Method for improving embryo implantation

Abstract

A method of increasing embryo implantation in a subject is disclosed, the method comprising administering to a uterine cavity of the subject an amount of a formulation comprising copper and / or zinc effective to stimulate endometrial production of leukemia inhibitory factor (LIF) and / or vascular endothelial growth factor (VEGF). Alternatively, a device may be inserted into the uterine cavity of the subject for a period of time effective to stimulate the endometrium to produce LIF and / or VEGF, where the device comprises copper and / or zinc. The methods are suitable for women treated by any of assisted reproductive techniques, such as those involving embryo transplantation, such as in vitro fertilization (IVF) and variants including IVF-ICSI (Intrauterine Single Sperm Injection) and in vitro maturation (IVM) therapy, and techniques involving intrauterine artificial insemination (IUI) therapy. However, the methods described are also applicable to women who wish to improve pregnancy prospects by natural fertilization.

Description

[0001] Divisional application

[0002] This application is a divisional application of application number 201980042077.6, application date May 29, 2019, and invention name “Method for Improving Embryo Implantation”. Technical Field

[0003] The present disclosure relates to the field of assisted reproductive technology, such as in vitro fertilization (IVF). More specifically, the present disclosure relates to methods, formulations, and devices for improving implantation of transplanted embryos in a subject.

[0004] Priority document

[0005] This application claims priority to Australian provisional patent application number 2018901900 filed on May 29, 2018, entitled “Method for enhancing embryo implantation”, the contents of which are incorporated herein by reference in their entirety. Background Art

[0006] Although the great progress of IVF treatment in the past three decades has greatly promoted the understanding of how to optimize in vitro embryo development, there has been little progress in the method for optimizing or improving the successful implantation of transplanted embryos. Even today, the transplantation of genetically normal embryos with good morphology into the uterus of a "fertile" recipient (i.e., a woman without her own reproductive potential barrier, such as pure male factor infertility) still cannot guarantee successful implantation, with up to half of these "optimal" embryos failing to implant (Dahdouh et al., 2015). Therefore, compared with species (such as mice and rabbits) with more than 95% successful embryo implantation, human IVF can be considered to be very inefficient (Valbuena et al., 2017). In addition, many women who receive IVF treatment have transplanted multiple high-quality embryos but have never successfully become pregnant, which shows the main obstacle to implantation in this recurrent implantation failure (RIF) cohort. In addition, many women do not have children because their embryos have failed to implant successfully, rather than being unable to produce embryos (Martin, 1995), which of course means potential implantation defects.

[0007] To date, the only treatments known to increase implantation in RIF involve destruction of the endometrium through dilation and curettage (also known as “endometrial scraping”) (Nastri et al., 2015) or insertion and subsequent removal of a contraceptive intrauterine contraceptive device (IUCD) (Mao et al., 2017). While it is not fully understood how this procedure improves implantation, it has been reported that the inflammatory response associated with endometrial destruction leads to the release of cytokines and growth factors known to enhance embryonic development (Liang et al., 2015). However, these current treatments for RIF have several drawbacks. First, they are typically performed at least one menstrual cycle before the actual embryo transfer, allowing the damaged endometrium to be shed with menstruation and have time to repair itself. Disruption of the endometrium (dilation and curettage) during the same menstrual cycle as embryo transfer has been reported to impair implantation, as this procedure disrupts the integrity of the endometrium and is associated with heavy bleeding in the uterine cavity (Karimzade et al., 2010). Second, undergoing an endometrial biopsy or IUCD insertion is painful for the recipient. Therefore, an ideal treatment would be one that is relatively painless, has no side effects, and can be performed during the same menstrual cycle as the intended embryo transfer.

[0008] It is unclear why human reproduction is less efficient than in most other animal species. However, significant interspecies differences in early reproductive events may contribute to this anomaly and provide some insights into how implantation in humans might be improved. In prolific breeders such as rodents, rabbits, and pigs, the cervix remains open before and after copulation, allowing large amounts of seminal plasma and sperm to infiltrate the endometrium, initiating the production of growth factors and cytokines and, in addition, leading to a pronounced post-coital uterine inflammatory response (Schjenken and Robertson, 2014; Robertson and Sharkey, 2016). This inflammatory response to semen remodels the uterine lining and promotes embryonic growth and development. Surgical removal of male accessory glands (prostate, seminal vesicles), thereby preventing uterine exposure to seminal plasma, leads to a significant reduction in the uterine inflammatory response and impaired implantation, highlighting the reproductive importance of this response (Schjenken and Robertson, ibid.; Robertson and Sharkey, ibid.). However, the female uterine cavity is exposed to relatively small numbers of sperm and associated seminal plasma proteins because sperm must pass through a cervical mucus "filter" before reaching the uterus (Schjenken and Robertson, supra). This significantly different reproductive physiology between species means that females are less likely to be exposed to semen after sexual intercourse and to elicit an inflammatory response that could enhance implantation compared to animals that ejaculate intrauterine ejaculation.

[0009] In addition to seminal plasma, other triggers of uterine inflammation have been reported. For example, zinc and copper metal ions are known to be cytotoxic to endometrial cells in culture (Wu et al., 2012) and produce an intrauterine inflammatory response when exposed to IUCDs (Sadovsky et al., 1975; Stanford and Mikolajczyk, 2002). Although it is known that this IUCD-associated endometrial inflammation is contraceptive, thereby impairing successful embryo implantation (Kelly et al., 1969; Sheppard, 1987; Stanford and Mikolajczyk supra), the present inventors hypothesize that shorter exposure to copper and / or zinc ions for a shorter time or at lower doses may produce a less intense inflammatory response, which may be able to upregulate the production of beneficial cytokines (such as vascular endothelial growth factor (VEGF) and leukemia inhibitory factor (LIF)), but not be sufficient to cause the harmful endometrial destruction commonly observed in IUCD users.

[0010] The implantation process of the embryo is a complex cascade of events that requires the precise interaction between the paracrine (e.g., hCG and cytokines) of the embryo itself on the endometrium by the action of ovarian steroids (e.g., estrogen and progesterone) and the endometrial secretion (e.g., hCG and cytokines) (Norwitz et al., 2001). After ovulation and subsequent elevation of serum progesterone, endometrial glandular epithelial cells are converted into highly active secretory cells that synthesize various substances (e.g., nutrients, growth factors, and cytokines) that can improve the development of the embryo and alter endometrial function. The production of LIF and VEGF by the uterine epithelium is two key cytokines that play a key role in assisting implantation (Norwitz et al., supra).

[0011] Evidence supporting a key role for LIF in implantation

[0012] There is a lot of evidence that LIF plays a key role in embryo implantation, such as:

[0013] 1. In LIF “knockout” mice (i.e., mouse models that do not produce LIF), normal embryos are produced, but they fail to implant in the uterus (Stewart et al., 1992; Chen et al., 2002). However, transplantation of these LIF- / - embryos into wild-type (LIF-producing) mice, or artificial delivery of exogenous LIF to LIF- / - mice by injection on day 4 of gestation, restores normal implantation ability (Stewart et al., supra; Chen et al., supra). Furthermore, tissue-specific ablation of LIF receptors in the mouse uterine epithelium also results in implantation failure (Cheng et al., 2017). All of these findings indicate that LIF activity is absolutely essential for implantation in mice.

[0014] 2. The peak production of LIF by the endometrium occurs during the luteal phase of fertile women, the exact time when the embryo normally begins to implant (Charnock-Jones et al., 1994; Cullinan et al., 1996).

[0015] 3. Endometrial LIF production is reduced in women with repeated IVF implantation failure (Mikolajczyk et al., 2007; Choi et al., 2016), suggesting that LIF plays a crucial role in the implantation process.

[0016] 4. It is reported that the addition of LIF to cultured mouse embryos promotes endometrial development to the "hatched" blastocyst stage and increases the growth of trophoblast cells in vitro (Lavranos et al., 1995). Similarly, it is reported that exposing mouse embryos to LIF in vitro and subsequently transferring them using a transcervical technique similar to human IVF improves implantation and pregnancy rates (Mitchell et al., 2002).

