A microfluidic chip, apparatus and method for in vitro fertilization

Abstract

A microfluidic chip (100) for in vitro fertilization, comprising a microfluidic circuit (102) having an oocyte feed branch (110), a spermatozoon feed branch (120), a fertilization area (130) and an embryo processing branch (140), the microfluidic chip (100) further comprising at least one remotely controllable microstructure (10) that is mobile between the fertilization area (130) and at least one of the oocyte feed branch (110), a spermatozoon feed branch (120) and the embryo processing branch (140).

Description

A microfluidic chip, apparatus and method for in vitro fertilizationTECHNICAL FIELD

[0001] The present disclosure relates to the field of in vitro fertilization (IVF), and more particularly to a microfluidic chip for IVF, an apparatus for IVF and an IVF method.TECHNOLOGICAL BACKGROUND

[0002] In vitro fertilization has developed over the past decades, however the underlying principles generally remain the same: preparing one or more oocytes, preparing one or more spermatozoa, and bringing them close to each other in a controlled in vitro environment to provoke fertilization and obtain an embryo.

[0003] Nowadays, the techniques used for IVF still require an operator manipulating the gametes or the embryo, generally by a micropipette. These manipulations require access to the gametes and the embryo and therefore must take place outside of the controlled environment. This result in several drawbacks: the cells risk being damaged while outside the controlled environment, the amount of consumables needed is high, manipulations into and out of the controlled environment are time-consuming, errors and contaminations may occur, etc.

[0004] Therefore, there is a need for new in vitro fertilization techniques.SUMMARY

[0005] In this respect, the present disclosure relates to a microfluidic chip for in vitro fertilization, comprising a microfluidic circuit having an oocyte feed branch, a spermatozoon feed branch, a fertilization area and an embryo processing branch, the microfluidic chip furthercomprising at least one remotely controllable microstructure that is mobile between the fertilization area and at least one of the oocyte feed branch, a spermatozoon feed branch and the embryo processing branch.

[0006] A microfluidic circuit is a circuit for circulation of one or more fluids, wherein the dimension of the channels is generally between 1 and 500 micrometers (pm), preferably between 5 and 200 pm. A microfluidic circuit is used to manipulate microquantities of the fluids, e.g. between 10’9to 1018liters. A microfluidic chip forms a support for the microfluidic circuit. The microfluidic circuit may be engraved in the microfluidic chip.

[0007] The oocyte feed branch is a part of the microfluidic circuit where one or more oocytes may be introduced and optionally prepared. Likewise, the spermatozoon feed branch is a part of the microfluidic circuit where one or more spermatozoa (also referred to as sperm herein) may be introduced and optionally prepared. The fertilization area is a part of the microfluidic circuit where fertilization occurs between at least one oocyte and sperm. The fertilization may be assisted, as will be described below. Finally, the embryo processing branch is a part of the microfluidic circuit where the embryo is processed after fertilization, e.g. grown in a culture medium. While defined in a functional manner, the oocyte feed branch, the spermatozoon feed branch, the fertilization area and the embryo processing branch may at least partially overlap. For instance, fertilization may occur directly in the embryo processing branch, in which case the fertilization area overlaps with the embryo processing branch.

[0008] As used herein, for conciseness and unless the context indicates otherwise, "a", "an", and "the" are intended to refer to "at least one" or "each" and to include plural forms as well. Conversely, generic uses of the plural form may include singular elements.

[0009] As used herein, gametes collectively refer to oocytes and spermatozoa, and cells include at least gametes, zygotes, and embryos. In the following, for the sake of conciseness, "embryo" is understood asencompassing a fertilized oocyte at any stage of its development, including a zygote.

[0010] At least one remotely controllable microstructure is provided in the microfluidic chip, desirably in the microfluidic circuit. The microstructure may assist movements of the gametes or the embryo from one part of the microfluidic circuit to another. For doing so, the microstructure can be moved between the fertilization area and at least one of the oocyte feed branch, a spermatozoon feed branch and the embryo processing branch, in a direction from and / or to the fertilization area. Methods for remotely controlling the microstructure will be detailed below. Remote control means that there is no mechanical contact, either direct or indirect, between the microstructure and the remote controller (or remote manipulator).

