capability or means of actively directing its progression in the vastly larger volume of the uterus. In addition, cunent IVF transfer techniques (elaborated below) cause unfavorableconditions for implantation, responsible for both reduced implantation as well as to increased risk of unwanted implantation sites.

[0007] The following risks of unsafe / unwanted implantation sites are up to 5-6 times more frequent in IVF treatments then in spontaneously conceived pregnancies: (i) Ectopic pregnancy (EUP) - a pregnancy not implanted in the uterine mucosa, e.g., fallopian tube, ovary and cervix. These locations pose serious health risk to the mother, being the leading cause of mortality (and morbidity) in the first trimester. Ectopic pregnancies do not lead to a viable pregnancy and need to be terminated. The treatment of EUP might include surgery or even the use of chemotherapy agents incorporating additional health risk, (ii) Placenta previa -when implantation occurs close to the uterine cervix, the placenta, which starts to develop at the implantation site, might cover the cervical opening- these placentas are termed “ placenta previa .” Pregnancies complicated by placenta previa might not deliver vaginally and require delivery caesarian section. These pregnancies are also at risk for substantial maternal hemorrhage and preterm labor, (iii) Placenta accreta - when implantation occurs in adjacent to uterine scar tissue (whether due to previous surgery, infection or other) placental tissue, usually confined to uterine mucosa (endometrium), might invade into the uterine muscular tissue or further to adjacent tissues such as the bladder. Such cases harbor increased risk for uterine rupture, severe morbidity by damaging adjacent organs or life-threatening bleeding. Treatment might require hysterectomy (i.e., removal of the uteres entirely).

[0008] By mapping described challenges and cunent procedure it is evident that implantation stage is a pivot point and that Embryo Transfer (ET, transferring the embryo into the uterus) is a key improvable phase.

[0009] In cunent ET technique, the blastocyst is injected, in a delicate and operator-dependent manner, into the uterine cavity via a catheter at a speed of 12 m / sec, creating unfavorable conditions for implantation. Many factors influence the implantation success: if ET injection is too fast, the media volume too big, transfer is too close / far to the uterine wall / fundus (aimed to ~lcm) or too traumatic (even minor trauma or bleeding), the chances for proper implantation dramatically decrease.

[0010] In addition, as described above, for the implantation to be successful, embryo must adhere to an implantation site in a space that is substantially larger than its own.

[0011] Although attempts to improve the ET step in the IVF procedure were made in the past, currently no technology (aside from ultrasound) has managed to break through and become a standard of use. Nowadays, the ET phase remains a major pitfail in the IVF procedure, and the only part of IVF treatment without significant advancement since 1978.

[0012] Most current proposed interventional techniques to improve ET interfere with endometrial-embryo interaction, whether by applying an additional medium between the two or changing implantation site alltogether (myometrium instead of endometrium). Other use cumbersome equipment which, in itself, hinders implantation, e.g., due to uterine / endometrial irritation or other.

[0013] However, despite asserting to improve implantation rates, none of the prior art methods and devices were actually proven to improve implantation rates, thus rendering the ET step as the ongoing pitfail of the IVF procedure, which requires further improvement.

[0014] Contrary to the known art, the present invention provides an apparatus and method for optimizing the implantation process with minimal to no interference with the natural process, while preventing / limiting implantation in unwanted sites.SUMMARY OF INVENTION

[0015] According to an aspect of the presently disclosed subject matter, there is provided an apparatus configured to facilitate delivery of an embryo (such as a fertilized ovum) to an implantation site on an endometrium of a patient, the apparatus comprising one or more walls defining a cavity, the walls comprising a rim configured for adhering to the endometrium and defining an opening to the cavity;the walls comprising one or more throughgoing apertures, each sized to allow passage therethrough of intrauterine environmental molecules, and to prevent passage therethrough of a blastocyst;the material of the walls being configured to degrade, e.g., being biodegradable, enzymatically degradable, hydrolytically degradable, etc., in the intrauterine environment within a predetermined range of time.

[0016] The cavity may be sized so as allow free movement therewithin of a blastocyst.

[0017] The intrauterine environmental molecules may comprise nutrients, signaling molecules, and / or hormones.

[0018] The intrauterine environmental molecules may comprise human chorionic gonadotropin, progesterone-R, and / or estrogen-R.

[0019] The throughgoing apertures may each be sized no smaller than to allow passage therethrough of a sphere having a diameter of about 50 pm, i.e. , they may be larger than about 50 pm, thereby allowing passage therethrough of intrauterine environmental molecules.

[0020] The throughgoing apertures may each be sized no larger than to allow passage therethrough of a sphere having a diameter of about 150 pm, i.e., they may be smaller than about 150 pm, thereby preventing passage therethrough of the embryo. The throughgoing apertures may each being sized no larger than to allow passage therethrough of a sphere having a diameter of about 100 pm.

[0021] The lower limit of the predetermined range of time may allow for implantation of the embryo in the endometrium to occur before the walls degrade.

[0022] The upper limit of the predetermined range of time may allow for the walls to degrade before the embryo is of a size which exceeds that of the cavity.

[0023] The rim may comprise an adhesive agent.

[0024] The rim may comprise a gripping structure configured to facilitate the adhering to the endometrium.

[0025] The apparatus may be formed as an elongate member with the opening being formed along its length.

[0026] The walls may define a half cylindrical shape.

[0027] The rim may comprise a flange extending away from the opening and being configured for adhering to the endometrium.

[0028] The material of the walls may be flexible.

[0029] The material of the walls may be a shape memory material.

[0030] The material of the walls may comprise a hydrogel comprising a cross-linked polymer.

[0031] The polymer may comprise one or more selected from a group including hyaluronic acid, a salt of hyaluronic acid, polyethylene glycol, albumin, human serum albumin, a modified albumin, cationized bovine serum albumin, cationized human serum albumin, and combinations thereof.

[0032] The polymer may comprise a functional group.

[0033] The polymer may be crosslinked with a crosslinker selected from a group including a pentaerythritol based-crosslinker, an aldehyde, bisdiazobenzidine, phenol 2,4-disulfonyl chloride, l,5-difluoro-2,4-dinitrobenzene, urea, 3,6-bis(mercurimethyl)-dioxane urea, dimethyl adipimidate, N,N'-ethylene-bis-(iodo-acetamide), an acrylate-based linker, a di-halogen, a di-acid, a di-alcohol, a diamine, and combinations thereof.

[0034] The polymer may comprise hyaluronic acid or a salt thereof, functionalized with at least one allyl group and crosslinked with a pentaerythritol based-crosslinker.

[0035] The polymer may comprise hyaluronic acid or a salt thereof, functionalized with at least one acrylate moiety or methacrylate moiety, and crosslinked.

[0036] The polymer may comprise polyethylene glycol functionalized with at least one allyl group and crosslinked with a pentaerythritol based-crosslinker.

[0037] The material of the walls may comprise at least one active ingredient.

[0038] The active ingredient may comprise a progestogen. According to some examples, the progestogen is progesterone.

[0039] The at least one active ingredient may comprise a hormone, a local regulator, a growth factor, a cytokine, an extracellular matrix component, and / or an immune cell.

[0040] At least one of the active ingredients may be selected from a group including human chorionic gonadotropin, estrogen, progesterone-R, estrogen-R, early pregnancy factor, platelet-activating factor,luteinizing hormone, follicle-stimulating hormone, prostaglandin E2, prostaglandin F2a, cytokines, glycogen, HB-EGF, integrals, selectins, laminin, fibronectin, T-lymphocytes, macrophages, lectin concanavalin A, thromboxane B2, leukotriene C4, leukotriene B4, ILla, ILlb, IL6, CSF1R, CSF-1, TNF-a, LIF, GnRH, VEGF, IGFBP 1-6, IGF-1, IGF-2, TGF bl, TGF a, EGF, IFN-gamma, PDGF, prolactin, GH, renin, prorenin, endorphin, endothelin, corticotropin releasing hormone, uteroglobin, Lipocortin 1, parathyroid hormone like protein, and fibroblast growth factor.

[0041] The material of the walls may be a porous material, wherein the throughgoing apertures are provided by the porosity of the material.

[0042] According to another aspect of the presently disclosed subject matter, there is provided a kit comprising:an apparatus as described above; anda delivery tube having an inner diameter suitable for passage therethrough of the apparatus, and a length sufficient to reach into a uterus of a patient.

[0043] The kit may further comprise an embryo injection means.