[0017] 5. In contrast, it has been reported that blocking LIF action in vitro using LIF neutralizing antibodies (Mitchell et al., supra) or polyglycosylated LIF antagonists (Lalitkumar et al., 2013) inhibits implantation potential in mice and humans, respectively. In addition, it has been shown that delivery of LIF neutralizing antibodies into the uterine cavity on day 5 of gestation significantly impairs implantation in rhesus monkeys (Sengupta et al., 2006).

[0018] 6. Endometrial hypoplasia (thin endometrium), which may be caused by insufficient endometrial vascular perfusion (Jinno et al., 2001), is a known cause of implantation failure. There is significant evidence that LIF is involved in angiogenesis in the endometrium and placenta (Alfer et al., 2017).

[0019] In summary, endometrial LIF production increases after ovulation and appears to assist implantation by enhancing embryonic development, while also initiating events in the endometrium that appear to be critical for initial embryo attachment and invasion (i.e., implantation). Furthermore, low endometrial LIF production is associated with reduced IVF success rates and infertility of unknown etiology. Furthermore, exposure to copper in the form of copper-containing intrauterine devices (IUCDs) has previously been reported to reduce endometrial LIF expression and impair implantation (Güney et al., 2007). However, the effects of shorter-term copper (or zinc) exposure on endometrial LIF production remain unclear.

[0020] Evidence supporting a key role for VEGF in implantation

[0021] There is also a lot of evidence that VEGF plays a key role in embryo implantation, including:

[0022] 1. VEGF production peaks in the endometrium during the mid-luteal phase of fertile women, the exact time when embryo implantation normally begins. However, this mid-luteal peak in VEGF production is not seen in women with repeated IVF implantation failure (Jee et al., 2009), suggesting that VEGF plays a crucial role in the implantation process.

[0023] 2. VEGF triggers endometrial edema by increasing vascular permeability and enhancing the production of vasodilators (Rockwell et al., 2002). Edema of endometrial tissue (decidua) brings the uterine surface into close proximity with the blastocyst and appears to be important in establishing receptivity (the positioning phase for implantation).

[0024] 3. Genetic polymorphisms that impair VEGF gene expression (e.g., VEGF+405CC polymorphism) have been found to be more common in women with recurrent IVF implantation failure (Boudjenah et al., 2012), and VEGF+405CC polymorphism has also been associated with lower implantation rates in “potentially fertile” women undergoing IVF due to pure male factor infertility (Boudjenah et al., 2014).

[0025] 4. Blockade of VEGF activity by administration of VEGF neutralizing antibodies has been shown to prevent implantation in both primate and rodent pregnancy models (Rockwell et al., supra; Ghosh and Sengupta, 2005).

[0026] 5. Application of VEGF to embryos in culture has been shown to increase their developmental speed / cell division rate (Hannan et al., 2011; Binder et al., 2014).

[0027] 6. Exposure of human uterine epithelial cells to VEGF in vitro increases their adhesion to common extracellular matrix (ECM) proteins found on the embryonic surface (i.e., fibronectin and collagen), potentially assisting the initial attachment of the embryo to the uterine epithelium (Binder et al., supra).

[0028] 7. It has been reported that glandular expression of VEGF in early luteal endometrial biopsies was significantly higher in women who successfully conceived during IVF compared with those who did not conceive (Jinno et al., supra; Seo et al., 2011).

[0029] 8. It has been reported that in women experiencing repeated IVF implantation failure, levels of VEGF in endometrial secretions obtained from uterine lavage (irrigation of the uterine cavity with sterile saline during the mid-luteal phase) were significantly lower than those seen in fertile controls (Hannan et al., supra), suggesting that VEGF may assist implantation.

[0030] 9. Culturing mouse embryos in VEGF increased their adhesion to extracellular matrix (fibronectin)-coated plates in vitro and also increased the successful implantation of these VEGF-exposed embryos when transferred into pseudopregnant recipients (Binder et al., supra).

[0031] 10. VEGF is known to play a key role in the development of new capillaries (angiogenesis) and vascular perfusion of tissues, among which the development of thin endometrium is associated with low capillary density and endometrial stromal VEGF expression (Miwa et al., 2009).

[0032] In summary, available evidence suggests that endometrial-derived VEGF plays a key role in promoting implantation by accelerating preimplantation embryo development, enhancing adhesive interactions between the embryo and the uterine epithelium (improving binding to ECM proteins), and potentially increasing endometrial stromal edema. A deficiency in VEGF action (e.g., through poor production by epithelial cells or by blocking VEGF action) impedes implantation. Therefore, it has been proposed that therapies that increase endometrial VEGF production could increase the successful implantation rate of human embryos during IVF treatment. Although copper has previously been reported to trigger increased endometrial VEGF production in IUCD users (Xin et al., 2004; Rafi et al., 2013), it remains unknown whether a “short burst” of copper ion exposure or copper delivery outside of the “foreign body” setting of an IUCD triggers a similar VEGF response without causing endometrial disruption, as seen in IUCD users (Sadovsky et al., 1975; Stanford and Mikolajczyk, 2002). Similarly, prior to the experiments described below, the effects of short-term zinc exposure on endometrial VEGF production were unknown.

[0033] The present disclosure relates to the field of assisted reproductive technology, such as IVF and intrauterine insemination (IUI), and more particularly to a method, formulation, and apparatus for improving implantation of a transplanted embryo in a subject. Methods (and formulations and apparatus) involving the use of copper ions and, optionally, zinc ions, may provide a simple and relatively inexpensive treatment method for improving embryo implantation, particularly in women who have experienced recurrent implantation failure (RIF) following IVF or other assisted reproductive technology treatments. Summary of the Invention

[0034] In a first aspect, the present disclosure provides a method for improving embryo implantation in a subject (i.e., a woman or other female animal), wherein the method comprises administering to the uterine cavity of the subject a formulation comprising copper and / or zinc in an amount effective to stimulate the endometrium to produce leukemia inhibitory factor (LIF) and / or vascular endothelial growth factor (VEGF).

[0035] In a second aspect, the present disclosure provides a method for improving embryo implantation in a subject, wherein the method comprises inserting a device comprising copper and / or zinc into the uterine cavity of the subject for a period of time effective to stimulate the endometrium to produce leukemia inhibitory factor (LIF) and / or vascular endothelial growth factor (VEGF).

[0036] The method is particularly suitable for use in women being treated with any of the assisted reproductive techniques, and particularly those involving embryo transfer, such as in vitro fertilization (IVF) and variations including IVF-ICSI (intracytoplasmic sperm injection).

[0037] In a third aspect, the present disclosure provides a formulation for improving embryo implantation in a subject, the formulation being suitable for administration to the uterine cavity and comprising an amount of copper and / or zinc effective to stimulate the endometrium to produce leukemia inhibitory factor (LIF) and / or vascular endothelial growth factor (VEGF), and wherein the formulation optionally comprises one or more pharmaceutically acceptable carriers and / or excipients.

