WO2025051890A1 - Open recoverable intra-uterin device with adhesive means - Google Patents

Abstract

The present invention relates to an open recoverable intra-uterine device comprising adhesive means and used particularly for culturing gametes and/or embryos in utero.

Description

OPEN RECOVERABLE INTRA-UTERIN DEVICE WITH ADHESIVE MEANS

FIELD OF THE INVENTION

The present invention relates to an open recoverable intra-uterine device with adhesive means for fixing one or more elements selected from the group consisting of embryos, male and/or female gametes, fertilized oocytes, unfertilized eggs, or combinations thereof, used particularly for culturing gametes and/or embryos in utero.

BACKGROUND OF THE INVENTION

In natural conception, floating embryo arrives in the uterine cavity from the Fallopian tube where fertilization happened, at the fourth day of its development (morula) and around 24h before the beginning of implantation. This short period of time before implantation may play a role in the decrease of expulsion risk from the uterine cavity.

In in vitro fertilization (IVF), however, generally, the embryo is transferred into the uterine cavity at day 2 or 3, i.e., it is present in the uterine cavity 48 to 72 hours before its presence during the natural conception. Consequently, it remains much more time in a floating state which may contribute to a higher risk to be expelled from the uterine cavity. Different reasons are usually proposed to be implicated in this risk of expulsion including uterine peristalsis and contractions, low site of deposition and negative pressure generated when removing the transfer catheter. Several strategies as bed rest, fibrin sealant (US 6 196 965) and mechanical closure of the cervix were proposed to avoid this problem of embryo expulsion. Unfortunately, all these strategies failed to resolve this problem.

The main reason why the embryo is transferred at day 2 or 3 is the consequence of a persistent technical difficulty to optimize culture medium in vitro. Indeed, in vitro culture of mammalian embryos is a fundamental process in assisted reproductive technology (ART). Generally, in vitro culture conceived for using in ART, only mimics complexity of in vivo and in utero microenvironment. However, the natural milieu of gametes and embryos also contains redundant and overlapping reactive oxygen species (ROS) -protective systems that cannot even be mimicked in vitro (Menezo Y et al. 2016).

Dialogue between mother and embryo through an important intercellular communication network with extracellular vesicles has recently been proposed to play a crucial role in implantation (Godakurama et al. 2022, Javier Gonzalez Fernandez 2023). Whereas extracellular vesicles (EVs) such as apoptotic bodies, microvesicules (MVs), and exosome (EXOs) are released from all cells in the body and are accumulated in all fluid spaces (Beetier et al. 2023), in the reproductive field, they are secreted from the embryo (trophectoderm) and from the maternal endometrium (epithelial cells) measuring between 100 to 1000 nm. They contain lipids, proteins, RNAs (IncRNA, mRNA, small non-coding RNA, rRNA, miRNA) and DNAs. Recently, it was also suggested that endometrium itself may be implicated in the preparation of embryo to implant. First, miR-30d containing EXOs is secreted by endometrium and becomes internalized by embryonic trophoblast cells. It has been shown to induce genes involved in embryonic implantation (Vilella et al., 2015). It was also reported that mtDNAs present in MVs which were secreted by endometrial cells, seems to be involved in the embryonic ATP production. All these possible effects from the endometrium on the embryo highlights that the reproductive tract EVs cargo has an important role in reproductive events, which is missing in current in vitro media.

In addition, the influence of culture conditions on epigenetic chromatin remodelling during preimplantation development has been demonstrated. Most studies use a Mouse Embryo Assay (MEA) to assess appropriate DNA methylation. One of these studies demonstrated a loss of imprinted methylation among all culture systems using commercial media (used in IVF) compared with in vivo-derived embryos. No commercial human IVF culture media would pass the Mouse Embryo Assay (MEA) if appropriate methylation/epigenetics were taken into account (Market -Velker BA et al. 2010). In a recent study it was demonstrated that considered in in vitro culture media are implicated in this alteration of epigenetic remodelling of chromatin in human ART (Yves Menezo et al., 2020).

In conclusion, the exposition of IVF-derived embryos to sub-optimal in vitro conditions may lead to epigenetic errors with possible adverse short (mosaicism, invasive phenotype) and long term (Developmental Origin of Health and Disease-DOHaD) or even transgenerational consequences (Ventura et al., 2015). In the last 15 years it has been demonstrated in bovine model that the post-fertilization environment affects embryo quality in terms of gene expression, cryotolerance and metabolism (Wrenzycki et al., 2005; Lonergan et al. 2006; Duranthon et al., 2008) with even a change in the DNA methylation profile according to the time of in vitro sub- optimal exposition (Salilew-Wondim et al., 2015).

Considering the difficulty to render the conditions of in vitro culture closer to the conditions in utero, notably after gamete intrafallopian transfer (GIFT) and (zygote intrafallopian transfer (ZIFT) with the invasive laparoscopy, the research efforts for improving in vitro fertilization (IVF) are now directed to selection with artificial intelligence of the best in vitro derived embryo to be implanted. In addition, preimplantation genetic test to detect aneuploidies (PGT-a) is used as means to improve implantation.

However, all of these technics failed to resolve the problem of embryo quality implantation in the uterine cavity. Thus, it still remains a need to improve preimplantation embryo developments’ conditions to produce embryos more functional with better quality and ability of implantation. Some studies were directed to the development of means allowing to perform an in vivo/in utero culture. Particularly, three different devices for embryo in vivo preimplantatory development are known.

One of them (developped by Invocell®) is a vaginal device. It consists in a box as a real container without any link and communication with the environment and containing oocytes and sperm and placed into the vagina. Only temperature, no light and presence of patient’s movement are considered to be « in vivo ».