[0044] According to another aspect of the presently disclosed subject matter, there is provided a method for delivering an embryo to an implantation site on the endometrium of a patient, the method comprising the steps of:providing an apparatus as described above;inserting the apparatus into the uterus and adhering it to the implantation site such that the cavity of the apparatus is open to the implantation site via the opening; andinserting the embryo into the cavity of the apparatus when the rim is adhered to the endometrium.

[0045] Inserting the embryo into the cavity of the apparatus may comprise injecting the embryo through the walls of the apparatus.

[0046] According to another aspect of the presently disclosed subject matter, there is provided a method for delivering an embryo to an implantation site on the endometrium of a patient, the method comprising the steps of:providing an apparatus as described above;providing the embryo in the cavity of the apparatus; andinserting the apparatus with the embryo in its cavity into the uterus and adhering it to the implantation site such that the cavity is open to the implantation site via the opening.

[0047] The embryo may be temporarily attached to a cavity -facing surface of the wall of the apparatus prior to insertion of the apparatus into the uteres.

[0048] According to another aspect of the presently disclosed subject matter, there is provided an apparatus 100 for insertion, i.e., designed to be inserted, into a uterus of a patient and adherence to an endometrium 102, the apparatus 100 being made of a biocompatible and biodegradable hydrogel comprising a crosslinked polymer, and comprising a cavity with surrounding walls 610, 612 and an opening 616, wherein (i) the rim of the opening 616 is designed to adhere to the endometrium 102; (ii) the cavity is of a size compatible with a fertilized ovum / embryo 100; and (iii) the hydrogel is designed to biodegrade after a predefined period.

[0049] According to another aspect of the presently disclosed subject matter, there is provided a kit comprising (i) an apparatus 100 as defined above; and (ii) a delivery tube 106, such as a catheter, having an inner diameter suitable for passage of the apparatus 100, and a length sufficient to reach into a uterus.

[0050] According to another aspect of the presently disclosed subject matter, there is provided a method for delivering a fertilized ovum / embryo 104 to an implantation site on the endometrium 102 within the uterus of a patient, thereby increasing successful pregnancy rates, utilizing the apparatus or kit of the presently disclosed subject matter.

[0051] The method may comprise the steps of: (a) providing an apparatus 100 as defined above, wherein the apparatus does not comprise the fertilized ovum / embryo 104, or a kit as defined above comprising the apparatus 100; (b) inserting the apparatus 100 into the uterus and enabling its adherence / attachment to the endometrium 102 at the implantation site; and (c) following adherence of the opening’s 616 rim of the apparatus 100 to the endometrium 102, inserting the fertilized ovum / embryo 104 into the cavity of the apparatus 100, wherein: (i) the fertilized ovum / embryo 104 is free to contact the endometrium 102 via the opening 616, and implant therein; (ii) the apparatus 100 enables passage / diffusion of materials and fluids from the uterine-cavity to the cavity of the apparatus 100; and (iii) the apparatus 100 biodegrades and dissolves after a predefined period.

[0052] The method may comprise the steps of: (a) providing an apparatus 100 as defined above, wherein the apparatus further comprises the fertilized ovum / embryo 104, or kit as defined above comprising the apparatus 100; and (b) inserting the apparatus 100 into the uterus and enabling its adherence / attachment to the endometrium 102 at the implantation site, wherein: (i) following adherence of the opening’s rim of the apparatus 100 to the endometrium 102, the fertilized ovum / embryo 104 is free to contact the endometrium 102 via the opening 616, and implant therein; (ii) the apparatus 100 enables passage / diffusion of materials and fluids from the uterine-cavity to the cavity of the apparatus 100; and (iii) the apparatus 100 biodegrades and dissolves after a predefined period.BRIEF DESCRIPTION OF DRAWINGS

[0053] Figs. 1A-1C illustrate the process of transferring a fertilized ovum into a uterus using the apparatus according to the presently disclosed subject matter;

[0054] Figs. 2A-2F illustrate the tip of a catheter through which an apparatus according to certain embodiments of the presently disclosed subject matter is transferred into a uterus;

[0055] Figs. 3A-3C illustrate an embodiment of a transport system for delivering an apparatus according to the presently disclosed subject matter into a uterus;

[0056] Figs. 4A-4D illustrate another embodiment of a transport system for delivering an apparatus according to the presently disclosed subject matter into a uteres;

[0057] Figs.5A-5C illustrate an example of a cylindrical apparatus according to certain embodiments of the presently disclosed subject matter;

[0058] Fig. 6A-6B illustrate another example of a cylindrical apparatus according to certain embodiments of the presently disclosed subject matter;

[0059] Figs. 7A-7B illustrate a half-cylindrical apparatus according to certain embodiments of the presently disclosed subject matter;

[0060] Figs.8A-8B illustrate an alternative half-cylindrical apparatus according to certain embodiments of the presently disclosed subject matter;

[0061] Fig. 9 is an illustration of an exemplary degradable crosslinked polyethylene glycol (PEG) and a degradable linker used for the fabrication of an apparatus according to some examples of the presently disclosed subject matter;

[0062] Fig. 10 shows 'H-NMR spectrum of NE 2-127;

[0063] Fig. 11 shows 'H-NMR spectrum of NE 2-130;

[0064] Fig. 12 shows 'H-NMR spectrum of NE 2-133II (a duplicated experiment);

[0065] Fig. 13 shows 'H-NMR spectrum of NE 2-134;

[0066] Fig. 14A shows 'H-NMR spectrum of NE SG 2-145II;

[0067] Fig. 14B shows 'H-NMR spectrum of NE SG 2-145II (a duplicated experiment) after second precipitation;

[0068] Fig. 15 shows 'H-NMR spectrum of an overlay of methacrylic acid, NE-2-146I and NE-2-146II;

[0069] Fig. 16 shows products prepared by HA-90MA based resin (Tables 4A, 4B);

[0070] Figs. 17A and 17B show products prepared by HA-90MA and HA-7MA (Tables 5A, 5B);

[0071] Fig. 18A shows products prepared by HA-40MA and HA-30MA with Progesterone (Tables 8A, 8B);

[0072] Fig. 18B shows products prepared by HA-40MA and HA-30MA with Progesterone (Tables 8A, 8C);

[0073] Fig. 18C shows products prepared by HA-40MA and HA-30MA with Progesterone (Tables 8A, 8D);

[0074] Fig. 19A shows products prepared by HA-80MA with Progesterone (Tables 9A, 9B); and

[0075] Fig. 19B shows products prepared by HA-80MA with Progesterone (Tables 9A, 9C).DETAILED DESCRIPTION

[0076] To date, no specific optimal implantation site has been identified but rather unwanted areas are mapped and known. With this in mind, the aforementioned topic is under continued medical research and it is possible that in the future a specific individualized optimal implantation site will be identifiable on a patient by patient basis. In such a case, a site-specific implantation device, such as the instantly claimed apparatus 100, would be of upmost importance both in research, e.g., testing and validating research assumptions, and in implementing research conclusion by delivering an embryo 104 to pre-recognized locations.

[0077] Thus, in order to facilitate implantation of the fertilized ovum 104 in the uterus while minimizing interference with natural process of implantation and ovum-endometrial interactions, the present invention provides an injectable apparatus 100 designed to be inserted into a uteres and designed to pass through a catheter / insertion means 106, optionally while containing / holding a fertilized ovum 104 and media, wherein the apparatus 100 maximizes ovum-endometrial interaction by limiting the volume in which the ovum is contained while preventing implantation in unwanted sites and without blocking or interfering with ovumendometrium contact and interaction.

[0078] Accordingly, in a first aspect, the present invention provides an apparatus 100 for insertion into a uterus of a patient and adherence to an endometrium 102, i.e. , the inner epithelial layer, along with its mucous membrane, of the mammalian uterus, the apparatus 100 being essentially made of a biocompatible and biodegradable hydrogel comprising a crosslinked polymer, and comprising a cavity with surrounding walls 610, 612 and an opening 616, wherein: (i) the opening’s rim is designed to adhere to the endometrium 102; (ii) the cavity is of a size compatible with a fertilized ovum / embryo 104, which enables, upon adherence of the rim of the opening 616 to the endometrium 102, free movement of the ovum / embryo 104 therein; contacting of the ovum / embryo to the endometrium 102; and implanting of the ovum / embryo 104 in the endometrium 102; and (iii) the hydrogel is designed to biodegrade after a predefined period.

[0079] According to the present invention, the apparatus 100 disclosed is designed for insertion into a mammalian uterus and adherence to an endometrium of the uterus. The term “patient” as used herein thus refers to a mammal, such as human, livestock and other domestic animals including cows, sheep, goats, horses, and dogs. In a particular embodiment, the term “patient” refers to a human female.