[0038] In some preferred embodiments, the copper and / or zinc are provided in the form of a solution, such as a solution comprising copper chloride (CuCl2) and / or zinc chloride (ZnCl2). BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG1 provides graphical results showing the effects of metal ions provided by CuCl2 (A) and ZnCl2 (B) solutions on LIF production by cultures of the human Ishikawa endometrial adenocarcinoma cell line (Ishikawa cells);

[0040] FIG2 provides graphical results showing the effect of metal ions provided by CuCl2 (A) and ZnCl2 (B) solutions on VEGF production by cultures of Ishikawa cells;

[0041] Figure 3 shows the results of an experiment evaluating the amount of copper and zinc ions released from metal implants into Ishikawa cell cultures in αMEM or G2 medium. Metal implants (A) copper sheet implants (Cu) and (B) copper-zinc sheet bimetallic implants (CuZn) were placed in the cell culture for 2 and 5 minutes;

[0042] FIG4 provides graphical results showing the effects of metal ions released from various metal implants (i.e., copper sheet implants (Cu), copper zinc sheet bimetallic implants (CuZn), zinc sheet implants (Zn), zinc aluminum sheet bimetallic implants (ZnAl), and zinc gold sheet bimetallic implants (ZnAu)) on (A) LIF production and (B) VEGF-A production by Ishikawa cells. Measurements were taken from the culture supernatant after 8 hours of culture. Cells were exposed to the metal implants for 2 minutes;

[0043] FIG5 provides graphical results comparing stimulation of (A) LIF production and (B) VEGF production by Ishikawa cells cultured in "conditioned medium" (i.e., including copper and zinc ions); the LIF production experiment also included a positive control in which Ishikawa cells were cultured in medium exposed to a copper-zinc bimetallic implant (CuZn) for 2 minutes;

[0044] FIG6 provides graphical results obtained after culturing Ishikawa cells exposed to metal implants (i.e., copper implants (Cu), zinc implants (Zn), and copper-zinc bimetallic implants (CuZn)) for 2 and 5 minutes for 8 hours. These figures show the effects of metal implant exposure on LIF production (A, B) and VEGF production (C, D);

[0045] Figure 7 Examples of images showing embryo implantation growth of day 3 mouse embryos after culture in the following media are provided: (A) fresh G2 medium (control), (B) G2 medium conditioned with Ishikawa cell culture, and (C) G2 medium conditioned with Ishikawa cell culture exposed to a copper-zinc bimetallic implant for two minutes; and

[0046] Figure 8 shows the embryo implantation growth (embryo attachment surface area, mm) of mouse embryos cultured in the following culture medium on day 3. 2 Results of the following experiments: fresh G2 medium (control), G2 medium conditioned with Ishikawa cells, and G2 medium conditioned with Ishikawa cells exposed to a copper-zinc bimetallic implant for two minutes. Results were evaluated at 66 hours (A) and 90 hours (B). DETAILED DESCRIPTION

[0047] In the experiments described below, the present inventors found that copper ions (Cu 2+ ) and / or zinc ions (Zn 2+) produced increased amounts of leukemia inhibitory factor (LIF) and vascular endothelial growth factor (VEGF), both of which are believed to play an important role in embryo implantation. In addition, when day 3 embryos were grown in a "conditioned" medium including metal ions, the surface area for trophoblast cell growth was significantly increased, indicating that embryo health and implantation potential were improved. Therefore, the present disclosure relates to novel methods (as well as formulations and devices) that involve using copper ions and optionally zinc ions to increase endometrial production of LIF and / or VEGF to improve embryo implantation in women (and other female animals), including women who have experienced recurrent implantation failure (RIF) after IVF or other assisted reproductive technology treatments.

[0048] In a first aspect, the present disclosure provides a method for improving embryo implantation in a subject (i.e., a woman or other female animal), wherein the method comprises administering to the uterine cavity of the subject a formulation comprising copper and / or zinc in an amount effective to stimulate the endometrium to produce leukemia inhibitory factor (LIF) and / or vascular endothelial growth factor (VEGF).

[0049] The methods are suitable for women undergoing treatment with any of the assisted reproductive technologies, such as those involving embryo transfer, such as in vitro fertilization (IVF) and variations including IVF-ICSI (intracytoplasmic sperm injection) and in vitro maturation (IVM), and those involving intrauterine insemination (IUI) therapy. However, the methods are also suitable for women who wish to improve their prospects of pregnancy by conceiving naturally.

[0050] The methods described may be particularly useful for women who exhibit hypoplastic or "thin" endometrium (e.g., an endometrial thickness of 7 mm or less), which is a recognized cause of infertility and implantation failure after embryo transfer (Kasius et al., 2014; Yuan et al., 2016). In this regard, it is believed that by stimulating the endometrium to produce LIF and / or VEGF, which are believed to stimulate endometrial angiogenesis, the methods described can be performed to improve angiogenesis and thereby treat implantation failure associated with thin endometrium. Therefore, the present disclosure also extends to methods for treating women experiencing hypoplastic endometrium. Women experiencing hypoplastic endometrium can be easily identified by standard ultrasound imaging (e.g., transvaginal ultrasound).

[0051] Furthermore, while the present disclosure focuses on females, one skilled in the art will recognize that the methods (and formulations and devices) disclosed herein are also applicable to other non-human female subjects (i.e., other female animals), such as, for example, livestock (e.g., cattle, horses, and sheep), wild animals (e.g., pandas, large cats such as tigers and lions, elephants, etc.), and companion animals (such as dogs and cats), particularly when assisted reproductive techniques (particularly those involving embryo transfer) are employed to assist in achieving pregnancy.

[0052] In some embodiments, the formulation used in the method of the first aspect comprises copper and optionally zinc.

[0053] In other embodiments, the formulation comprises copper or copper and zinc.

[0054] In still other embodiments, the formulation comprises zinc.

[0055] Copper and / or zinc can each be present in the formulation in one or more of the following forms: elemental (e.g., pure), alloy, complex, oxidized, salt (including esterified salts and other salts formed from organic acids), or solution. Preferably, the copper and / or zinc is present in a form that can provide a source of copper / zinc ions in utero. Such forms may include nanoparticles. The formulation can be in the form of, for example, a liquid, semisolid, or solid dosage form.

[0056] In some embodiments, copper and / or zinc are provided in the form of a solution, such as a solution comprising copper chloride (CuCl2) and / or zinc chloride (ZnCl2), a solution comprising copper sulfate (CuSO4) and / or zinc sulfate (ZnSO4), copper gluconate and / or zinc gluconate, a solution of copper chlorate (Cu(ClO4)2) and / or zinc chlorate (Zn(ClO3)2), a solution comprising copper nitrate (Cu(NO3)2) and / or zinc nitrate (Zn(NO3)2), or a solution comprising copper bromide (CuBr) and / or zinc bromide (ZnBr2), or a mixture thereof (e.g., a solution of CuCl2 and copper gluconate or a solution of CuCl2 and ZnSO4). Such solutions can provide a source of copper / zinc ions in utero.

[0057] In other embodiments, copper and / or zinc are provided in the form of a semisolid (e.g., gel or foam). For example, a gel comprising CuCl2 and / or ZnCl2, or a gel comprising nanoparticles of copper and / or zinc or nanoparticles of CuCl2 and / or ZnCl2. The gel can, for example, provide a short-term depot dosage form in the uterus. As will be appreciated by those skilled in the art, a biodegradable gel depot can also be formed in situ using well-known injectable in situ depot-forming drug delivery systems. Such a gel can provide a source of copper / zinc ions in the uterus that can achieve controlled and / or sustained release of copper / zinc ions. In some specific embodiments of suitable gel formulations, copper and / or zinc can be formulated with hydroxyethyl cellulose and glycerol, and in one example, copper and / or zinc can be formulated with hydroxyethyl cellulose and glycerol. (IQ Medical Ventures BV; Rotterdam, The Netherlands).

[0058] In still other embodiments, the copper and / or zinc are provided in a solid dosage form. For example, the copper and / or zinc can be provided as small soluble crystals or other particles (e.g., powders or nanoparticles of the metal). For example, crystals or other particles of CuCl and / or ZnCl. Such crystals or particles can be administered in a suitable carrier liquid or gel (e.g., which can be prepared shortly before administration), or in the form of soluble tablets or capsules administered by a suitable uterine delivery device. Such crystals or other particles can provide a source of copper / zinc ions in the uterus.

[0059] It should be understood that the preparation is not an IUCD.

[0060] Furthermore, it will be appreciated that the formulation used in the method of the first aspect is preferably not provided as any solid form or within any device that requires surgical removal from the uterus following treatment. In this regard, the dosage form of the above-described embodiments will, in many (if not all) cases, allow for relatively non-invasive and "comfortable" treatment by allowing it to be administered in a simple manner (e.g., by using a device such as a flexible catheter that is "loaded" with the formulation and passed through the cervical canal and into the uterine cavity) without the need for subsequent removal of the formulation.