The second one is an intra-uterine device disclosed in the application WO 03/011200. It consists in a permeable silicone capsule permitting a communication between its inner and outer part. The post ICSI (intra-cytoplasm sperm injection) oocytes are placed into this capsule and transitory placed into the uterus less than 2 hours later as a mini-intrauterine device (IUD) offering to early preimplantatory embryo a natural environment. The principle of this device is to allows the exchanges between the uterine medium and the embryo/gametes encapsulated in the device via the wall of the device made from a permeable material. However, even if several molecules contained in the uterine medium are able to cross the permeable wall, the exchange of molecules and circulation of the uterine medium is insufficient and the embryos/gametes placed in the devices are not in optimal culture conditions.

In addition, a permeable porous membrane is fragile and handling of this capsule, in particular when loading and unloading encapsulated elements, or when implanting the capsule in or recovering it from the uterus, could damage the wall of the capsule and possibly cause the loss of the encapsulated elements.

To improve in utero culture conditions and to optimize the embryos/gametes preimplantation development, a third device was provided (described in the international application W02007074409), wherein instead to a wall made from permeable material, the device was provided with a wall having several openings with a particular size susceptible to allow molecules of the uterine medium to enter into the device but blocking the exit of embryos/gametes placed in the device. However, even if several molecules of the uterine medium are able to enter the device, there are other one which remain outside and also, the endometrial cells and other cells necessary to the preimplantation development of the embryo, whose size is bigger than the size of the openings cannot penetrate into the device and the culture conditions are thus not optimal. Finally, the high viscosity of the uterine fluid may explain why exchanges between microenvironment and uterine elements are not optimal with this device.

From the above, it appears that the current methods and devices do not allow to solve the problems occurring during the period of preimplantation of embryo and thus increasing the quality of gametes and embryos and embryo implantation rate after its transfer into uterine cavity. Particularly, the devices known in the art which are designed to mimic in utero conditions or to put the embryo into contact with the uterine medium appear not to be optimal. Therefore, there is still a need to provide methods and/or devices allowing to improve the condition of preimplantation in order to optimize the quality of gametes and embryos and to increase the chance of embryo implantation and development in the uterine cavity and thus improving the ART.

SUMMARY OF THE INVENTION

As it appears from the background part, developing an in vitro preimplantation embryo culture which mimics closely the in vivo/in utero conditions seems to be a hard work. In addition, the devices already provided for putting into contact embryo/gametes with the uterine medium do not allow to obtain an optimal quality of embryo and consequent rate of implantation. This is the reason why the current studies are rather directed to an improvement of the methods of selecting embryo before the implantation into the uterine cavity instead to the improvement of culture conditions of preimplantation stage, let alone to the improvement of means allowing in vivo/in utero culture.

Surprisingly, in the above-mentioned conditions, the inventor of the present invention decided to continue the effort of improvement of transitory in vivo/ in utero culture for embryo/oocyte development in order to decrease the in vitro culture time before definitive transfer in the uterine cavity, since this appears to him to be the best route for improving the methods of assisted fertilization and particularly, the functionality of embryo in order to improve the process of implantation. Maintaining the principle of using the uterine cavity as an incubator and the uterine medium as a natural culture medium, the inventor found out a solution allowing to put into very close contact (as during the natural conception), the embryo/gametes with all elements (molecules and cells) of the uterine medium without any restriction. This solution consists in an open recoverable intra-uterine device which comprises a support having a surface coated with adhesive means retaining the embryos/gametes and a removable cover part. When inserted into the uterine cavity, the removable cover part is removed and the embryos/gametes fixed on the support of the device are entirely exhibited to the uterine medium as the embryos/gametes during the natural fertilization but remain solidly fixed on the surface of the support of the device. When the device is retrieved from the uterine cavity embryos/gametes are still fixed on the surface of the support of the device and the removable cover part recovers them again.

The present invention thus relates to open recoverable intra-uterine device (1 ) comprising:

- a solid support (2) coated with at least one adhesive compound for fixing one or more elements selected from the group consisting of embryos, male and/or female gametes, fertilized oocytes, unfertilized eggs, or combinations thereof, and

- a removable cover part (4).

Thus, since the cover part (4) of the device (1 ) of the invention may be removed when the device is introduced in the uterine cavity it allows the uterine fluid even with its high viscosity with all its components (molecules and cells) to be in cellular contact with the elements fixed on the support (2).

The main advantage of the device of the invention is the possibility to put into direct contact the elements fixed on the support (2) with the uterine fluid without releasing these elements in the uterine cavity because they are solidly fixed on the support (2). At this manner, after predetermined period of time, these elements may be recovered. They are safely recovered since the removal cover part (4) is able to recover them again when the device is retrieved from the uterine cavity, passing by the cervix and the vagina.

Therefore, the device (1 ) of the present invention is characterized in that the elements fixed by the adhesive means are not released in the intra-uterine cavity. This is in contrast to the intrauterine devices known in the art which are notably used for transferring embryos/spermatozoids during the assisted fertilization.

The contact of the fixed elements with the uterine fluid, which may comprise fluids coming both from the uterine cavity and from the fallopian tubes, can favor the development of the embryos. Furthermore, exchange of cells can facilitate subsequent re-implantation and nidation of the embryo in the uterus.

The phenomena of interaction with the uterine fluid and all its components, enhanced compared to the state of the art, can be of a kind to favor the development of the elements fixed on the support (2) of the device (1 ), and in particular the development of the embryo or the fertilization of the implanted eggs.

In addition, the open recoverable intra-uterine device (1 ) of the invention is able i) to improve embryo quality and invasive phenotype and euploidy level; ii) to respect this high vulnerability period of embryo development with genome reprogramming; iii) to increase involvement of future parents, mother’s uterine cavity being used as natural biological incubator; iv) to ensure close contact with endometrial cells and uterine fluid offering real cross-talk between the mother and the embryo; v) to offer a period of rescue and wellness to embryos before or after all sophisticated handlings such as ICSI, vitrification or in vitro culture, and vi) to perform complete in vivo culture of several oocytes with later intrauterine insemination or a prolonged period of in vivo culture until the hatching process which may be considered as an in vivo assisted hatching and adhesion process when limited number of embryos are cultivated in this in vivo and natural microenvironment.