[0080] The term “biocompatible” as used herein, e.g., with respect to the hydrogel composing the apparatus 100, refers to a property of a material (e.g., the polymer, crosslinker, glue) that does not cause substantially harmful response to the patient when introduced into the patient. For example, it means that when materials or apparatuses that are foreign to a patient are used, they do not induce substantially harmful reactions suchas inflammatory reaction and / or immune reactions. Biocompatible materials which may be used for the present disclosure include biodegradable or biosafety materials. Non-limiting examples of biocompatible polymers include hyaluronic acid (HA), also referred to as hyaluronan, or a salt thereof, polyethylene glycol (PEG), polylactide (PLA), polyglycolide (PGA), poly (lactic-co-gly colic acid) (PLGA), polyorthoester, polyanhydride, polyamino acid, polyhydroxybutyric acid, polycaprolactone, polyalkylcarbonate, ethyl cellulose, chitosan, starch, guargum, gelatin, collagen, albumin such as human serum albumin (HSA), and modified albumin such as a cationized bovine serum albumin (CB SA) and a cationized human serum albumin (CHSA).

[0081] The term “biodegradable” as used herein means that the apparatus 100 degrades under physiological conditions, more specifically under the physiological conditions in the uteres, i.e., under specific pH, temperature and enzymatic activity characterizing a uterus of the patient referred to. For instance, in a human female uterus, the pH ranges from about 4.42 to about 7.94 (depending on the region), and the temperature is about 36.5-38.5°C. One non-limiting example of an enzyme that can be found in the female uterus is hyaluronidase, which is an enzyme that degrades hyaluronic acid (HA).

[0082] The term “biodegradable polymer” as used herein refers to a polymer which is capable of being degraded into a low molecular weight material(s) through a degradation process such as a hydrolytic or enzymatic reaction. In certain embodiments, the crosslinked biodegradable polymer essentially composing the apparatus 100 is hyaluronan, optionally partially hydrolyzed, or a salt thereof, e.g., an alkaline metal or alkaline earth metal salt of hyaluronan such as sodium hyaluronate, potassium hyaluronate, and calcium hyaluronate.

[0083] According to the invention, the apparatus 100 is essentially made of a biocompatible and biodegradable hydrogel comprising a crosslinked polymer, i.e., may comprise, in addition to the crosslinked polymer, other components required so as to, e.g., stabilize the apparatus or attach it to the endometrium upon insertion into the uterus. The term “essentially” as used herein thus means that content of the crosslinked polymer within the material composing the apparatus 100 is about 20% by weight or more, e.g., about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98%, or more, by weight. The rest of the crosslinked polymer content is made of mainly water (and the crosslinker).

[0084] In certain embodiments of the apparatus 100, the adherence of the rim of the opening 616 to the endometrium 102 enables the fertilized ovum / embryo 104, after adherence of the opening’s rim to the endometrium 102, to reach / contact the endometrium 102 and implant therein. This is also enabled due to the fact that the cavity of the apparatus 100 is sufficiently large to enable free movement and / or rolling of the fertilized ovum / embryo 104 therewithin.

[0085] In certain embodiments, the size of the apparatus 100 is determined according to the size of the fertilized ovum / embryo 104 and according to the size of the uterus. For instance, for human use, in which the diameter of the fertilized ovum / embryo at the time of insertion into the uterus for implantation is about 100-200pm, the volume of the apparatus’s cavity is at least about 0.05mm3, such as 0.075, 0.085, 0.09, 0.095, 0.1, 0.125, 0.15, 0.175, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2 mm3or more. Alternatively, for cows’ use, for example, in which the diameter of an embryo is about l-2mm, the volume of the apparatus’s cavity is at least about 3mm3, such as 3.25, 3.5, 3.75, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 mm3or more.

[0086] In addition, the external dimensions of the apparatus 100 are designed to refrain from irritating the uteres. In specific embodiments, the apparatus 100 fits a catheter that is used for its insertion into the uterus, such as a 5.31-2.8FR catheter having a 1.77-0.93mm diameter. In specific embodiments, the length and / or diameter of the apparatus 100 does not exceed 5 mm.

[0087] In certain embodiments of the apparatus 100, the hydrogel enables, e.g., upon insertion of the apparatus 100 into the uterus, passage / diffusion of materials such as nutrients, signaling molecules, and fluids from the uterine-cavity into the cavity of the apparatus 100 (and vice-versa). In specific embodiments, the hydrogel permits passage of proteins, cytokines, and signal molecules of a molecular weight of up to about 1000 kDa, e.g., 900, 750, 500, 250, 100, 75, 50, 25, 10, 5, 1 kDa or less.

[0088] To avoid interference with the embryo’s growth, the apparatus 100 is designed to biodegrade within a predefined period. Accordingly, in certain embodiments of the apparatus 100, the hydrogel biodegrades after implantation of the fertilized ovum / embryo 104 in the endometrium 102 and does not interfere with the development and growth of the embryo. In certain embodiments, the biodegradable polymer-based hydrogel biodegrades within 4 to 10 days, such as within 4, 5, 6, 7, 8, 9, or 10 days, after insertion into the uterus. The degradation rate of the apparatus 100 mainly depends on the hydrogel composing the apparatus 100, i.e. , on the crosslinked polymer constituting the hydrogel, and the physiological conditions within the uterus of the mammalian referred to, but may depend on other parameters such as the size of the apparatus 100 as well.

[0089] In certain embodiments, the apparatus 100 is made of biocompatible material and is designed to adhere to the uterine cavity at the insertion I placement point for a predefined period of time until implantation of the fertilized ovum / embryo 104 in the endometrium occurs, and is designed to biodegrade thereafter. In certain embodiments, the biocompatible material comprising the apparatus 100 causes as little interference and uterine / endometrium irritation as possible. Moreover, after it biodegrades, the residues I degradants are also biocompatible and non-toxic, and do not cause any irritation to the developing embryo, the uterus or the female patient.

[0090] In certain embodiments, the apparatus 100 degrades after a predefined period, e.g., within about 4 days to about 10 days, to allow implantation of the fertilized ovum / embryo 104 in the endometrium 102. The degradation may be complete or partial, in both cases to a point of having no structural integrity or ability that might apply force on the developing embryo or limit its growth.

[0091] In specific embodiments, the apparatus 100 is made of a material that is: (i) biocompatible and adheres to the endometrial wall 102 until implantation of the embryo 104 is complete, and is shaped and sized to hold and retain a fertilized ovum / embryo 104. In further specific embodiments, the apparatus 100 comprises apertures, e.g., preparing the apertures during printing / fabricating the apparatus, and / or is made of a porous material enabling passage of materials, nutrients, signaling molecules, etc. (Table 1 below provide an exemplary list thereof), and fluids from the uterine-cavity / endometrium to the fertilized ovum / embryo 104; and (ii) designed to adhere to the uterine cavity at the insertion / placement point for a predefined period of time until implantation of the fertilized ovum 104 in the uterine wall occurs, and is designed to degrade thereafter.TABLE 1: LIST OF MATERIALS, NUTRIENTS, SIGNALING MOLECULES AND SIZES THEREOFTABLE 1: LIST OF MATERIALS, NUTRIENTS, SIGNALING MOLECULES AND SIZES THEREOF

[0092] In further specific embodiments, the rim of the opening 616 of the apparatus 100 comprises and / or is coated-with an adhesive agent, such as biocompatible glue or binding molecules, that adheres to the endometrium 102 until implantation of the fertilized ovum / embryo 104 is complete, e.g., about 2 to 5 days after insertion, while withstanding forces of normal daily activities.

[0093] In certain embodiments, the apparatus 100 is made of a material that can withstand sterilization, so that it can be sterilized prior to use.

[0094] In certain embodiments, the apparatus 100 is made of a porous material that enables passage of materials and fluids from the uterine / endometrial cavity (the inner space of the uteres) to the fertilized ovum / embryo 104 within the cavity of the apparatus 100. In further or alternative embodiments, the structure of the apparatus 100 comprises apertures 614 for enabling passage of such materials and fluids from the uterine / endometrial cavity to the fertilized ovum 102 within the apparatus’s cavity. In specific embodiments, the apparatus 100 is made of a porous material and also comprises apertures 614. In any case, the material and / or the apertures 614 permit passage of protein, cytokines and signal molecules or any other substance (having roughly ~100-1000kD).