[0061] The formulation may comprise one or more pharmaceutically acceptable carriers and / or excipients, which may vary depending on the dosage form to be employed. For example, for solutions of copper and / or zinc salts, the formulation may comprise water or physiological (isotonic) saline. For gels comprising copper and / or zinc salts, the formulation may comprise biocompatible and biodegradable polymer excipients (e.g., lactide / glycolide polymers and hydroxyethyl cellulose, etc.); in such gels, copper and / or zinc may be present in the form of esterified salts, particularly salts comprising saturated fatty acids such as decanoic (capric) acid (e.g., copper (2) decanoate).+ )), or salts of other organic acids, such as salts of D-gluconic acid (e.g., copper gluconate). As will be appreciated by those skilled in the art, the formulation may further comprise one or more substances selected from the group consisting of preservatives (e.g., sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid), binders (e.g., starch, gelatin, natural sugars (such as glucose and anhydrous lactose), and natural and synthetic gums (such as acacia), hydroxyethylcellulose, carboxymethylcellulose, and polyethylene glycol), lubricants (e.g., sodium oleate, sodium stearate, magnesium stearate, etc.), humectants (e.g., glycerol), antioxidants, suspending agents, stabilizers, coating agents, and solubilizers, as required and / or desired for a particular dosage form.

[0062] In addition, the formulation may contain one or more other substances that stimulate the production or activity of LIF, VEGF, or other cytokines, growth factors, etc. (e.g., integrins) that may be beneficial to embryo implantation. For example, the formulation may also contain an effective amount of benzoic acid, which is believed to increase the expression of LIF and integrins V3 and V5 to promote embryo implantation (Korean Patent Publication No. 20160108690; the entire contents of which shall be deemed to be incorporated herein by reference).

[0063] The formulation is administered to the subject in an amount effective to stimulate the endometrium to produce leukemia inhibitory factor (LIF) and / or vascular endothelial growth factor (VEGF).

[0064] In some embodiments, the amount is typically an amount that provides at least 500 ppb of copper ions to the uterine fluid and / or an amount that provides at least 50 ppb of zinc ions to the uterine fluid. However, preferably, the formulation is administered to the subject in an amount that provides at least 1250 ppb (more preferably, at least 2500 ppb) of copper ions to the uterine fluid and / or at least 100 ppb (more preferably, at least 250 ppb) of zinc ions to the uterine fluid. As will be readily understood by those skilled in the art, uterine fluid samples can be obtained by aspiration using an embryo transfer catheter (Ametzazurra et al., 2009) and their metal ion content assessed using mass spectrometry. However, the appropriate amount of the formulation (i.e., providing at least 500 ppb of copper ions to the uterine fluid and / or providing at least 50 ppb of zinc ions to the uterine fluid) can be tested in other ways by adding different amounts of the formulation and amount to "model" uterine fluid (e.g., a volume of 5 ml of a suitable medium, such as αMEM or G2 medium or a suitable embryo transfer medium) at 37°C and assessing the metal ion content by mass spectrometry in samples of the medium collected at appropriate time points (e.g., 2 minutes and / or 5 minutes after the addition of the formulation). It is believed that the "release" of copper and / or zinc ions into the culture medium will be substantially equivalent to the release that would occur in the uterus. Therefore, the detection of an amount of copper ions of at least 500 ppb in the test sample can indicate the amount of the formulation (containing copper) that is suitable for administration. Similarly, the detection of an amount of zinc ions of at least 50 ppb in the test sample can indicate the amount of the formulation (containing zinc) that is suitable for administration.

[0065] In some embodiments, the amount of the agent effective to stimulate the endometrium to produce leukemia inhibitory factor (LIF) and / or vascular endothelial growth factor (VEGF) can be an amount that provides copper in an amount ranging from about 0.025 μg to about 12.5 μg (preferably about 0.25 μg to about 10 μg, more preferably about 0.5 μg to about 5 μg, and most preferably about 0.05 μg to about 2.5 μg) and / or zinc in an amount in an equivalent range of about 0.05 μg to about 12.5 μg (preferably about 0.25 μg to about 10 μg, more preferably about 0.5 μg to about 5 μg, and most preferably about 0.05 μg to about 2.5 μg) to the endometrial fluid.

[0066] In some embodiments of the formulation comprising a CuCl2 solution, the formulation comprises CuCl2 at a concentration of at least about 20 μM, preferably at least about 50 μM. In particular, the formulation preferably comprises a CuCl2 solution at a concentration in the range of about 20 μM to about 200 μM, more preferably in the range of 50 μM to 150 μM. Such a formulation can be administered to a subject in a volume of about 100 to 1000 μl, more preferably about 150 to 500 μl.

[0067] In some embodiments of the formulation comprising a ZnCl2 solution, the formulation comprises ZnCl2 at a concentration of at least about 20 μM, preferably at least about 50 μM. In particular, the formulation preferably comprises a ZnCl2 solution at a concentration in the range of about 20 μM to about 150 μM, more preferably in the range of 50 μM to 125 μM. Such a formulation can be administered to a subject in a volume of about 100 to 1000 μl, more preferably about 150 to 500 μl.

[0068] When the subject is treated by any one of the assisted reproductive technologies related to embryo transplantation, the preparation is preferably applied to the subject before embryo transplantation and in the same menstrual cycle (in the case of IVF, it will be considered as the same IVF cycle). Preferably, the preparation is enough to stimulate the endometrium to produce a certain level of LIF and / or VEGF to improve the time of embryo implantation before embryo transplantation. Preferably, the subject is given a single administration of the preparation before embryo transplantation. In some embodiments, the preparation is no more than 14 days before embryo transplantation, preferably no more than about 10 days before embryo transplantation, and more preferably no more than about 5 days before embryo transplantation. In some embodiments, the preparation is applied to the subject 5 days before embryo transplantation, or 4 days before embryo transplantation, or 3 days before embryo transplantation, or 2 days before embryo transplantation, or 1 day before embryo. In other embodiments, the preparation is administered on the same day of embryo transfer; For example, shortly before embryo transfer (for example, within 3 hours, or 60 minutes, or 30 minutes, or 10 minutes, or 5 minutes or 1 minute of embryo transfer) or less preferably immediately after embryo transfer (for example, within 10 minutes of embryo transfer) the preparation is administered. Administering the preparation while embryo transfer can also be a selection. For example, the embryo can be transplanted to the experimenter in the form of a preparation that, in such embodiments, can also comprise components and substances that are typically included in standard embryo transfer culture medium (for example, found in EmbryoGlue). TM Components and substances in embryo transfer medium; Vitrolife, Gothenburg, Sweden). Alternatively, the preparation for simultaneous administration with embryo transfer can be provided by coating or impregnating an embryo transfer catheter (or at least a portion of the distal portion of an embryo transfer catheter inserted into the uterine cavity, or a portion thereof) in the preparation; in such an embodiment, it may be preferable if a portion of the distal portion of the catheter, which is a short distance (e.g., 1 to 3 cm) from the tip of the catheter (where the embryo is expelled), is coated or impregnated in the preparation.

[0069] Although described in the preceding paragraphs, in some preferred embodiments of the method of the first aspect, the formulation is not administered at the time of embryo transfer (i.e., simultaneously) or after embryo transfer. That is, the method of such embodiments comprises administering the formulation only before embryo transfer, preferably a single administration.

[0070] For women experiencing endometrial hypoplasia, the formulation is applied to the uterine cavity at least once during the relevant menstrual cycle, and preferably during the period between cessation of menstruation and ovulation or the onset of the increase in progesterone associated with ovulation. In some embodiments, the formulation may be administered multiple times during this period (e.g., once daily for several consecutive days).