FIGURES

In the appended figures, provided by way of non-limiting example:

FIG.1 shows a diagrammatic perspective view of one embodiment of the open recoverable intrauterine device (1 ) of the invention;

FIG.2 shows a diagrammatic perspective view of one embodiment of the open recoverable intrauterine device (1 ) of the invention when the cover part (4) is partially removed; FIG.3 shows a diagrammatic perspective view of wells (3);

FIG.4 shows immunohistochemistry (IMH) staining of adult ovarian mice tissue. DAPI was used to stain nucleus and anti-ZP2 antibody coupled with a fluorescent secondary antibody (donkey anti-rat 594) was used to stain zona pellucida.

FIG.5 shows immunohistology image of embryo: post-incubation with primary antibody anti-ZP2 antibody and secondary fluorescent antibody donkey anti-rat 594 to stain the zona pellucida (grey). DAPI (dark grey) was used to stain the nuclei;

FIG.6 shows schema of the four tested conditions on mice embryos. 181 zygotes were separated into four groups and were treated with KSOM medium (#1 ), primary antibody (#2), primary antibody then Dynabeads™ (#3) or with Dynabeads™ alone (#4). Embryo development was assessed every 24h;

FIG.7 shows immunohistology image after addition of secondary fluorescent antibody and DAPI. Conditions without primary antibody (#1 and #4) were not stained. Conditions #2, #3 and #5 (magnets tested every day) were stained with the secondary fluorescent antibody.

DETAILED DESCRIPTION OF THE INVENTION

Other features and advantages of the open intra-uterine device of the invention will become more apparent in the course of the following description.

The open recoverable intra-uterine device (1 ) shown in FIG.1 is particularly used in assisted fertilization techniques to implant and temporarily maintain male and female gametes (in vivo fertilization) and/ or embryos (preimplantation development) in a uterine cavity.

In principle, in an in vitro assisted fertilization method, this intra-uterine device enables one or more elements (same or different) to be fixed on the support (2) at the start of development, for example the embryo in the start of embryo development. The device is introduced into the uterine cavity for a predetermined period (from a few hours to a few days), and thereafter it is recovered to extract the embryo(s) or fertilized oocytes to monitor the development and/or in diagnosis purpose before intra-uterine implantation.

The open recoverable intra-uterine device (1 ) of the invention comprises a support (2) coated with adhesive means for fixing elements (embryos, male and/or female gametes, fertilized oocytes, unfertilized eggs) and a removable cover part (4).

The open recoverable intra-uterine device (1 ) of the invention may have any shape adapted to be inserted into a device used for intrauterine supply, for example a catheter. Preferably, the device (1 ) of the invention has a rectangular or ellipsoid shape.

The support (2) of the open recoverable intra-uterine device (1 ) according to the present invention has a length comprised between 5 mm and 20 mm, preferably between 10 mm and 15 mm and more preferably, the length is 15 mm. The width of the support (2) is comprised between 0.5 mm and 1.5 mm, preferably between 0.7 mm and 1 mm and more preferably, the width is 1 mm. The depth of the support is comprised between 1 mm and 10 mm, preferably between 4 mm and 8 mm and more preferably, the depth is 5 mm.

According to one preferred embodiment shown in FIG.1 , the device (1 ) has a rectangular shape and comprises a solid support (2) and a removable cover part (4) having the same shape. In this embodiment, the length of the support is 15 mm, the width is 1 mm and the depth is 5 mm.

In addition, according to a preferred embodiment, the solid support (2) comprises one or more wells (3) in its inner part, i.e. dig in the material of the support. These wells are coated with adhesive means able to fix one or more elements selected from the group consisting of embryos, male and/or female gametes, fertilized oocytes, unfertilized eggs, or combinations thereof. When the support comprises wells, the wells are considered as a part of the support.

According to one embodiment, the shape of the wells is preferably a circular shape (as the wells (3) on FIG. 1 , 2 and 3) but they can have any other shape allowing to receive the elements to be loaded in the well. For example, the wells may have a rectangular, a square, an oval or a conical shape.

FIG 3 is a perspective view of a circular well (3). According to this preferred embodiment, the wells (3) of the device (1 ) of the invention have preferably a diameter comprised between 0.1 mm and 0.5 mm, more preferably between 0.1 and 0.3 and even more preferably, the diameter of the wells is 0.15 mm. The diameter of the wells may vary in accordance with the shape of the wells. For example, when the wells have a conical shape, the wide part of the wells which is opened on the surface of the support, has the same diameter as those for the circular walls disclosed above.

The solid support (2) of the device (1 ) is constituted from a biocompatible material. Generally speaking, the materials used must be tested to be non-toxic and stable in use. This material may be selected from the group consisting of a polymer, a ceramic, a glass, an elastomeric material, a stainless steel, a metal material, a titanium and a titanium alloy, preferably a titanium or a titanium alloy. In particular, the support (2) can be produced in a porous polymer, of the polyethersulfone (PES), polyacrylate, acry-late copolymer or polyvinylidene type.