[0095] Moreover, the apertures 614 reduce the area of the cavity -facing surface of the apparatus 100. This may reduce the chances that the fertilized ovum / embryo 104 adheres to the apparatus before it has implanted in the endometrium 102.

[0096] As noted above, opening’s rim is designed to adhere to the endometrium 102. This can be achieved by any number of ways. In certain embodiments, the apparatus 100 itself is made of a hydrogel that is characterized by having adherence capabilities to the endometrium 102 (i.e ., the biodegradable polymer-based hydrogel is characterized by having adherence capabilities to the endometrium 102). In alternative, or added, embodiments, the rim of the opening 616 is comprises an adhesive agent, for example by being coated therewith or by being formed from a material exhibiting adhesive qualities, such as a dedicated biocompatible glue or binding molecules. In specific embodiments thereof, the coating of the opening 616 with such an adhesive agent is carried out before insertion of the apparatus 100 into the uterus, e.g., immediately before insertion or during manufacture thereof. In alternative specific embodiments, the coating of the opening 616 with such an adhesive agent is carried out after insertion of the apparatus 100 into the uterus and before orduring contacting the endometrium 102, e.g., using a dedicated means for delivering such an adhesive agent to the opening 616 which is in the uterus. In further specific embodiments, such means for delivering such an adhesive agent constitutes part of the delivery system used for transferring the apparatus 100 into the uteres.

[0097] According to some examples, the rim may be formed to mechanically adhere to the endometrium, e.g., comprise a gripping structure configured to latch onto the endometrium to provide mechanical adhesion thereto, for example as is known in the art. The rim may be configured for mechanical adhesion and / or comprise an adhesive agent as described above, mutatis mutandis.

[0098] In further or alternative embodiments, the rim comprises a flange (see 618 in Fig. 8B) which extends in a direction away from the opening defined by the rim. The surface of the flange, at least in part, may be configured for adhering to the endometrium, for example as described herein. Accordingly, by increasing the contact surface area between the rim of apparatus 100 and the endometrium, the adhesion of the apparatus to the implantation site is increased.

[0099] In specific embodiments, the adherence of the apparatus to the endometrium 102 is designed to withstand forces of normal daily activities, such as walking, running, swimming, light exercise, etc.

[0100] In certain embodiments of the apparatus 100 of any of the embodiments above, passage of materials, such as nutrients, proteins, and liquids is carried out via the hydrogel matrix. The passage of such materials might not be possible just through the bulk hydrogel itself due to the limitation of the physical properties of the hydrogel itself, and some molecules might require dedicated passage apertures within the walls of the apparatus 100. Accordingly, in certain embodiments of the apparatus 100 of any of the embodiments above, in order to facilitate and / or improve such passage, and / or to enable passage of larger molecules that cannot pass through the hydrogel, the surrounding walls 610, 612 of the apparatus 100 further comprise apertures 614 enabling passage of materials such as nutrients, signaling molecules (e.g., as exemplified in Table 1 above), and fluids from the uterine-cavity into the cavity of the apparatus 100.

[0101] In specific embodiments, the apertures 614 have a diameter smaller than that of the fertilized ovum / embryo 104. For instance, when the apparatus 100 is intended for human use, the apertures 614 may have a diameter of up to about 100 pm, and when the apparatus 100 is intended for cow use, the apertures 614 may have a diameter of up to about 2-3 mm, thereby enabling passage of relatively larger molecules, e.g., up to lOOkDa, or up to lOOOkDa, while preventing the fertilized ovum / embryo 104 from exiting the cavity before it has the chance to implant in the endometrium 102.

[0102] In certain embodiments, the size and number of apertures 614 affects the biodegradation time of the apparatus 100. For instance, when more apertures 614 are present and / or when the apertures 614 are larger, the overall surface area of the apparatus 100 may be increased, and full biodegradation of the apparatus may thus be faster, and vice-versa.

[0103] The apparatus 100 can be in any shape and size, and may be designed to contain a cavity sufficient to freely retain a fertilized ovum / embryo 104 while maintaining an external size that minimizes irritation of the uterus. Accordingly, in certain embodiments, the apparatus 100 of any of the embodiments above is domeshaped, cylindrical, or half-cylindrical.

[0104] The shape and size of the apparatus 100 can vary according to need and desire, and may be shaped as dome-like, a half cylinder, or any other volume occupying shape consistent with after-mentioned criteria. The shape and volume of the apparatus 100 are designed to hold and retain a fertilized ovum / embryo 104, which defines the minimum volume of the cavity thereof, e.g., a volume of at least about ~3-5nL when referring to human embryo. The shape and volume of the apparatus 100 are also designed to refrain from interrupting the uterus and to prevent clogging or obstruction of fluid passage within the uteres. In specific embodiments, the apparatus 100 fits a 5.31-2.8FR catheter having a 1.77-0.93mm diameter. In specific embodiments, the length of the apparatus 100 does not exceed 5 mm.

[0105] According to examples in which the apparatus 100 has a generally half-cylindrical shape, e.g., as illustrated in Figs. 7A and 7B, the ratio of the area of the rim which adheres to the endometrium to the mass of the apparatus is high compared to other shapes, for example dome-shaped. Accordingly, adhesion of the apparatus may be increased. Moreover, as the apparatus 100 is typically inserted via a tube, whose diameter is constrained by phy siological considerations vis-a-vis the patient, an apparatus 100 having an elongate shape such as the half-cylindrical one illustrated in Figs. 7A and 7B may be provided with a larger volume of its cavity compared to other shapes, for example dome-shaped.

[0106] In certain embodiments, the apparatus 100 is constructed with or without an additional supporting element, e.g., for enhancing structural strength / integrity and / or for increasing the surface area that comes in contact with the endometrium 102 for improving adhesion (see, e.g., Figs. 8A & 8B).

[0107] The apparatus 100 is designed to be inserted into the uterus. The insertion may be performed using a narrow catheter having a diameter that may optionally be smaller than that of the apparatus 100. As such, the apparatus 100 will need to be able to bend, pressed and / or condensed to pass therethrough. Accordingly, in certain embodiments of the apparatus 100, the hydrogel is flexible. In specific embodiment thereof, the hydrogel is a shape memory material that returns to its original shape, e.g., after it is folded into a catheter 106 and released in the uterus.

[0108] In certain embodiments, the apparatus 100 is made of flexible shape memory material that returns to its original shape, e.g., after it is folded into a catheter and released in the uterus.

[0109] The apparatus 100 can be made of any suitable, biocompatible material or materials. In certain embodiments of apparatus 100 of any of the embodiments above, the polymer is selected from: hyaluronic acid (HA) having a molecular weight of from about 10 kDa to about 10 MDa, or a salt thereof; polyethyleneglycol (PEG) having a molecular weight of from about 10 kDa to about 1000 kDa, alginate, alginate-based compositions, albumin such as human serum albumin (HSA), modified albumin such as a cationized bovine serum albumin (CBSA) and a cationized human serum albumin (CHSA), dextran, chitosan, heparin, starch, glycogen, amylose, amylopectin, cellulose, xylan, natural and synthetic polysaccharides, etc., optionally functionalized, i.e., having a functional group(s), or any combination thereof. In specific embodiments, the polymer is a biodegradable polymer that can undergo biodegradation in the uterus.

[0110] The term “functional group” refers to any suitable chemical group that may be used to functionalize the biodegradable polymer so as to facilitate crosslinking thereof, either using a crosslinker, i.e., crosslinking agent, or a photo-initiator. Examples of suitable functional groups include, without being limited to, allyl group, and photoreactive groups such as acrylate or methacrylate groups, aryl azides such as phenyl azide, orl / zo-hydroxyphenyl azide, meto-hydroxyphenyl azide, tetrafluorophenyl azide, orl / zo-nitropphenyl azide, and meto-nitropphenyl azide, azido-methyl-coumarins, benzophenones, anthraquinones, certain diazo compounds, diazirines, and psoralen derivatives. Examples of crosslinkers that may be used for crosslinking the polymers include, without being limited to, a pentaerythritol based-crosslinker such as 2,2-bis(((3-mercaptopropanoyl)oxy)methyl)propane-l,3-diyl bis(3-mercaptopropanoate) (see, e.g., Fig. 9), an aldehyde such as glutaraldehyde or formaldehyde, a coordinating metal such as aluminum chromium, titanium, and zirconium, bisdiazobenzidine, phenol 2,4-disulfonyl chloride, l,5-difluoro-2,4-dinitrobenzene, urea, 3,6-bis(mercurimethyl)-dioxane urea, dimethyl adipimidate, or N,N'-ethylene-bis-(iodo-acetamide), an acrylate or methacrylate based linker such as acrylate esters, di-acrylate esters, di-halogens, di-acids, di-alcohols, and diamines, or any combination thereof. In certain embodiments, the crosslinker is a biodegradable crosslinker that can undergo biodegradation in the uterus.