[0071] When the subject is treated by IUI, the preparation is preferably applied to the subject before IUI and within the same menstrual cycle (i.e., within the same menstrual cycle in which insemination is performed). Preferably, the preparation is applied to the subject before ovulation / insemination to stimulate the endometrium to produce a certain level of LIF and / or VEGF to increase the time for embryo implantation. Preferably, the subject is given a single administration of the preparation before insemination. In some embodiments, the preparation is applied to the subject no more than 9 days before ovulation and preferably no more than about 5 days before ovulation. In some embodiments, the preparation is applied 5 days before ovulation, or 4 days before ovulation, or 3 days before ovulation, or 2 days before ovulation, or 1 day before ovulation. In other embodiments, the preparation is applied on the same day of ovulation. Preferably, the preparation is not applied at the time of insemination (i.e., simultaneously) or after insemination. However, for women experiencing endometrial hypoplasia, the formulation is administered to the uterine cavity at least once during the relevant menstrual cycle; preferably during the period between the cessation of menstruation and ovulation or the onset of the increase in progesterone associated with ovulation (e.g., the formulation may be administered multiple times during this period (e.g., once daily for several consecutive days)).

[0072] When the subject wishes to achieve pregnancy through natural conception, the preparation is preferably applied to the subject before ovulation and within the same menstrual cycle. Preferably, the preparation is applied to the subject before ovulation, enough to stimulate the endometrium to produce a certain level of LIF and / or VEGF to increase the time of embryo implantation. In some embodiments, the preparation is applied to the subject no more than 9 days before ovulation and preferably no more than about 5 days before ovulation. In some embodiments, the preparation is applied 5 days before ovulation, or 4 days before ovulation, or 3 days before ovulation, or 2 days before ovulation, or 1 day before ovulation. In other embodiments, the preparation is applied on the same day of ovulation. For women who experience insufficient endometrial development, the preparation is applied to the uterine cavity at least once in the relevant menstrual cycle; preferably within the time period between menstruation cessation and ovulation or the start of an increase in progesterone accompanying ovulation (e.g., the preparation can be applied multiple times within this time period (e.g., once a day for several consecutive days)).

[0073] In a second aspect, the present disclosure provides a method for improving embryo implantation in a subject (i.e., a woman or other female animal), wherein the method comprises inserting a device comprising copper and / or zinc into the uterine cavity of the subject for a period of time effective to stimulate the endometrium to produce leukemia inhibitory factor (LIF) and / or vascular endothelial growth factor (VEGF).

[0074] The device (or "uterine device") suitable for use in the method of the second aspect is generally elongated in form (e.g., similar to a surgical probe or embryo transfer catheter). Thus, the device may include a distal portion and a proximal base portion, and the step of inserting the device into the uterine cavity includes inserting at least a portion of the distal portion into the uterine cavity. When the distal portion is inserted into the uterine cavity, the proximal base portion remains outside the subject, allowing insertion and retrieval of the device. The portion of the distal portion inserted into the uterine cavity, or a portion thereof, may contain or be provided with copper and / or zinc and may generate copper and / or zinc ions to stimulate the endometrium to produce LIF and / or VEGF, while the remainder of the device may comprise stainless steel or a biocompatible polymer. In some embodiments, the portion of the distal portion inserted into the uterine cavity, or a portion thereof, may contain or be coated with copper and / or zinc metal, which may generate copper and / or zinc ions by corrosion (i.e., the copper and / or zinc is provided as elemental metal and, upon contact of the device with uterine fluid after insertion into the uterine cavity, may have an oxidized form of the metal on its surface and generate metal ions by corrosion). In other embodiments, the copper and / or zinc metal can be provided in the form of nanoparticles to increase the surface area available for corrosion, thereby effectively accelerating the rate of delivery of metal ions to the uterine fluid. In a specific embodiment, the device can be an embryo transfer catheter (e.g., a catheter used to transfer embryos (suspended in a small amount of transfer medium) to women undergoing IVF treatment), wherein the distal portion or a portion thereof that is inserted into the uterine cavity contains or is provided with copper and / or zinc. When the device is used in a subject undergoing any of the assisted reproductive techniques involving embryo transfer, this embodiment enables the delivery of metal ions to the uterine fluid at the time of embryo transfer, if desired. Furthermore, by enabling the delivery of copper and / or zinc ions without increasing the volume of the uterine cavity fluid, the use of a device as described can avoid any potential disadvantages associated with the administration of liquid formulations (e.g., solutions of CuCl2 and / or ZnCl2). This can be a significant advantage in embodiments where metal ion delivery is to be performed at the time of embryo transfer, as the use of excessive embryo transfer medium during embryo transfer has been associated with reduced IVF success rates (Sigalos et al., 2017).

[0075] The device is inserted into the uterine cavity for a period of time effective to stimulate the endometrium to produce leukemia inhibitory factor (LIF) and / or vascular endothelial growth factor (VEGF). Those skilled in the art will recognize that this period of time will be very short (i.e., to avoid the inhibitory effects of prolonged exposure of the uterus to copper and / or zinc ions on embryo implantation, such as is achieved with IUCDs). Typically, the period of time will be sufficient to provide at least 500 ppb (preferably at least 1250 ppb, and more preferably at least 2500 ppb) of copper ions to the uterine fluid and / or at least 50 ppb (more preferably at least 250 ppb) of zinc ions to the uterine fluid. In any case, this period of time will rarely, if ever, exceed 60 minutes and will typically not exceed about 10 minutes. More preferably, the period of time will be about 1 minute, or about 2 minutes, or about 3 minutes, or about 4 minutes, or about 5 minutes. However, it will be understood that the rate of release of copper and / or zinc ions into the uterine fluid may vary (which in turn may require increasing or decreasing the insertion time). For example, contact of copper and / or zinc metals with uterine fluid will cause them to corrode, thereby releasing metal ions into the solution. By placing other metals in contact with the copper and / or zinc metals, the release of copper and / or zinc ions can be slowed or accelerated, depending on the relative position of the metals on the current series (Zipper et al., 1977).

[0076] When the experimenter is treated by any one of the assisted reproductive technologies relating to embryo transplantation, the device is preferably inserted into the experimenter's uterine cavity before embryo transplantation and in the same menstrual cycle (in the case of IVF, will be considered as identical IVF cycle). Preferably, the device is enough to stimulate the endometrium to produce a certain level of LIF and / or VEGF to improve the time of embryo implantation before embryo transplantation. Preferably, the device is inserted into the experimenter's uterine cavity before embryo transplantation. In some embodiments, no more than 14 days, preferably no more than about 10 days and more preferably no more than about 5 days before embryo transplantation before embryo transplantation, insert the device. In some embodiments, 5 days before embryo transplantation, or 4 days before embryo transplantation, or 3 days before embryo transplantation, or 2 days before embryo transplantation, or 1 day before embryo, insert the device. In other embodiments, the device is inserted on the same day as the embryo transfer; for example, shortly before the embryo transfer (e.g., within 3 hours, or 60 minutes, or 30 minutes, or 10 minutes, or 5 minutes, or 1 minute of the embryo transfer) or less preferably immediately after the embryo transfer (e.g., within 10 minutes of the embryo transfer). Furthermore, as described above, where the device is an embryo transfer catheter, delivery of copper and / or zinc ions to the uterine fluid can be achieved at the time of embryo transfer.

[0077] In a third aspect, the present disclosure provides a formulation for improving embryo implantation in a subject, the formulation being suitable for administration to the uterine cavity and comprising an amount of copper and / or zinc effective to stimulate the endometrium to produce leukemia inhibitory factor (LIF) and / or vascular endothelial growth factor (VEGF), and wherein the formulation optionally comprises one or more pharmaceutically acceptable carriers and / or excipients.

[0078] In some embodiments, the formulation comprises copper and optionally zinc. In other embodiments, the formulation comprises copper or copper and zinc. In yet other embodiments, the formulation comprises zinc.

[0079] The copper and / or zinc may each be present in the formulation in one or more of the following forms: elemental (e.g., pure), alloy, complex, oxidized, salt (including esterified salts and other salts formed from organic acids), or solution. Preferably, the copper and / or zinc is present in a form that provides a source of copper / zinc ions in utero. The formulation may be in the form of, for example, a liquid, semisolid, or solid dosage form.