As mentioned above, the device (1 ) of the invention also comprises a removable cover part (4). According to one embodiment shown on FIG. 2, the cover part (4) is partially removed. This removable cover part aims to protect the element fixed on the support (2) of the device or loaded in the wells (3) during the insertion of the device (1 ) in the means (for example a catheter, particularly a catheter which allows to maintain the cover part folded on the device during the insertion in the vagina and before attending the uterine cavity) adapted to be inserted into uterine cavity, wherein the device (1 ) of the invention is released. When the device (1 ) is released from said means, the removable cover part (4) is partially removed such as the elements fixed on the support (2) remain fully exhibited to uterine medium. After a predetermined period of exposure, the removable cover part (4) is replaced on the support (2) in order to protect the fixed elements when the device is recovered from the uterine cavity. At this manner, the use of a removable cover part (4) which may be folded on the surface of the support (2) when the device (1 ) is inserted in the supplying means and removed when the device (1 ) reaches the uterine cavity, allows the elements fixed on the support (2) and/or on the wells (3) to be exhibited to the uterine medium and all its components (molecules and cells) without any restriction. This allows to put these elements in direct contact with the uterine medium exactly at the same manner as during natural fertilization. In addition, the use of removable cover part (4) allows to safely recover these elements before recovering them with the device (1 ).

The material used for manufacturing the removable cover part (4) may be any material which allows to be removed and then replaced without provoking damages on the elements fixed on the support. For example, this material may be a shape memory material.

In the context of the present invention, a shape memory material refers to a material which can be deformed, generally when cold but returns to its pre-deformed ("remembered") shape, generally when heated. Particularly, the shape memory material used for manufacturing the removable cover part (4) of the device of the present invention is selected from shape memory alloy, shape memory polymers or shape memory composites. The shape memory material is a biocompatible material. Preferably, the shape memory material is a shape memory biocompatible polymer.

The removable cover part (4) of the open recoverable intra-uterine device (1 ) according to the present invention has preferably the same dimensions as the support (2). Preferably, the cover part has a length comprised between 5 mm and 20 mm, more preferably between 10 mm and 15 mm and even more preferably, the length is 15 mm. The width of the removable cover part (4) is comprised between 0.5 mm and 1.5 mm, preferably between 0.7 mm and 1 mm and more preferably, the width is 1 mm. The depth of the removable cover part (4) is comprised between 1 mm and 10 mm, preferably between 4 mm and 8 mm and more preferably, the depth is 5 mm.

According to a preferred embodiment shown on FIG. 1 and FIG. 2, the removable cover part (4) has a rectangular shape with a length of 15 mm, a width of 1 mm and a depth of 5 mm.

As indicated above, the removable cover part (4) is partially removed when the device is inserted in the uterine cavity in order to put into direct contact the element fixed on the support (2) and the uterine medium and then, when the device is recovered, the removable cover part (4) is folded on the support (2) during the passage of the device in the cervix and the vagina.

According to one embodiment, a one extremity of the removal cover part (4) is attached to the well of the supplying means so as when the device (1 ) is ejected from the supplying means in the uterine cavity, the removal cover part (4) is removed from the support (2) allowing thus to exhibit the elements attached on the support (2) to the uterine medium. Then, when after predetermined period the device (1 ) is removed from the uterine cavity, the device (1 ) is replaced in the supplying means and the removable cover part (4) is replaced (folded) on the surface of the support (2) to cover the fixed elements. The open intra-uterine device (1 ) of the present invention, compared to other intra-uterine devices disclosed in the relating art is particular in that the protection of the embryo/gametes fixed on the support (2) of the device must be assured two times: 1 ) when the device is inserted in the uterine cavity and 2) when the device is recovered from the uterine cavity with the fixed embryos/gametes after predetermined period of time. This is not the case with other intra-uterine devices in the art since their purpose is to only leave the embryo in the uterine cavity but not to recover it. Therefore, the removable cover part (4) must be designed so as to allow to exhibit the embryo/gamete in the uterine cavity and to cover them when they are introduced and recovered from the uterine cavity.

Because after predetermined period of time the device (1 ) with the fixed element has to be recovered from the uterine cavity, it is essential that the elements are fixed at the robust manner but at the same time this fixation has to be reversible since they are later replaced in a catheter allowing to introduce them in the uterine cavity for implantation.

The solid support is thus coated with adhesive means (or also called herein “adhesive compounds”). In the context of the present invention the term “adhesive means” refers to means that allow the elements recited above (embryos/gametes etc.) to be adhered, to be attached or to be fixed on the support (2) of the device. Therefore, the terms “adhesive”, “adherent”, “attached” and “fixed” and terms derived from these ones are used interchangeably herein. The adhesive means used in the open recoverable intra-uterine device of the invention are selected from any means able to ensure strong but reversible attachment of the loaded elements. Particularly, the adhesive means are selected from biochemical adhesive compounds or from mechanical, particularly mechanical-immuno-adhesive means (Novo S. et al., 2013).

According to one embodiment, the biochemical adhesive compounds are selected from the group consisting of compounds having the ability to bind receptor(s) specific to zona pellucida glycoproteins (ZP), cyanoacrylates, fibrine glue, biosensors mimicking the structure of grasshoppers’ feet and polysilicon barcodes for adhesion to the zona pellucida.

The biochemical adhesive compounds coating the solid support (2) and/or the wells (3) are preferably selected according to their ability to bind receptor(s) specific to zona pellucida glycoproteins (ZP) that surrounds the mammalian oocytes (3D) until hatching of the blastocysts (at day 5 in the human embryo). Structural basis of egg coat-sperm recognition at fertilization may help to find the best receptor present on the sperm’s head to coat the support (2) and/or the wells (3) of the device (1 ) of the invention to increase adherence of embryos/gametes on it. Compounds binding ZP on oocytes and/or embryos which may be used for coating the support and/or the wells of the device of the invention are selected from:

- beta-1 ,4-galactosyltransferase (GalT1 ) with its ability to bind N-acetylglucosamine residues on ZP;

- multimeric zona recognition complex (MZRC) which is an aggregation of several receptor molecules at focal points on the sperm surface compatible with the zona adhesion as found in functionally competent spermatozoa after the maturation process;

- ZP3 R (sp56) receptors; - heads of capacitated spermatozoa directly coating the surface of on the support/wells of the device (1 );

- formation of a monolayer that act as a linker, called self-assembled monolayer (SAM);

- antibodies binding specifics proteins of ZP, particularly ZP-2;

- lectins which are implicated in processes like cell-cell recognition or cellular adhesion such as wheat germ agglutinin (WGA) which recognizes specifically N-acetylglucosamine (GlcNAc) and sialic acid, carbohydrates that are abundant on the surface of the plasma membrane and zona pellucida (3C) of oocytes and embryos.