[0111] In specific embodiments of the apparatus 100, the hydrogel is essentially made of HA or a salt thereof, functionalized with at least one allyl group and crosslinked with a pentaerythritol based-crosslinker such as 2,2-bis(((3-mercaptopropanoyl)oxy)methyl)propane-l,3-diyl bis(3-mercaptopropanoate). In alternative specific embodiments, the hydrogel is essentially made of HA or a salt thereof, functionalized with at least one photoreactive groups such as an acrylate or methacrylate moiety, i.e., acrylate ion, (CH2=CHCOO“), or methacrylic anhydride, and crosslinked. In some embodiments, the biodegradable polymer is HA functionalized by conjugation to methacrylic anhydride (MA). In some embodiments, the ratio of HA:MA in the conjugation reaction is about 1:0.5 - l:20HA:MA. In some embodiments, the ratio ofHA:MA in the HAMA conjugation reaction is selected from about 1:0.5, 1:1, 1:2, 1:5, or 1:13, and combinations thereof. In some embodiments, the ratio of HA:MA in the conjugation reaction is about 1:13.

[0112] In some embodiments, the conjugate is at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% conjugated.

[0113] Such functionalized HA polymers may be crosslinked, e.g., using a photo-initiator, such as lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). In other alternative specific embodiments, the hydrogel is essentially made of PEG functionalized with allyl group and crosslinked with a pentaerythritol based-crosslinker such as 2,2-bis(((3-mercaptopropanoyl)oxy)methyl)propane-l,3-diyl bis(3-mercaptopropanoate) (see, e.g., Fig. 9).

[0114] In some embodiments, the hydrogel further includes at least one active ingredient, which may be released from the hydrogel by diffusion or by hydrolysis of the matrix. Such active ingredients may serve to obtain a medical effect, and / or function as blockers in the hydrogel for enhancing the 3D printing resolution and / or quality. In some embodiments, the active ingredients includes a hormone, a local regulator, a growth factor, a cytokine, an extracellular matrix component, or an immune cell, such as those presented in Table 1. In some embodiments, the active ingredients include progesterone.

[0115] In certain embodiments, the apparatus 100 of any of the embodiments above is stored in dried form and is hydrated prior to use. Alternatively, the apparatus 100 is stored in a ready-for-use condition (i.e., “wet”). In further alternative embodiments, the apparatus 100 is fabricated immediately before use, e.g., using a 3D-printer.

[0116] In certain embodiments, the apparatus 100 of any of the embodiments above further comprises a fertilized ovum / embryo 104 within the cavity. In specific embodiments, the fertilized ovum / embryo 104 is free within the apparatus’s cavity.

[0117] In certain embodiments of the apparatus 100 according to any of the embodiments above, when the fertilized ovum / embryo 104 is placed in the apparatus 100 prior to its insertion into the uterus, the fertilized ovum / embryo 104 is temporarily attached to an inner surface of the cavity, e.g., by glue or binding molecules, during the transfer of the apparatus 100 into the uterus. This might be needed to prevent the fertilized ovum / embryo 104 from “falling out” of the apparatus 100 during its insertion into the uterus. This temporary attachment may be achieved by using a unique physical structure of the apparatus 100 and / or by using glue or binding molecules. Notably, after transfer and attachment of the apparatus 100 to the endometrium 102, the fertilized ovum / embryo 104 is automatically released from the apparatus 100 (without external intervention) and is free to move within the cavity I confined volume between the apparatus 100 and endometrium 102. In specific embodiments of the apparatus 100, the inner surface of the cavity is coated or comprises a glue or binding molecules designed to anchor the fertilized ovum / embryo 104 and prevent its rolling out the apparatus 100 during transfer into the uteres.

[0118] The apparatus 100 can be fabricated in any suitable way, such as 3D-printing, molding, etc. In certain embodiments, the fertilized ovum / embryo 104 is placed / inserted into the apparatus 100 after its fabrication and before insertion into the uterus. In alternative embodiments, the fabrication / assembling of the apparatus100 is performed around the fertilized ovum, such that the final apparatus 100 includes I holds the fertilized ovum 104, and is ready for transferring it into the uterus.

[0119] In a second aspect, the present invention provides a kit comprising: (i) an apparatus 100 according to any of the embodiments above; and (ii) a delivery tube 106, such as a catheter, having an inner diameter suitable for passage of the apparatus 100 that is optionally folded, and a length sufficient to reach into a uteres.

[0120] In specific embodiments, the kit further comprises a fertilized ovum / embryo injection means, such as a transfer needle 208 for injecting the fertilized ovum / embryo 104 into the cavity of the apparatus 100 after its adherence to the uterus. Optionally, such injection means is an integral part of the delivery tube 106.

[0121] In further or alternative specific embodiments, the kit further comprises means for applying an adhesive onto the rim of the apparatus’s opening 616 for adhesion thereof to the endometrium 102. This can be done either prior to insertion of the apparatus 100 into the uterus or after insertion immediately prior to attachment of the apparatus 100 to the endometrium 102.

[0122] In certain embodiments, the kit comprises more than one apparatus 100, such as 2, 3, 4, 5, or more apparatuses 100.

[0123] Notably, although the description relates to the presence of a single fertilized ovum / embryo 104 in each apparatus 100, it is to be understood that the apparatus according to any of the embodiments herein may comprise more than a single fertilized ovum / embryo 104, such as 2, 3, 4, 5, or more fertilized ovum / embryos 104.

[0124] In a third aspect, the present invention provides a method for implanting or assisting-implantation of a fertilized ovum 104 at a desired location on the endometrium 102 within the uterus of a patient, without forming a barrier around the fertilized ovum 104 that requires removal, and without changing I interfering the “natural” embryo-endometrium interaction and the contact of the embryo 104 with the endometrial cavity fluid and its soluble molecules.

[0125] By delivering and limiting the area of embryo implantation, as well as by limiting the volume in which the ovum / embryo moves / rolls in, the apparatus 100 may facilitate optimization of embryo-endometrial interaction by increasing the frequency and amount of interaction between the embryo and endometrium, which increases implantation chances, while also ensuring a safe implantation site. This is possible due to the fact that the fertilized ovum / embryo 104 is placed in the safe implantation site and cannot move therefrom due to the presence of the apparatus 100 that prevents the embryo 104 from drifting away from the desired implantation area and facilitates implantation at the selected site (illustrated in Figs. 1A & IB). The fact that the fertilized ovum / embryo 104 is restricted to move and roll around at an area defined by the apparatus 100 increases the amount of times the fertilized ovum / embryo 104 meets the endometrium 102 thereby increases meeting frequency and increases implantation chances. After embryo-endometrial interaction completion(i.e. , implantation of the embryo 104 in the endometrium 102), the apparatus 100 dissolves, leaving behind only the implanted embryo 104 (illustrated in Fig. 1C).

[0126] Accordingly, in certain embodiments, the present invention provides a method for delivering a fertilized ovum / embryo 104 to an implantation site on the endometrium 102 within the uterus of a patient, thereby preventing implantation at unwanted or unsafe sites and thus increasing successful pregnancy rates. The method may be carried out when the apparatus 100 of any of the embodiments above already holds / contains a fertilized ovum / embryo 104, or not.

[0127] Accordingly, in specific embodiments of the method, when the apparatus 100 does not hold / contain a fertilized ovum / embryo 104 prior to its insertion into the uteres, the method comprises the steps of: (a) providing an apparatus 100 or a kit according to any of the embodiments above, wherein the apparatus does not comprise the fertilized ovum / embryo 104; (b) inserting the apparatus 100 into the uterus and enabling its adherence / attachment to the endometrium 102 at the implantation site; and (c) following adherence of the opening’s 616 rim of the apparatus 100 to the endometrium 102, inserting the fertilized ovum / embryo 104 into the cavity of the apparatus 100, wherein: (i) the fertilized ovum / embryo 104 is free to contact the endometrium 102 via the opening 616, and implant therein; (ii) the apparatus 100 enables passage / diffusion of materials and fluids from the uterine-cavity to the cavity of the apparatus 100 (and vice-versa from the cavity to the uterine-cavity); and (iii) the apparatus 100 biodegrades and dissolves after a predefined period thereby not interfering with development and growth of the embryo. In specific embodiments thereof, the step of inserting the fertilized ovum / embryo 104 into the apparatus 100 after adherence / attachment of the apparatus 100 to the endometrium 102 is carried out by injecting the fertilized ovum / embryo 104 using a needle / tube / catheter 208 through the walls 610, 612 of the apparatus 100.