[0080] In some preferred embodiments, the copper and / or zinc are provided in the form of a solution, such as a solution containing copper chloride (CuCl2) and / or zinc chloride (ZnCl2). Such a solution can provide a source of copper / zinc ions in the uterus. Preferably, the solution is an isotonic saline solution of CuCl2 and / or ZnCl2.

[0081] In other embodiments, the copper and / or zinc are provided in a semisolid form (e.g., a gel or foam). For example, a gel comprising CuCl2 and / or ZnCl2, or a gel comprising nanoparticles of copper and / or zinc or nanoparticles of CuCl2 and / or ZnCl2. The gel can, for example, provide a short-term depot in the uterus, thereby providing a source of copper / zinc ions in the uterus, which can achieve controlled and / or sustained release of the copper / zinc ions (e.g., a gel wherein the copper and / or zinc is formulated with hydroxyethylcellulose and glycerol).

[0082] In still other embodiments, the copper and / or zinc are provided in a solid dosage form, such as small soluble crystals or other particles (e.g., powders or metal nanoparticles), for example, crystals or other particles of CuCl and / or ZnCl, which can be administered in a suitable carrier liquid or gel (e.g., which can be prepared shortly before administration), or in the form of a soluble tablet or capsule. Such crystals or other particles can provide a source of copper / zinc ions in utero.

[0083] Preparation can comprise one or more pharmaceutically acceptable carriers and / or excipients, such as those mentioned above. In addition, preparation can also comprise and be selected from following one or more materials: preservative, adhesive, lubricant, antioxidant, suspending agent, stabilizer, coating agent and solubilizing agent, such as those mentioned above. In addition, preparation can comprise stimulation LIF, VEGF or other cytokines, growth factors etc. (such as integrin) that may be useful to embryo implantation or one or more other materials of activity. Preparation can also comprise component and material that are usually contained in standard embryo transfer culture medium.

[0084] In some preferred embodiments, the formulation comprises a CuCl2 solution comprising a CuCl2 concentration of at least about 20 μM, preferably at least about 50 μM. In particular, the formulation preferably comprises a CuCl2 solution having a concentration in the range of about 20 μM to about 200 μM, more preferably in the range of 50 μM to 150 μM. In some other preferred embodiments, the formulation comprises a ZnCl2 solution comprising a ZnCl2 concentration of at least about 20 μM, preferably at least about 50 μM. In particular, the formulation preferably comprises a ZnCl2 solution having a concentration in the range of about 20 μM to about 150 μM, more preferably in the range of 50 μM to 125 μM.

[0085] When the formulation is provided as a liquid (e.g., a formulation comprising a CuCl2 and / or ZnCl2 solution), the formulation can be provided in the form of a package or device suitable for administering the formulation to the uterine cavity; for example, a flexible catheter "loaded" with the formulation suitable for passing through the cervical canal and into the uterine cavity. Such a catheter can be similar to the catheter used to transfer embryos (suspended in a small amount of transplantation medium) to women undergoing IVF treatment. The catheter can be composed of a biocompatible polymer and hermetically sealed for transport and storage containing the liquid formulation, which can be disposed in a reservoir or bulb located at or near the tip of the catheter. The tip of the catheter can include a fragile portion that can be broken before use. Otherwise, the tip can be sealed with an easily removable cap or cover. The amount of formulation contained within the catheter can be as low as 100 to 1000 μl, or more preferably, the volume ranges from about 150 to 500 μl.

[0086] In a fourth aspect, the present disclosure provides use of a copper and / or zinc preparation for improving embryo implantation in a subject, wherein the preparation is suitable for administration to the uterine cavity of the subject in an amount effective to stimulate the endometrium to produce leukemia inhibitory factor (LIF) and / or vascular endothelial growth factor (VEGF).

[0087] In a fifth aspect, the present disclosure provides the use of copper and / or zinc in the manufacture of a formulation for improving embryo implantation in a subject, wherein the formulation is suitable for administration to the uterine cavity of the subject in an amount effective to stimulate the endometrium to produce leukemia inhibitory factor (LIF) and / or vascular endothelial growth factor (VEGF).

[0088] In the fourth and fifth aspects of the use, the subject may be a woman being treated with any of the assisted reproductive techniques, particularly those involving embryo transfer, such as in vitro fertilization (IVF), and variations including IVF-ICSI (intracytoplasmic sperm injection) and in vitro maturation (IVM) treatments. However, the subject may also be a woman undergoing intrauterine insemination (IUI) therapy, or a woman wishing to improve her pregnancy prospects by conceiving naturally. The subject may also be selected from other non-human female subjects, such as, for example, livestock.

[0089] Copper and / or zinc can each be present in the formulation in one or more of the form of an element (e.g., pure), an alloy, a complex, an oxidation, a salt (including esterified salts and other salts formed from organic acids), or a solution. Preferably, the copper and / or zinc are present in a form that can provide a source of copper / zinc ions in the uterus. The formulation can be in the form of, for example, a liquid, semisolid, or solid dosage form. In some embodiments, the copper and / or zinc are provided in the form of a solution, such as a solution containing copper chloride (CuCl2) and / or zinc chloride (ZnCl2). Such a solution can provide a source of copper / zinc ions in the uterus.

[0090] The formulation may comprise one or more pharmaceutically acceptable carriers and / or excipients, such as those described above. Furthermore, the formulation may further comprise one or more substances selected from the group consisting of preservatives, adhesives, lubricants, antioxidants, suspending agents, stabilizers, coating agents, and solubilizing agents, such as those described above. Furthermore, the formulation may comprise one or more other substances that stimulate the production or activity of LIF, VEGF, or other cytokines, growth factors, and the like (e.g., integrins) that may be beneficial for embryo implantation.

[0091] In some preferred embodiments, the formulation comprises a CuCl2 solution comprising a CuCl2 concentration of at least about 20 μM, preferably at least about 50 μM. In particular, the formulation preferably comprises a CuCl2 solution having a concentration in the range of about 20 μM to about 200 μM, more preferably in the range of 50 μM to 150 μM. In some other preferred embodiments, the formulation comprises a ZnCl2 solution comprising a ZnCl2 concentration of at least about 20 μM, preferably at least about 50 μM. In particular, the formulation preferably comprises a ZnCl2 solution having a concentration in the range of about 20 μM to about 150 μM, more preferably in the range of 50 μM to 125 μM.

[0092] The methods and formulations of the present disclosure are further described below by the following non-limiting examples and figures.

[0093] Example

[0094] Example 1 Effects of Metal Ions on LIF and VEGF Production in Vitro

[0095] Materials and methods

[0096] Human Ishikawa endometrial adenocarcinoma cell line (Sigma-Aldrich; St Louis, MO, United States of America) was used to simulate the endometrial response to copper and zinc ions (Cu 2+ and Zn 2+ ) response. Cells were grown in T75 flasks (Nalge Nunc International, Rochester, NY, United States of America) at 37°C in an atmosphere of 5% CO2 in air in Ishikawa medium consisting of αMEM (Sigma-Aldrich), 2 mM glutamine (Sigma-Aldrich), and 1% non-essential amino acids (Sigma-Aldrich) supplemented with 5% fetal bovine serum (Invitrogen Corp, Carlsbad, CA, United States of America), without antibiotics. The medium was refreshed every 48 hours. Cells were subcultured using 0.25% trypsin / EDTA (Invitrogen). For metal ion exposure experiments, cells were seeded on 2-well chamber slides (Ibidi, Martinsried, Germany). After endometrial cells attached and reached 80-90% confluence, they were exposed to copper and zinc metal ions using two methods (saline solution or metal implant) as described below.

[0097] Metal ions in solution

[0098] Copper chloride or zinc chloride (Sigma-Aldrich) was dissolved in Ishikawa medium to produce salt solutions at various concentrations ranging from 0.2 μM to 200 μM, which were then applied to Ishikawa cells seeded in 2-well chamber slides for an 8-hour period. After 8 hours of exposure, the culture medium was removed and centrifuged at 1000 g for 10 minutes at 4°C to pellet any cell debris, and the supernatant was then stored in an Eppendorf tube at -80°C. The levels of LIF and VEGF in the supernatant were then determined using a multiplex immunoassay system (elisakit.com, Scoresby, VIC, Australia).