In addition, the biochemical adhesive means are selected from:

- cyanoacrylates: such as N-butyl cyanoacrylate and 2-Octyl cyanoacrylate (also called Dermabond disclosed in Min et al., 2011 );

- fibrin glue;

- biosensors mimicking the structure of grasshoppers’ feet providing a glue-free adhesion solution;

- polysilicon barcodes for adhesion to the zona pellucida (Oriol Penon et al. 2012).

Fixing an embryo or gamete on a solid support at robust and reversible manner without altering their normal development is a quate difficult.

The inventor surprisingly found that robust and reversible attachment of said elements, particularly oocytes and/or embryos may be obtained when combining some biochemical adhesive means with mechanical means. Such an attachment is particularly advantageous since it does not alter the quality of the attached elements as demonstrated by the examples below.

Thus, according to one embodiment, the adhesive means are mechanical-immuno-adhesive means.

Preferably, the mechanical means is a support or a stand and biochemical means is an antibody capable of binding at least one protein of zone pellucid of embryos, female gametes, fertilized oocytes or unfertilized eggs. Preferably, the support or strand is a bead and the mechanical- immuno-adhesive means is a bead coated with said antibody.

In the context of the present invention, the term “support” or “stand” is used to design a mechanical means which is a part of the adhesive means. In this context, the “support” is not the same as the support (2) which is included in the structure of the device (1 ) of the invention and on which surface are fixed the adhesive means.

The support or stand, particularly the bead may be manufactured from any material provided that it is able to be coated with an antibody anti zone pellucid. For example, such a material is a material having an attractive force. According to a preferred embodiment the bead coated with an antibody anti zone pellucid is a magnetic bead which is retained on the solid support (2) and/or the surface of the wells (3) by a means providing a magnetic force.

According to this embodiment, the magnetic beads are fixed on the surface of the solid support (2) and/or on the surface of the wells (3) of the device via a metal element providing a magnetic force. According to another embodiment, a means creating a magnetic field may be used to fix the magnetic beads on the solid support of the device (1 ).

According to a preferred embodiment, the support (2) and/or wells (3) are coated with magnetic beads coated with antibody anti-zone pellucid selected from the group consisting in anti-ZP-1 , anti- ZP-2 and anti-ZP-3, particularly ZP-2.

The Examples disclosed below describe an in vitro assay using a mouse model which demonstrated the good adhesion of magnetic beads coupled by antibodies anti zona pellucida (ZP-2) without any alteration of embryo development neither by the use of this adhesive means nor by the created magnetic field.

Particularly, the inventor developed a protocol to bind antibodies (primary) to the zona pellucida of embryo in a non-toxic manner. The binding of magnetic beads (for example Dynabeads™) to primary antibodies was challenging as beads were heavy and required constant motion to avoid sinking to the bottom of the dish. The inventor found out that by adding magnetic beads directly on top of the embryos, it is possible to slowly retrieve them with a magnet. To attract the magnetic beads at the bottom of the dish a motion may be created by gently stirring the beads.

In addition, the inventor surprisingly found that coupling directly the anti-ZP2 antibodies to the magnetic beads and maintain a continuous motion, assures the binding of the magnetic beads throughout the zona pellucida in a more homogenous and efficient manner, which results in a higher magnetic retrieval yield.

Therefore, according to a preferred embodiment, the adhesive means is magnetic bead and the anti-ZP antibody is anti-ZP-2 antibody.

According to another preferred embodiment, the biding is performed under gently stirring allowing to create a motion of the beads.

In another embodiment, the adhesive means are mechanical means such as 3D printed structures able to retain the above recited elements, particularly during their exhibition in the uterine cavity.

A particular mechanical means is for example a cage wherein the openings formed by the bars crossing have a size smaller than the size of the element putted into the cage. Therefore, the element, when loaded in the cage cannot be released in the uterine cavity but the uterine fluid with all substances necessary to the in-utero culture can penetrate the cage and thus being into direct contact with the loaded element.

This cage is provided with a removable door allowing to load the element before introducing the device (1 ) in the uterine cavity and to remove the element from the cage after retrieving the device (1 ) from the uterine cavity.

The cage may be manufactured from a nontoxic biocompatible material. This material may be selected from the group consisting of a polymer, a ceramic, a glass, an elastomeric material, a stainless steel, a metal material, a titanium and a titanium alloy. According to one embodiment, the cage maybe manufactured from shape memory material allowing thus to load/remove the elements by the openings formed by the crossing bars. In this case, the presence of a door is not necessary.

In addition, the cage may have any shape adapted to the inserted element. For example, the cage may have a square shape, a rectangle shape, a circular shape, an oval shape or conical shape.

The cage is fixed on the support (2) of the device (1 ) or when present, in the wells (3) of the device (1 ) by any means allowing a strong fixation. According to one embodiment, the cage may be extruded from the material of the support (2).

As indicated above, the elements attached on the surface of the solid support (2) and/or the wells (3) of the device of the invention are selected from the group consisting of embryos, male and/or female gametes, fertilized oocytes, unfertilized eggs, or combinations thereof.

Particularly, these elements are obtained from mammal selected from the group consisting of bovine, ovine, porcine, horses and human. More particularly, the mammal is a human being.