[0128] In alternative specific embodiments of the method, wherein the apparatus further comprises / holds / contains a fertilized ovum / embryo 104 prior to its insertion into the uterus, the method comprises the steps of: (a) providing an apparatus 100 with the fertilized ovum / embryo 104 within it, or kit comprising such an apparatus 100; and (b) inserting the apparatus 100 into the uterus and enabling its adherence / attachment to the endometrium 102 at the implantation site, wherein: (i) following adherence of the rim of the opening 616 of the apparatus 100 to the endometrium 102, the fertilized ovum / embryo 104 is free to contact the endometrium 102 via the opening 616, and implant therein; (ii) the apparatus 100 enables passage / diffusion of materials and fluids from the uterine-cavity to the cavity of the apparatus 100 (and vice-versa from the cavity to the uterine-cavity); and (iii) the apparatus 100 biodegrades and dissolves after a predefined period thereby not interfering with development and growth of the embryo. In specific embodiments thereof, the fertilized ovum / embryo 104 is positioned / placed within the apparatus 100 prior to insertion of the apparatus 100 into the uterus. In certain such embodiments, the method for delivery of anovum / embryo to a uterus or for assisting implantation of a fertilized ovum / embryo within a uterus of a female patient.

[0129] In certain embodiments of the method according to any of the embodiments above, the insertion of the apparatus 100 into the uterus is: (i) carried out under visualization, such as an ultrasound or an optic fiber; and / or by using a catheter (that is optionally part of the kit).

[0130] In certain embodiments, the catheter used has a diameter that fits the size of the apparatus, such as a 1.77-0.93mm diameter when using an apparatus that fits a human embryo, or a larger sized catheter when using a larger apparatus, e.g., for cows.

[0131] In certain embodiments of the method according to any of the embodiments above, the adherence / attachment of the apparatus 100 to the endometrium 102 is carried out using an adhesive agent such as glue / adhesion molecules that is applied onto the apparatus 100 either: during manufacture; before insertion into the uterus / or inside the uteres prior to attachment of the apparatus 100 to the endometrium 102.

[0132] Fig. 1A illustrates insertion / transfer of an apparatus 100 into the uterus using a designated catheter 106. After transferring the apparatus 100 and placing it on the endometrium 102 at a desired implantation site, the catheter 106 is removed leaving behind only the apparatus with the fertilized ovum 104 (Fig. IB). After a predefined period, in which the fertilized ovum / embryo 104 undergoes implantation in the endometrium 102, the apparatus 100 disassemble and dissolved leaving the fertilized ovum / embryo 104 implanted in the endometrium 102 without any barrier (Fig. 1C).

[0133] Figs. 2A-2F illustrate how the apparatus 100 is delivered using a catheter 106: during preparation, an apparatus 100 without a fertilized ovum / embryo is placed / inserted into a catheter 106, optionally in a folded form (as illustrated in Fig. 2A). Once the edge / opening of the catheter 106 is positioned at a desired implantation site, the apparatus 100 is pushed outwardly using, e.g., a wire, stick, needle or any other suitable means, to bring the apparatus 100 into contact with the endometrium. Optionally, the rim of the opening 616 of the apparatus comprises an adhesive. However, if not, it is possible (if needed) to add such an adhesive to assist the attachment of the apparatus 100 to the endometrium 102. As illustrated in Figs. 2D & 2E, while the apparatus 100 exits the catheter 106, it unfolds and returns to its original shape. Once attached in place, a fertilized ovum / embryo 104 is injected into the cavity of the apparatus 100, e.g., using a needle 208, followed by dissociation / disconnection of the apparatus 100 and extraction of the catheter 106, leaving behind only the apparatus 100 attached to the endometrium and holding the fertilized ovum / embryo 104. In alternative embodiments, the apparatus 100 is positioned at the end / tip of the catheter 106, such that after the catheter 106 is inserted and positioned at a desired implantation site, the apparatus 100 is simply detached / dislodged therefrom (and not pushed as described above).

[0134] The invention will now be illustrated by the following non-limiting examples.EXAMPLES

[0135] The required specifications of the apparatus 100, which combine the need for precise structural features, i.e., size, shape, internal cavity volume, porosity (to allow transfer of biological molecules and fluids), and short biodegradation time, demand a unique balance between stability and degradability. To address these challenges, the first goal was to develop a robust methodology for the preparation of dome-like structures that can be used to encapsulate a fertilized ovum.

[0136] Initial attempts to make such structures from biocompatible alginate-based materials, resulted in limited ability to control their shape, and their degradation time scale was significantly longer than desired. Later we used PEG-based monomers and crosslinkers that can be covalently conjugated upon irradiation to form shapeable crosslinked polymeric gels. We assumed that introducing hydrolysable moieties (such as esters) as part of the polymeric network will allow to fine tune the biodegradation rates of the formed hydrogels- see Fig. 9. Preliminary results showed that indeed by tuning the molecular structures of the PEG-based monomers, crosslinked hydrogels were formed, which showed suitable degradation rates.

[0137] Next, the porosity of the hydrogels was tested to ensure that nutrients and proteins that are present in the uteres will be able to pass through the hydrogel and reach the encapsulated blastocyst / fertilized ovum to allow its normal development. It was shown that the hydrogels had limited porosity, and that molecules bigger than IkDa could not diffuse through the polymeric hydrogel.

[0138] To address this challenge, i.e., lack of porosity of the bulk hydrogel, a 3D printer was used to design the upper part of the encapsulating apparatus 100 as a porous mesh. This allows creation of apertures 614 in the walls of the apparatus while precisely define the apertures’ sizes (e.g., about 10-100 micron) so that the required growth factors will be able to pass through the mesh, while the fertilized blastocyst / fertilized ovum (-200 micron) will not be able to escape through the apertures 614. To test this strategy, the suitability of PEG-based materials was tested when printed in a 3D printer. Taking advantage of the high precision of 3D printing, we could design pours mesh composed from degradable crosslinked gel, which allow large nutrients / proteins to reach the encapsulated blastocyst / fertilized ovum within the apparatus 100.

[0139] In addition to demonstrating the ability to design and fabricate porous structures, cellular toxicity of the hydrogels was tested by allowing them to degrade in cell medium, which was then used for growing cells. No apparent toxicity was observed in cells cultured in medium that contained the degraded hydrogels in comparison to cells grown in regular medium, both in terms of the number of cells and their shape.CELL ATTACHMENT ASSAY

[0140] After preparing a dome-shaped apparatus 100 with the desirable structure and porosity, a spheroidsendometrial cell attachment assay was established.

[0141] For spheroids generation, Jeg3 cells, a human trophoblast cell line, were cultured on a bactoagar coated 10 cm dish for 48 hours in a humid atmosphere containing 5% CO2 at 37°C. The resulting spheres were stained using calcein-AM and were monitored using G-BOX gel-imager, in order to test their viability and to count them.

[0142] Ishikawa, human endometrial cells were cultured on a 6-wells dish. Then, 3-5 labeled spheroids of 50-200 pm in diameter, similar in size to an implanting blastocyst, were transferred to the upper surface of the confluent monolayer of Ishikawa cells in each well. Then, each spheroid in three of the wells was covered with the apparatus 100, while in the other wells the spheroids were not covered as a control. This was conducted under a microscope. In order to set down the device for the purpose of the experiment a handle was added to the dome-shaped apparatus 100.

[0143] The spheroids were then co-cultured with the endometrial cells for 6 hours at 37°C. At the end of incubation, the apparatuses 100 were removed and non-adherent spheroids were removed by washing and shaking the culture plates. Counting of the attached spheroids was performed manually under a fluorescent microscope, by examining the stability of each spheroid upon tapping the stage.

[0144] The percentage of attached spheroids relative to the total number of spheroids used to initiate the coincubation experiments (adhesion percentage) was calculated. In wells where the spheres were covered with the dome-shaped apparatus 100, 78% of the spheres remained adherent at the end of the experiment, opposed to 69% of the spheres in wells without the apparatus 100. Thus, using the apparatus 100 has been proven not to cause any negative effect on the implantation of the embryo to the endometrium, and even presented an increase of about 9% in the attachment efficiency.HYALURONIC ACID (HA) vs. PEG

[0145] Due to limited prior safety data for intrauterine usage of PEG in pregnancy, Hyaluronic acid (HA) will be used for the next stage towards the development of the degradable apparatus. HA is a major component of the human extracellular matrix, giving elasticity and hydrophilicity to tissues, and therefore displays very good cell adhesion, biocompatibility, and biodegradability by hyaluronidase. HA is particularly abundant in the female reproductive tract, both in the oviduct and in the uteres, and it is reported to have an invaluable role in the process of embryo implantation. Several HA formulations have been approved by the FDA.