[0099] Metal implants

[0100] Implants composed of various metals are generated using metal wire (copper) or various types of metal sheets (copper, zinc, aluminum and gold). In some cases, implants are composed of two metals (bimetallic implants), particularly copper and zinc (CuZn), zinc and aluminum (ZnAl) and zinc and gold (ZnAu) sheets. After heat sterilization, the implant is brought into direct contact with the Ishikawa cells and then inoculated on a 2-well chamber slide in Ishikawa culture medium or G2 embryo culture medium (Vitrolife) using sterilized surgical tweezers for a period of 1 minute to 5 minutes before removal. Ishikawa cell supernatants are collected 4 to 8 hours later, centrifuged at 1000 g for 10 minutes at 4 ° C, and then frozen at -80 ° C for subsequent evaluation of metal ions and cytokines by mass spectrometry.

[0101] result

[0102] In the case of metal ion solutions, it was found that copper ions significantly increased LIF production above baseline when the CuCl2 concentration exceeded 2 μM (see Figure 1A ), where LIF production was 420% higher than baseline at a concentration of 200 μM. No toxicity related to LIF production was observed within the tested dose range within an 8-hour incubation time. Zinc ions also significantly increased LIF production at ZnCl2 concentrations ranging from 50 μM to 150 μM compared to the control (see Figure 1B However, when administered at doses exceeding 150 μM, zinc ions appeared to cause toxicity, and a decrease in LIF production was observed ( Figure 2A Regarding VEGF production, copper chloride solution significantly increased VEGF production at concentrations above 20 μM, reaching a peak at 100 μM CuCl2. On the other hand, zinc chloride did not produce a significant increase in VEGF compared to the control, but appeared to reduce VEGF production at a concentration of 150 μM ( Figure 2B).

[0103] In the case of the Cu wire / Cu sheet and CuZn bimetallic sheet implants, mass spectrometry confirmed the release of Cu and Zn ions into the conditioned αMEM / G2 medium within a relatively short time (see Figure 3A and Figure 3B ; NOTE: In cases where copper implants are used and Figure 3A In the results shown in , a small amount of zinc was detected due to the presence of zinc in the αMEM medium. As expected, the presence of zinc (in the CuZn bimetallic implant) slowed the release of copper ions into the solution ( Figure 3B ), in which zinc appears to act as a sacrificial anode. In addition, it was observed that, like CuCl2 and ZnCl2 solutions, copper and zinc ions from silk and sheet metal implants increased the production of LIF and VEGF by endometrial cell cultures in multiple replicates ( Figure 4A and Figure 4B ). Copper or copper-zinc implants respond much better than any other metal combination.

[0104] Further experiments were performed to determine whether this increase in LIF and VEGF production (i.e., upregulation) was related to the release of metal ions, the associated generation of chemistatic currents during in situ corrosion, or the physical presence of the metal implant (a "foreign body" response). These experiments involved adding 1.5 ml of Ishikawa αMEM medium to each well of a 2-well chamber slide containing no cells, and thereafter adding a CuZn bimetallic implant for a period of 2 minutes (after which it was removed using sterile forceps). The resulting "conditioned medium" was then added to a 2-well slide containing Ishikawa cells and incubated for a period of 8 hours, thereby exposing these cells to the metal ions but not to the physical presence of the implant or any chemistatic currents generated by implant corrosion. These experiments showed that conditioned medium containing copper and zinc ions was still able to induce an increase in LIF and VEGF production that was above that produced in control unexposed cultures (see Figure 5A and Figure 5B ).

[0105] Example 2 Effects of Metal Ions on Embryo Development and Attachment

[0106] Materials and methods

[0107] Ishikawa cells were seeded onto 2-well chamber slides in Ishikawa medium until they reached 80-90% confluence. Then, 5 minutes before exposure to metal implants, the medium was changed to G2 embryo medium, and the Ishikawa cells were exposed to copper, zinc, or copper-zinc bimetallic implants for a duration of 2 or 5 minutes (or remained unexposed (control)), and the resulting G2 medium was collected after 8 hours. The supernatant was then analyzed as described in Example 1 above to confirm that metal implant exposure resulted in upregulation of endometrial LIF and VEGF production (see Figures 6A to 6D ).

[0108] To determine whether metal ions can improve embryo implantation, the mouse embryos after compaction were exposed to "conditioned medium" before growth assays were performed. In particular, nucleated oocytes were collected from superovulated female F1 mice approximately 21 to 22 hours after hCG administration. After removing the cumulus cells, the oocytes were cultured in 20 μl drops (10 embryos / drop) of G1 medium under paraffin oil (Vitrolife) at 37°C, 6% CO2, 5% O2, and 89% N2. After 48 hours, the compacted and morula-stage embryos were randomly transplanted into the following cultures: (i) control G2 medium (Vitrolife) that was not exposed to Ishikawa cells; (ii) separate cultures in 2 μl of control Ishikawa-exposed G2; or (iii) separate cultures in G2 medium exposed to a 2-minute copper-zinc bimetallic implant in 2 μl of drops. After 48 hours, the ability of the blastocysts to grow was assessed in the following manner. First, a flat-bottomed 96-well tissue culture plate (BD Biosciences, Franklin Lakes, NJ, United States of America) was coated with fibronectin (10 μg / ml; BD Biosciences), rinsed twice with sterile PBS, and incubated in 4 mg / ml bovine serum albumin (Sigma-Aldrich). Each well was then rinsed and then filled with G2 medium supplemented with 5% FCS and balanced at 37°C for 3 hours under paraffin oil (Ovoil; Vitrolife) before adding the blastocyst. The hatched and hatching blastocysts from each corresponding treatment were placed in the prepared wells (1 embryo per well) and incubated for 90 hours. During the culture period, growth was checked and images were taken using an inverted microscope (Nikon Eclipse TS100-F) equipped with a heating stage set to 37°C at 66 and 90 hours after being transplanted to the growth plate. The extent of growth for each treatment was obtained by measuring the growth area in each image taken throughout the experiment using ImageJ. All images were collected and analyzed at matching magnifications.

[0109] Results and discussion

[0110] Although no difference in embryonic development rate was observed when conditioned medium from copper- and zinc-exposed cultures was added to day 3 mouse embryos, the surface area of ​​trophoblast cells growing onto fibronectin-coated plates was very significantly increased after 3 to 4 days (see Figure 7 、 8A and 8B), thereby demonstrating improved embryonic health and implantation potential.

[0111] This latter observation is important for two reasons. First, it demonstrates the ability of copper and zinc ions to mediate upregulation of endometrial LIF / VEGF production to enhance the implantation process. Second, it demonstrates that, although copper and zinc ions have traditionally been considered toxic to preimplantation embryonic development (Brinster and Cross, 1972; Webb et al., 1973; Holland and Pike, 1978; Erbach et al., 1995), this toxicity is not significant with the short-term exposures used in these experiments. Thus, despite nearly half a century of teaching that IUCDs containing copper or zinc provide effective contraception (Stanford, 2002), it is clear from the results described herein that short-term delivery of copper and / or zinc ions to the endometrium can actually enhance the implantation process.