As used herein, the term “embryo” refers to mammalian fertilized oocyte (zygote), wherein the first mitosis has not been yet performed.

The male and female gametes are haploid reproductive male and female cells respectively.

As used herewith the term “fertilized oocyte” refers to mammalian female gamete fertilized by a mammalian male gamete, i.e. the fertilized oocyte corresponds to a zygote (diploid cell). At this early post-fertilization stage, the fertilized oocyte may be transferred via the device (1 ) of the invention into the uterine cavity for a period of 24 hours or 2 to 5 days (for attending embryo stage of development) and then, it may be retrieved to be analyzed. After such an analysis, if appropriate, the formed embryo is transferred via an appropriate device (catheter) into the uterine cavity in order to proceed to its implantation in the uterine wall.

The present invention also relates to a method of preparing an open retrievable intra-uterine device for placing one or more elements selected from the group consisting of embryos, male and/or female gametes, fertilized oocytes, unfertilized eggs, or combinations thereof comprising the steps of: providing said element under the appropriate form to be attached on the surface of the solid support (2) and/or the wells (3) of the device, providing the open retrievable intra-uterine device (1 ) suitable for receiving said element(s) according to the invention, attached said element(s) on the surface of the solid support (2) and/or the wells (3) of the device (1 ).

The open recoverable intra-uterine device of the invention furthermore cooperates with accessories (means) for placing the device in the uterine cavity. For example, a transfer catheter having an inside diameter of 1 cm to 1.3 cm may be used. A catheter of this kind is adapted to pass through the cervical canal, which generally has a diameter of about 1 .5 cm.

As indicated above, the device of the invention can be implanted passing through the cervix as a standard IUD (intra-uterine device) for contraception into the uterus and removed after a defined time of incubation.

The device of the present invention may also use as implantation site in the Fallopian tube. This implantation needs to use a surgical procedure as coelioscopy with general anaesthesia or culdoscopy with local anaesthesia. Such in vivo intra Fallopian embryo culture is similar to GIFT or ZIFT except the fact that using of the open recoverable intra-uterine device of the invention, the incubation time is under control and an unlimited number of embryos can be loaded which were retrieved and selected for transfer after a simple flushing procedure.

The device of the invention presents a number of advantages: said device is not likely to cause any trouble to the uterus (little or no tissue reaction, without inducing inflammatory or fibrotic reactions and or inappropriate uterine wall tissue damage or scarring) due to the fact that it is not implanted within the uterine wall, it does not require absolutely surgery or anaesthesia to be inserted and it can be inserted in a completely ambulatory fashion.

Furthermore, the device of the invention is designed such that it has a means of remaining within the uterus (i.e. small suture thread glued into the tip of the device for attachment inside or outside the uterus) and that can be easily retrieved (attached suture thread) at any time after uterine implantation.

As explained above, the open recoverable intra-uterine device of the invention is able to present gametes and/or embryos to the natural microenvironment of the uterine cavity without encapsulation (as the similar devices in the relating art). It permits to obtain for the gametes and the embryos an optimal micro-environment in a small volume. The device of the invention offers to developing embryos a closer biophysics-chemical environment with less osmolarity alteration and permits to the uterus to become a real transitory natural incubator offering the possibility of a complete physico-chemical and cellular complex cross talk to take place between the mother and embryos. Even, complete in vivo/in utero fertilization may take place into the uterine cavity with the performance of intrauterine insemination after the intrauterine transfer of this device fixing oocytes.

The device of the present invention resolves the problem of embryo(s) passage from the uterus through the inner cervical canal and their expulsion into the vagina.

Therefore, as mentioned above, the open recoverable intra-uterine device may be used successfully in assisted reproduction.

According to one aspect, the present invention thus relates to a method of assisted reproduction, wherein the method comprises the following steps:

- preparing an open recoverable intra-uterine device (1 ) of the invention, particularly as per the above-described method,

- placing the device in the uterine cavity for a predetermined period,

- at the end of this period, removing the device,

- monitoring the development of the element of interest, and

- reintroducing the element of interest into the uterine cavity without the device of the invention or conserving the element for further treatment.

As used herein, the “predetermined period” corresponds to the period or time necessary to postfertilization embryo development (conventionally performed in in vitro culture) before the definitive transfer of the embryo into uterine cavity. The “predetermined period” may also correspond to the time necessary for performing in utero fertilization (i.e. fertilization in the device of the invention) when oocytes and spermatozoids are contained in the device.

The device may be placed in the uterine cavity by the means described above.

More particularly, the device of the present invention may be used in in vivo fertilization by injecting prepared sperm and retrieval oocytes in the device for implanting it into the uterus. After a defined and controlled incubation time (2 hours for example) of in vivo and in utero culture, the sperm and oocytes are retrieved and zygotes and/or unfertilized oocytes are collected after a simple flushing procedure. Then, selection of zygotes for cryopreservation or in vitro culture of the remaining embryos to be transferred at day 3 is performed.

According to one embodiment, the device of the present invention may be also used in in vivo preimplantatory embryo development by injecting several embryos at different stage of development (i.e 6-8 cells) in the device followed by the implantation of the device into the uterine cavity during a controlled time (i.e 48 hours). After removal of the device from the uterus, the embryos at the blastocyst stage are flushed from the device and transferred into the uterine cavity using a conventional transfer catheter or delayed in a further cycle after freezing. According to another embodiment, the device of the invention may be used in in vivo embryo assisted hatching.

Of course, numerous modifications can be made to the embodiment described hereinabove without departing from the scope of the invention.

EXAMPLES

The purpose of the below described assays is to evaluate:

1 ) the capacity of anti-ZP antibody, particularly anti-ZP-2 antibody to bind the zona pellucida;

2) the anti-ZP2 - magnetic bead (Dynabeads™) system to bind and retrieve mouse embryos, and

3) early embryo development post-incubation with antibodies/magnetic beads.