[0146] To obtain HA-based hydrogels, it must be used at high concentration (>2 mg ml'1) or chemically modified.

[0147] A functional version of HA was made, in which the HA was conjugated to methacrylic anhydride (MA) at various weight ratios of HA:MA, namely 1:13 (NE 2-127, Table 2, Fig. 10), 1:1 (NE 2-130, Table3, Fig. 11), 1:2 (NE 2-133, Table 4, Fig. 12), 1:5 (NE 2-134, Table 5, Fig. 13), 1:5 (NE-SG 2-145, Table 6, Figs. 14A, 14B). As can be seen from Table 3, different ratios resulted in different conjugation percentages.TABLE 2: PERCENT CONJUGATION OF DIFFERENT CONJUGATES

[0148] For the conjugate SG 2-145, the pH was maintained at between 8 to 10 during the reaction. In the end of the day the reaction was entered to the fridge for overnight. After overnight, the pH was 6.87. This was followed by precipitation in EtOH, filtration, continue with the white solid, high vacuum for overnight. An NMR of the result following the second precipitation is shown in Fig. 14B.

[0149] Additional conjugates were tried. A summary of the HA:MA ratios and the resulting % conjugation is provided in Table 3 below.TABLE 3: PERCENT CONJUGATION OF ALL CONJUGATES

[0150] Further experimentation will be done to evaluate adhesive and porosity properties, as well as optimizing HA formulation to meet printability, safety features and biodegradability.DEVELOPMENT AND OPTIMIZATION OF A HA FORMULATION WITH ADHERENCE CAPABILITIES

[0151] Various HA-based formulations, as well as various crosslinking techniques will be tested to find a final formulation that meets the following requirements: (a) printability, i.e. , that can be used in a 3D-printer to prepare stable structures with an inner space at various sizes and shapes; (b) high resolution, i.e., enabling generating structures with apertures of up to 0.1 mm to enable passage of materials and nutrients into an oocyte residing within the stmcturc while preventing the oocyte from exiting the structure through the apertures; (c) stability and degradability in the presence of hyaluronidase (an enzyme residing in the uterus and that degrades HA) the balance between stability and degradability will be determined using (i) various crosslinking techniques: for instance, higher crosslinking will yield slower degradable constructs, and vice-versa', and (ii) architecture of the structure, namely, thickness and volume-surface ratio: for instance, the thicker the stmcturc is, the slower the degrading; and the more porous the structure is, the faster the degrading.

[0152] The above conjugates were further crosslinked by using a photo-initiator (lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) andN,N-aminophosphine (PNP)) under conditions described below, and progesterone was further added in some formulations during printing. The crosslinking was achieved by the 3D printing process.

[0153] The formulations are provided in Tables 4A, 5A, 6A, 7A, 8A, and 9A, and the respective crosslinking conditions are provided in Tables 4B, 5B, 6B, 6C, 7B, 8B, 8C, 8D, 9B, and 9C. Figures demonstrating products prepared with the respective formulations are added in parenthesis in each table title.TABLE 4A: HA-90MA BASED RESIN (FIG. 16)TABLE 4B: HA-90MA BASED RESIN - CROSSLINKING CONDITIONS (FIG.16)Stage washed with water, Post curing - 15 minTABLE 5A: HA-90MA AND HA-7MA (FIGS. 17A, 17B)TABLE 5B: HA-90MA AND HA-7MA - CROSSLINKING CONDITIONS (FIGS. 17A, 17B)Stage washed with water, Post curing - 15 minTABLE 6A: HA-13MA AND HA-7MA ITH PROGESTERONETABLE 6B: HA-13MA AND HA-7MA ITH PROGESTERONE - CROSSLINKING CONDITIONSStage washed with water, Post curing - 15 min TABLE 6C: HA-13MA AND HA-7MA WITH PROGESTERONE - CROSSLINKING CONDITIONSStage washed with water, Post curing - 15 minTABLE 7A: HA-40MA AND HA-30MA ITH PROGESTERONETABLE 7B: HA-40MA AND HA-30MA ITH PROGESTERONE - CROSSLINKING CONDITIONSStage washed with waterTABLE 8A: HA-40MA AND HA-30MA WITH PROGESTERONE (FIGS. 18A-18C)TABLE 8B: HA-40MA AND HA-30MA WITH PROGESTERONE - CROSSLINKING CONDITIONS (FIG. 18A)Stage washed with waterTABLE 8C: HA-40MA AND HA-30MA WITH PROGESTERONE - CROSSLINKING CONDITIONS (FIG.18B)Stage washed with water and dried with airTABLE 8D: HA-40MA AND HA-30MA ITH PROGESTERONE - CROSSLINKING CONDITIONS (FIG.18C)Stage washed with water, curing - 30 secPROGESTERONE (FIGS.19A, 19B)TABLE 9B: HA-80MA WITH PROGESTERONE - CROSSLINKING CONDITIONS (FIG.19A)Stage washed with isprOH, curing - 30 secTABLE 9C: HA-80MA WITH PROGESTERONE - CROSSLINKING CONDITIONS (FIG.19B)Stage washed with isprOHTESTING ADHERENCE TECHNIQUES

[0154] Various techniques will be used to improve adhesion capabilities of the apparatus to the tissue. Such techniques will include addition of various molecules and materials, as well as structural modifications. For instance:

[0155] (1) Increasing surface area at the perimeter of a dome-structured apparatus by adding margins- this will provide larger contact area between the apparatus and the tissue, and improve adherence.

[0156] (2) Addition of residues / adhesion molecules on the apparatus’ margins, e.g., that resemble the embryo’s adhesion molecules. None limiting examples of such adhesion molecules are: integrins, cadherins and selectins.

[0157] (3) Adding hydrophobic and / or mucoadhesive fibers to the apparatus, such as alginate and chitosan.

[0158] The adherence layer / material needs to meet at least the following requirements: (a) adhere to the uterine tissue for a prolong period of time (7 days till implantation which occurs about 2-5 days after insertion into the uterus)- this is in order to allow the apparatus to perform its function and limit the embryo ’ s movement for the entire desired period of time; (b) dissolve or evacuated from the uterine cavity only after 5 days from its introduction to allow the apparatus and evacuate, and thus to make room for the evolving fetus; (c) will not harm the integrity or composition of the uterine tissue in any way in order to prevent harm / damage to the continued development of pregnancy; (d) will not be toxic to the mother and fetus; (e) withstand physical forces applied on the uterus due to daily activities; (f) made of biocompatible material; and (g) the adhesive agent and its decomposition-materials will not cause significant inflammatory reaction or be toxic to the uterine tissue or fetus.DESIGNING THE DELIVERY SYSTEM

[0159] The main features that will be taken into consideration are: (a) diameter- should be about 0.93-1.77 mm (FR5.31-2.8); (b) simplicity of use; (c) an easy loading capability of the structure and embryo within the delivery system; (d) a single- or dual-phase insertion of the structure and embryo to facilitate adherence of the apparatus to the tissue; and (e) adapting the delivery system to the structure of the apparatus while avoiding damage to the apparatus and its properties (e.g. in terms of stability, porosity, degradation rate, size, etc.) Ex- vivo AND IN- VIVO MODELS

[0160] The safety and efficiency of the apparatus in embryo implantation will be carried out using ex-vivo and in-vivo models. The preparation of such models includes, e.g., preparation of: (a) endometrium cells -epithelial cell lines (ECC-1) and human endometrial stromal cell lines; and (b) fluorescent-labeled ghost blastocyst. This can be done, e.g., using human first trimester commercial trophoblast cells (SW-71) and formation of GFP-expressing blastocyst-like spheroids (BLS-GFP).

[0161] To exclude the possibility that the apparatus’ structure and / or degradation products will affect the implantation of the embryo, fluorescent-labeled ghost blastocysts will be implanted with and without the apparatus, and with and without the apparatus degradation products.

[0162] After implantation, the apparatus will be removed and adherence and implantation will be assessed, e.g., using a fluorescent microscope.

[0163] The same experiments will be repeated while tilting and rotating to test physical movements and pressures on the implementation process.