[0112] While not wishing to be bound by theory, it is believed that the differences observed between the experiments described herein indicate that the beneficial effects of copper on endometrial function (in terms of LIF / VEGF production) and the well-described deleterious effects of copper on endometrial health and function in IUCD users can be explained by the large differences in the "dose" of copper delivered to the endometrium. That is, multiple studies have examined the rate of copper delivery in IUCD users, where this rate varies depending on the surface area of ​​copper in the IUCD device and the time since initial insertion. In one such study, the average amount of copper delivered to the uterus was reported to vary between 13.3 μg and 116.7 μg per day (Timonen, 1976). Other studies reported average delivery rates of 26.7 μg (Chantler et al., 1984), 14 μg (Larsson et al., 1981), and 45 μg (Hagenfeldt, 1972) of copper per day. In the experiment of Example 1, a 4-fold increase in endometrial LIF production was observed at a concentration of 200 M CuCl2. If it is assumed that the female uterine cavity has a volume of approximately 150 μl (Casslen, 1986), then the amount of copper that would need to be applied in vivo to provide a corresponding dose of 200 μM is 1.91 g of copper. Furthermore, since a relative decrease in endometrial VEGF production was observed at concentrations exceeding 100 μM CuCl2 in vitro ( Figure 2A), the optimal amount of copper required to elicit a positive endometrial response in vivo is likely to be close to 1 g, a level that is only about 1% to 7% of the daily copper exposure of IUCD users (Hagenfeldt, supra; Timonen, supra; Larsson et al., supra; Chantler et al., supra). Therefore, it is believed that the dose of copper required to elicit a beneficial endometrial response (i.e., an increase in LIF / VEGF) that can enhance embryo implantation must be below 13.3 g, the lowest daily copper release rate reported by IUCD devices that can impair implantation (Copper T 100 mm Hg). 2 IUCD; Timonen, supra). In the experiments of Example 1, 20 M CuCl2 was demonstrated to be the lowest amount of copper that caused an increase in endometrial LIF and VEGF, equivalent to a 0.191 g dose of copper delivered to the uterus in vivo. Thus, in some preferred embodiments of the disclosed methods, the formulation / device is administered / inserted into a subject to deliver a dose of between 0.1 g and 10 g of copper to the uterine cavity. A similar dosage range may also be preferred for zinc.

[0113] In addition to upregulating endometrial LIF / VEGF production, copper and zinc ions can also improve the implantation process by destroying potentially harmful bacteria that may be present in the uterus. That is, while the uterus has traditionally been considered a sterile zone free of potentially pathogenic bacteria, it is now recognized that certain bacteria colonize the endometrium in some infertile women and that this is associated with impaired implantation potential (Moreno et al., 2016). Because both copper and zinc are known to have antimicrobial properties (Vincent et al., 2016; Siddiqi et al., 2018), it is possible that local application of copper or zinc ions (or a combination) to the uterine cavity may kill or inhibit potentially implantation-damaging bacteria in this group of patients, thereby improving their potential for both natural and IVF-assisted fertility.

[0114] Example 3 Prophetic Example of Treatment to Improve Implantation of Transplanted Embryos

[0115] An isotonic saline solution containing CuCl2 at a concentration of 150 μM and ZnCl2 at 100 μM was prepared.

[0116] A 35-year-old woman who had experienced recurrent implantation failure (RIF), specifically five failed IVF cycles with good-quality embryos, was referred to the clinic 5 days before the expected transfer of a further IVF embryo. A transfer catheter (e.g., Soft-Pass IVF) commonly used for intrauterine insemination therapy, loaded with 200 μl of CuCl2 / ZnCl2 solution, was used. TM Artificial insemination catheter; Cook Medical Inc., Bloomington, IN, USA), and the solution was applied to the uterine cavity.

[0117] On the scheduled date for embryo transfer, the woman returned to the clinic and received the high-quality embryo according to the usual protocol.

[0118] Throughout the specification and appended claims, unless the context requires otherwise, the words “comprise” and “include” and variations such as “comprising” and “including”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0119] The reference to any prior art in this specification is not, and should not be taken as, any form of suggestion that such prior art forms part of the common general knowledge.

[0120] It will be understood by those skilled in the art that the methods, uses, and / or formulations disclosed herein are not limited to the specific applications described. The methods, uses, and / or formulations described are also not limited to their preferred embodiments with respect to the specific elements and / or features described or recited herein. It will also be understood that the methods, uses, and / or formulations described may be rearranged, modified, and substituted numerous times without departing from the scope of the present disclosure as set forth and defined by the following claims.

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Claims

1. Use of copper ions in the preparation of a preparation for enhancing embryo implantation, stimulating endometrial angiogenesis, or improving pregnancy rate in a subject, wherein the preparation is administered to the uterine cavity of the subject, wherein the copper ions are provided in the form of a solution or a gel, (i) wherein the subject wishes to achieve pregnancy through natural conception, and the preparation is administered to the subject before ovulation and during the same menstrual cycle, or (ii) wherein the subject is undergoing assisted reproductive technology treatment involving embryo transfer, and the formulation is administered to the subject before and during the same menstrual cycle as the embryo transfer, or (iii) wherein the subject is undergoing intrauterine insemination (IUI) treatment, and the formulation is administered to the subject before and during the same menstrual cycle as the IUI, wherein the preparation is administered into the uterine cavity and provides copper ions to the uterine cavity, - an amount within the range of about 0.025 μg to about 12.5 μg per administration; or - for a period not exceeding 60 minutes, wherein the formulation comprises copper (Cu) in the range of 20 μM to 200 μM 2+ )ion.

2. The use according to claim 1, wherein the copper-containing preparation is provided in the form of a biodegradable gel.

3. The use according to claim 2, wherein the gel is formulated with hydroxyethyl cellulose and glycerol.

4. The use according to any one of claims 1 to 3, wherein the preparation is administered to the subject no more than 14 days before embryo transfer.

5. The use according to claim 4, wherein the preparation is not administered at the time of embryo transfer or after embryo transfer.

6. The use according to any one of claims 1 to 5, wherein the preparation is administered multiple times a day or for consecutive days in the same menstrual cycle.

7. The method according to any one of claims 1 to 6, wherein the subject is a female experiencing endometrial hypoplasia.

8. The use according to any one of claims 1 to 7, comprising inserting a device comprising a copper preparation into the uterine cavity of a subject for a period of time effective to stimulate the endometrium to produce leukemia inhibitory factor (LIF) and / or vascular endothelial growth factor (VEGF).

9. Use according to claim 8, wherein the device has an elongated shape and comprises a distal portion and a proximal base portion, and the step of inserting the device into the uterine cavity comprises inserting at least a portion of the distal portion into the uterine cavity.

10. The use according to claim 9, wherein the portion of the distal part inserted into the uterine cavity or a part thereof contains copper or is provided with copper, and produces copper ions to stimulate the endometrium to produce LIF and / or VEGF.

11. The use according to claim 8 or 9, wherein the device is an embryo transfer catheter.

12. The use according to any one of claims 8 to 11, wherein the device comprises copper ions and is inserted into the uterine cavity for a period of time effective to provide the uterine cavity with copper ions in the range of about 0.025 μg to about 12.5 μg per administration.

13. The use according to any one of claims 8 to 12, wherein the embryo transfer catheter is used to transfer the embryo into the subject and simultaneously deliver copper ions to the uterine cavity.

14. A formulation for enhancing embryo implantation in a female subject, the formulation being suitable for administration into the uterine cavity and comprising copper ions in an amount effective to stimulate the endometrium to produce leukemia inhibitory factor (LIF) and / or vascular endothelial growth factor (VEGF), and wherein the formulation optionally comprises one or more pharmaceutically acceptable carriers and / or excipients.

15. The formulation according to claim 14, wherein the formulation comprising copper ions is provided in the form of a biodegradable gel or in a form that provides in situ biodegradability in the uterus.

16. The formulation of claim 15, wherein the copper ions are formulated with hydroxyethylcellulose and glycerol.

17. The preparation according to claim 16, wherein the preparation further comprises one or more other substances that stimulate the production or activity of LIF, VEGF or other cytokines or growth factors that are beneficial to embryo implantation.

18. The formulation of claim 17, wherein the formulation comprises 20-200 μM copper ions.

19. A device adapted to pass through the cervical canal and into the uterine cavity of a subject, wherein the device comprises a formulation according to any one of claims 15 to 18.

20. The device according to claim 19, wherein the device has an elongated shape and comprises a distal portion and a proximal base portion, and wherein the distal portion or a portion thereof inserted into the uterine cavity comprises copper ions or is provided with copper ions and produces copper ions to stimulate the endometrium to produce LIF and / or VEGF.

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