Methods and materials

> Material needed for the embryo collection:

1 Eppendorf tube (for 1 mL KSOM)

5 Petri dishes labelled 1 to 5 (Add additional 4 Petri dishes for rinsing steps except for 5)

3 Eppendorf tubes labelled 1 °Ab; 2°Ab; BB

Primary antibody (1 °Ab) Rat anti-ZP2 (1 :50)

Dynabeads™ Protein G (ThermoFisher Scientific) (referred to as 2°Ab)

Blocking buffer (BB) composed of PBS-BSA 3% diluted in Tween 0.1%

KSOM medium culture

> Mixture preparation :

The Ab mixtures was always kept on ice.

1) Preparing BB mixture PBS-BSA 3% diluted in Tween 0.1% - Placing in a tube labelled BB

2) Preparing 1 °Ab mixture (1 :50) 1 :12.5 (4X) - 8pL of antibody in 92pL BB - Placing in a tube labelled 1 °Ab

3) Preparing 2° Ab mixture Dynabeads (1 :1000) 1 :2500- 0.1 pl of Dynabeads in 249pl BB. Placing in a tube labelled 2°Ab.

> Embryo collection

1 ) Preparing 5 different Petri dishes labelled 1 - 5 with drops of KSOM (Figure 6). 2) Collecting single-cell mice embryos and counting them (# of embryos:)

3) Separating the embryos into 4 groups. (If two-cell embryos, place them in another group (#5)) a. #1 : > embryos b. #2 : > embryos

C. #3 : > embryos d. #4 : > embryos

(■#5) : embryos)

> Incubation in primary antibody ( 1 Ab):

4) Placing 50 pl of mixture 1 °Ab on embryos in Petri #2/#3 (and #5).

5) Placing 50 pl of mixture BB on embryos in Petri #1 and #4.

6) Incubating embryos for 1 h at 37° C.

7) Taking Petri #2 and rinsing twice with KSOM -Adding 2 drops (50 pL) KSOM on Petri dish

#1 , and transferring the embryos through both drops, to allow rinsing. Transferring the embryos into a new Petri dish with KSOM, labelled #2B.

8) Repeating with 1 °Ab for conditions #3 and #5.

9) Repeating with BB instead of 1 °Ab and for conditions #1 and #4.

> Incubation in secondary antibody (2’Ab):

10) Placing 50 pl of mixture 2° Ab on embryos in Petri #3/#5 and #4.

11 ) Placing 50 pl of mixture BB on embryos in Petri #1 and #2.

12) Incubating for 30 min at 37° C.

13) Taking Petri #3B and rinsing with PBS (pH 7.4) ■ Adding 200 pl of PBS on Petri dishes, gently shaking and transferring the embryos to a new Petri dish with KSOM, labelled #3C.

14) Repeating for #5B and #4B

15) Taking pictures of all conditions.

16) Placing in an incubator at 37° C Y1

For #5: Placing magnet and observing attraction of embryos towards magnet - making a video.

Picture of embryo development was taken every 24h for 96h.

If cell development stops the embryos are kept on the same Petri dish. At the end of the experiment: the number of embryos fully developed vs. dead is recorded.

> Magnetic effect :

17) On day 5: magnets (one big and one small) were used on conditions #1

to transfer embryos from one side of the Petri dish to the other. Conditions #1 and #2 were maintained such as.

18) Counting the embryo attracted vs. non attracted to conditions #3 and #4.

Results

I.The anti-ZP2 antibody binds to the zona pellucida a) In order to test the anti-ZP-2 antibody, the inventor used a modified protocol on adult ovarian tissue. Anti-ZP-2 antibody was used as primary antibody and donkey anti-rat 594 (fluorescent dye) as secondary antibody to validate the binding of the primary antibody to the zona pellucida (FIG.4). The fluorescent staining of the zona pellucida led to the conclusion that anti-ZP-2 specifically binds to ZP-2 receptor. The primary antibody was thus validated (van Oss. et al. 1986). b) The same protocol was adapted on fixed mouse embryos in suspension using Eppendorf tubes. Centrifugation was done in between staining and washing steps. The protocol was done in technical duplicates. As shown on the FIG. 5, at the end of the experiment 90% of embryos from the control group were present and 93.3% from the tested embryos were present and expressed stained zona pellucida as expected. The protocol on embryos was thus validated.

a) First, beads alone were tested under a microscope. Post incubation of the beads, a magnet was placed next to the slide to attract the beads. The beads attraction to magnetic fields was observed. b) Then, embryos were incubated with anti-ZP-2 and Dynabeads™. Post-incubation, a magnet was placed next to the Petri dish : a weak attraction was observed. c) Finally, the inventor added magnetic beads directly on the embryos and added motion to the mixture. Post-incubation, a magnet was placed next to the Petri dish : an attraction was observed over time.

From the above, it was concludesd that the induction of a motion of the recation mixture containing the embryo, anti-ZP-2 antibodies and Dynabeads™ allows to improve the magnetic atraction of the beads by the magnetic field. 3. The bindin of antibodies does not interfere with the development of the embryos

To test if embryo developement may be altered following the binding of anti-ZP2 antibody, a total of 181 mice embryos at the single-cell stage were obtained by IVF, collected and separated into 4 experimental groups: 45 in the control group (#1 ) without any antibodies; 45 embryos incubated only with the primary antibody (1 °Ab) (#2); 46 embryos with both the 1 ° Ab and the Dynabeads (2°Ab) (#3); and 45 embryos for the last condition (#4) with only the 2°Ab. Of note, an additional group (#5) was done with the 28 embryos at the two-cell stage. #5 had the same condition as #3 but was used as a test group to use the magnet on the embryos following the experiment.