[0164] The same experiments will be repeated ex-vivo in a uterus.

[0165] Safety tests will be carried out to examine possible negative effects of the apparatus components and degradation products, as well as the different adherence molecule, on the development of the embryos. This will be done on mouse embryos by producing mice embryos using known IVF procedures, e.g., by: (a) harmonically stimulating female mice; (b) retrieving eggs and in vitro fertilizing them; (c) incubating the fertilized eggs I embryos in a medium that includes apparatus’ degradation products and such adherence molecule; and (d) examining distribution rate, vitality, and morphology of the embryos.TESTING THE ENTIRE SYSTEM

[0166] After determining safety features, various parameters of the apparatus, adherence preferences, and the delivery system, the resultant system will be tested ex-vivo and in-vivo.

[0167] Ex-vivo experiments will involve the use of ex-vivo uterus, and insertion of the apparatus with a fluorescent-labeled blastocyte into the uterus using the selected delivery system. The uterus will be tilted and shaken, and then the adherence of the blastocytes will be examined.

[0168] In-vivo experiments will involve the use of an animal model, such as sheep. After retrieving eggs and in vitro fertilizing them, the fertilized eggs I embryos will be returned into the female animal using the selected delivery system. Finally, pregnancy and implantation of the embryos will be determined using standard techniques (chemical, ultrasound, etc.).

[0169] It will be recognized that examples, embodiments, modifications, options, etc., described herein are to be construed as inclusive and non-limiting, i.e., two or more examples, etc., described separately herein are not to be construed as being mutually exclusive of one another or in any other way limiting, unless such is explicitly stated and / or is otherwise clear. Those skilled in the art to which this invention pertains will readily appreciate that numerous changes, variations, and modifications can be made without departing from the scope of the presently disclosed subject matter, mutatis mutandis.

Claims

CLAIMS1. An apparatus configured to facilitate delivery of an embryo to an implantation site on an endometrium of a patient, the apparatus comprising one or more walls defining a cavity, the walls comprising a rim configured for adhering to the endometrium and defining an opening to the cavity;the walls comprising one or more throughgoing apertures, each sized to allow passage therethrough of intrauterine environmental molecules, and to prevent passage therethrough of a blastocyst;the material of the walls being configured to degrade in the intrauterine environment within a predetermined range of time.

2. The apparatus according to claim 1 , wherein the cavity is sized so as allow free movement therewithin of a blastocyst.

3. The apparatus according to any one of the preceding claims, the intrauterine environmental molecules comprising nutrients, signaling molecules, and / or hormones.

4. The apparatus according to any one of the preceding claims, the intrauterine environmental molecules comprising human chorionic gonadotropin, progesterone-R, and / or estrogen-R.

5. The apparatus according to any one of the preceding claims, the throughgoing apertures each being sized no smaller than to allow passage therethrough of a sphere having a diameter of about 50 pm.

6. The apparatus according to claim 5, the throughgoing apertures each being sized no larger than to allow passage therethrough of a sphere having a diameter of about 150 pm.

7. The apparatus according to claim 6, the throughgoing apertures each being sized no larger than to allow passage therethrough of a sphere having a diameter of about 100 pm.

8. The apparatus according to any one of the preceding claims, wherein the lower limit of the predetermined range of time allows for implantation of the embryo in the endometrium to occur before the walls degrade.

9. The apparatus according to any one of the preceding claims, wherein the upper limit of the predetermined range of time allows for the walls to degrade before the embryo is of a size which exceeds that of the cavity.

10. The apparatus according to any one of the preceding claims, wherein the rim comprises an adhesive agent.

11. The apparatus according to any one of the preceding claims, wherein the rim comprises a gripping structure configured to facilitate the adhering to the endometrium.

12. The apparatus according to any one of the preceding claims, being formed as an elongate member with the opening being formed along its length.

13. The apparatus according to claim 12, the walls defining a half cylindrical shape.

14. The apparatus according to any one of the preceding claims, the rim comprising a flange extending away from the opening and being configured for adhering to the endometrium.

15. The apparatus according to any one of the preceding claims, wherein the material of the walls is flexible.

16. The apparatus according to claim 15, wherein the material of the walls is a shape memory material.

17. The apparatus according to any one of the preceding claims, wherein the material of the walls comprises a hydrogel comprising a cross-linked polymer.

18. The apparatus according to claim 17, wherein the polymer comprises one or more selected from a group including hyaluronic acid, a salt of hyaluronic acid, polyethylene glycol, albumin, human serum albumin, a modified albumin, cationized bovine serum albumin, cationized human serum albumin, and combinations thereof.

19. The apparatus according to any one of claims 17 and 18, wherein the polymer comprises a functional group.

20. The apparatus according to any one of claims 17 through 19, wherein the polymer is crosslinked with a crosslinker selected from a group including a pentaerythritol based-crosslinker, an aldehyde, bisdiazobenzidine, phenol 2,4-disulfonyl chloride, l,5-difluoro-2,4-dinitrobenzene, urea, 3,6-bis(mercurimethyl)-dioxane urea, dimethyl adipimidate, N,N'-ethylene-bis-(iodo-acetamide), an acrylate-based linker, a di-halogen, a di-acid, a di-alcohol, a diamine, and combinations thereof.

21. The apparatus according to claim 17, wherein the polymer comprises hyaluronic acid or a salt thereof, functionalized with at least one allyl group and crosslinked with a pentaerythritol based-crosslinker.

22. The apparatus according to claim 17, wherein the polymer comprises hyaluronic acid or a salt thereof, functionalized with at least one acrylate moiety or methacrylate moiety, and crosslinked.

23. The apparatus according to claim 17, wherein the polymer comprises polyethylene glycol functionalized with at least one allyl group and crosslinked with a pentaerythritol based-crosslinker.

24. The apparatus according to any one of the preceding claims, wherein the material of the walls comprises at least one active ingredient.

25. The apparatus according to claim 24, wherein the at least one active ingredient comprises a progestogen.

26. The apparatus according to claim 25, wherein the progestogen is progesterone.

27. The apparatus according to any one of claims 24 through 26, wherein the at least one active ingredient comprises a hormone, a local regulator, a growth factor, a cytokine, an extracellular matrix component, and / or an immune cell.

28. The apparatus according to any one of claims 24 through 27, wherein at least one of the active ingredients is selected from a group including human chorionic gonadotropin, estrogen, progesterone-R, estrogen-R, early pregnancy factor, platelet-activating factor, luteinizing hormone, follicle-stimulating hormone, prostaglandin E2, prostaglandin F2a, cytokines, glycogen, HB-EGF, integrins, selectins, laminin, fibronectin, T-lymphocytes, macrophages, lectin concanavalin A, thromboxane B2, leukotriene C4, leukotriene B4, ILla, ILlb, IL6, CSF1R, CSF-1, TNF-a, LIF, GnRH, VEGF, IGFBP 1-6, IGF-1, IGF-2, TGF bl, TGF a, EGF, IFN-gamma, PDGF, prolactin, GH, renin, prorenin, endorphin, endothelin, corticotropin releasing hormone, uteroglobin, Lipocortin 1, parathyroid hormone like protein, and fibroblast growth factor.

29. The apparatus according to any one of the preceding claims, the material of the walls being a porous material, wherein the throughgoing apertures are provided by the porosity of the material.

30. A kit comprising:an apparatus according to any one of the preceding claims; anda delivery tube having an inner diameter suitable for passage therethrough of the apparatus, and a length sufficient to reach into a uterus of a patient.

31. The kit according to claim 30, further comprising an embryo injection means.

32. A method for delivering an embryo to an implantation site on the endometrium of a patient, the method comprising the steps of:a) providing an apparatus according to any one of claims 1 through 29;b) inserting the apparatus into the uterus and adhering it to the implantation site such that the cavity of the apparatus is open to the implantation site via the opening; andc) inserting the embryo into the cavity of the apparatus when the rim is adhered to the endometrium.

33. The method according to claim 32, wherein inserting the embryo into the cavity of the apparatus comprises injecting the embryo through the walls of the apparatus.

34. A method for delivering an embryo to an implantation site on the endometrium of a patient, the method comprising the steps of:a) providing an apparatus according to any one of claims 1 through 29;b) providing the embryo in the cavity of the apparatus; andc) inserting the apparatus with the embryo in its cavity into the uterus and adhering it to the implantation site such that the cavity is open to the implantation site via the opening.

35. The method according to claim 34, wherein the embryo is temporarily attached to a cavity-facing surface of the wall of the apparatus prior to insertion of the apparatus into the uterus.

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