In order to test possible damage caused on embryo by the magnetic field of the magnet directly after the first incubation, condition #3 was done twice (once with single-cell embryos (#3) and once with 2-cells embryos (not shown)).

The experimental protocol is show on FIG. 6.

On Day 0: 181 Embryos were divided into 4 groups. Additional 28 2-cells-embryos were selected to reproduce condition (#3) and test the magnet directly after each experimental day (#5).

Groups #2, #3, #5 received the anti-ZP-2 antibody diluted in KSOM and were incubated 1 h at 37°C. Post incubation, the embryos were rinsed in KSOM drops twice and placed on a new Petri dish. Groups #3, #4 and #5 then received the beads diluted in KSOM and were incubated for 30min at 37° C. Post incubation, the embryos were rinsed twice and placed in new Petri dishes. Pictures were taken. All groups were placed overnight at 37° C. Condition #5 was used to test the magnet each day. Embryos were then placed at 37° C overnight as well.

On Days 1 , 2, 3 and 4: All embryos were observed and pictures were taken. Divided and non-divided embryos were reported (Table 1 ). The magnet was used on condition (#5) to evaluate embryo retrieval.

Table 1 : Embryo development assessment over time

Day 0 Day 1 Day 2 Day 3 Day 4

#1 100 95.6 100 100 100

#3 100 95.6 95.6 95.6 95.6

At the end of Day 4: donkey anti-rat 594 fluorescent antibodies were added to all conditions in order to evaluate 1 ) the binding of the anti-ZP2 to the zona pellucida and 2) the binding of the Dynabeads™ to the primary antibody.

As observed in FIG. 7, condition #2, #3 and #5 showed fluorescence. This suggests that Dynabeads™ probably detached from the primary antibody and migrate to the bottom of the dish, not properly bound to the primary antibody, allowing for the fluorescent secondary antibodies to bind correctly. The attachment of primary antibodies to Dynabeads™ is improved by inducing a continuous motion.

From the above it appears that neither binding of the anti-ZP2 antibody, nor presence of magnetic beads interfered with embryo development. Binding of the beads to the antibodies is performed preferably in a continuous movement. When embryos were covered with magnetic beads, embryo weak magnetic retrieval was observed, i.e., embryo is thus easily recovered and the antibodies anti-zona pellucida may be recovered too, notably by a protease treatment in vitro.

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Claims

1. An open recoverable intra-uterine device (1 ) comprising:

- a solid support (2) coated with at one or more adhesive means for fixing one or more elements selected from the group consisting of embryos, male and/or female gametes, fertilized oocytes, unfertilized eggs, or combinations thereof, and

- a removable cover part (4).

2. The open recoverable intra-uterine device (1 ) according to claim 1 , wherein the solid support (2) comprises one or more wells (3) coated with one or more adhesive means for fixing one or more elements selected from the group consisting of embryos, male and/or female gametes, fertilized oocytes, unfertilized eggs, or combinations thereof.

3. The open recoverable intra-uterine device (1 ) according to claim 1 or 2 having a shape adapted to be inserted into a device used for intrauterine supply, preferably, said device (1 ) has a rectangular or ellipsoid shape.

4. The open recoverable intra-uterine device (1 ) according to any one of claims 1 to 3, wherein the support (2) has a length comprised between 5 mm and 20 mm, a width comprised between 0.5 mm and 1.5 mm and a depth comprised between 1mm and 10 mm.

5. The open recoverable intra-uterine device (1 ) according to any one of claims 1 to 4, wherein the solid support (2) is constituted from a biocompatible material selected from the group consisting of a polymer, a ceramic, a glass, an elastomeric material, a stainless steel, a metal material, a titanium and a titanium alloy, preferably a titanium or a titanium alloy.

6. The open recoverable intra-uterine device (1 ) according to any one of claims 2 to 5, wherein the wells have a diameter comprised between 0.1 mm and 0.5 mm.

7. The open recoverable intra-uterine device (1 ) according to any one of claims 1 to 6, wherein the adhesive means are biochemical adhesive compound.

8. The open recoverable intra-uterine device (1 ) according to claim 7, wherein the biochemical adhesive compounds are selected from the group consisting of compounds having the ability to bind receptor(s) specific to zona pellucida glycoproteins (ZP), cyanoacrylates, fibrine glue, biosensors mimicking the structure of grasshoppers’ feet and polysilicon barcodes for adhesion to the zona pellucida.

9. The open recoverable intra-uterine device (1 ) according to any one of claims 1 to 6, wherein the adhesive means are mechanical-immuno-adhesive means.

10. The open recoverable intra-uterine device (1 ) according to claim 9, wherein the mechanical- immuno-adhesive means is preferably a stand coated by an antibody capable of binding at least one protein of zone pellucid of embryos, female gametes, fertilized oocytes or unfertilized eggs.

11. The open recoverable intra-uterine device (1 ) according to claim 10, wherein the stand is a bead.

12. The open recoverable intra-uterine device (1 ) according to claim 11 , wherein the bead is a magnetic bead which is retained on the surface of the support (2) by a means providing a magnetic force.

13. The open recoverable intra-uterine device (1 ) according to any one of claims 1 to 12, wherein the support (2) is coated with magnetic beads coated with antibody anti-zone pellucid selected from the group consisting of anti-ZP-1 , anti- ZP-2 and anti-ZP-3, preferably anti-ZP-2.

14. The open recoverable intra-uterine device (1 ) according to any one of claims 1 to 6, wherein the adhesive means are mechanical adhesive means, preferably a cage.

15. The open recoverable intra-uterine device (1 ) according to any one of claims 1 to 14, wherein the one or more elements fixed by one or more adhesive means coating the surface of the support (2) is from mammals elected from the group consisting of bovine, ovine, porcine, horses and human, preferably from human.