[0011] Thanks to the remotely controllable microstructure, no micropipette or other access-requiring instrument is needed anymore to perform the IVF. Accordingly, manipulation time is reduced, errors are avoided, and the microfluidic chip can remain in a controlled environment without interruption if desired. Moreover, since the preparation of the gametes, the fertilization and the embryo processing take place on the same microfluidic chip, the amount of consumables can be reduced, which further avoids errors and decreases costs and waste.

[0012] Optionally, the microfluidic chip is transparent, thus enabling observation of the IVF process.

[0013] Optionally, the fertilization area is hermetically enclosed from an outside of the microfluidic chip. Thus, the fertilization area may communicate with the outside of the microfluidic chip only through other parts of the microfluidic circuit, e.g. the oocyte feed branch, the spermatozoon feed branch, or the embryo processing branch. The hermetically enclosed fertilization area does not grant access to anyexternal instrument such as a micropipette and ensures that fertilization occurs in controlled conditions.

[0014] Optionally, the embryo processing branch comprises a plurality of chambers, each chamber being configured to receive one or more embryos. Therefore, a plurality of fertilizations can be performed, with each embryo or group of embryos growing in its / their own dedicated environment.

[0015] Optionally, the fertilization area is downstream of the oocyte feed branch and of the spermatozoon feed branch, and upstream of the embryo processing branch. In these embodiments, the fertilization area may be separated from the aforementioned branches. This facilitates monitoring of the IVF and decreases the risk of mixing cells.

[0016] Optionally, the oocyte feed branch comprises an oocyte decoronization conduit. Decoronization, also known as denudation, refers to the removal of the cumulus cells around the oocyte. The decoronization conduit may have a narrowing shape and / or sharp edges for decoronizing the oocyte when the oocyte travels through the decoronization conduit. Therefore, decoronization can occur in the microfluidic chip, thereby enhancing the ability of the microfluidic chip to host a full IVF cycle.

[0017] Optionally, the spermatozoon feed branch comprises a complex pathway, optionally including one or more obstacle or one or more maze. The complex pathway may be a compulsory pathway for sperm moving towards the fertilization area. The complex pathway, possibly including obstacles or mazes, helps separating motile spermatozoa from non-motile spermatozoa and / or poorly motile spermatozoa, based on the motile sperm's ability to swim through obstacles and / or mazes. This allows the most promising sperm to be efficiently selected

[0018] Optionally, the microstructure comprises a chamber for housing at least one of a spermatozoon, an oocyte and an embryo, andfurther comprises at least one handle for manipulation by a remote controller. Thanks to the chamber, the microstructure can capture a cell in order to move it from one point to another, e.g. between the fertilization area and one of the branches. The handle, or plurality of handles, may have the desired features to enable remote manipulation of the microstructure.

[0019] Optionally, the remote controller is an optical controller, a magnetic controller or an acoustic controller. An optical controller may use optical tweezers to trap and move the handles. A magnetic controller may use a magnetic field to move the magnetically-responsive handles. An acoustic controller may use acoustic tweezers to trap and move the handles.

[0020] Besides the features already mentioned, the remotely controllable microstructure may comprise at least one of the following features, taken alone or in technically possible combinations:- the chamber is provided with an entrance, a cross-section of the entrance being smaller than a cross-section of the chamber;- a cross-section of the chamber is less than or as large as three times the cross-section of the entrance, optionally less than or as large as twice the cross-section of the entrance;- the chamber is as large as or narrower than three times a width of the target cell to manipulate;- a length of the chamber is less than or as long as four times a width of the entrance, optionally less than or as long as three times the width of the entrance;- the microstructure is devoid of any articulated part;- the chamber comprises a vent opposite the entrance;- any path from the chamber to an outside of the chamber, other than the entrance, has at least one cross-section strictly narrower than the entrance;- the microstructure further comprises at least one pillar to support the chamber;- a material of the microstructure (e.g. of the handle) has a refractive index greater than a refractive index of the fluid medium in the microfluidic circuit, optionally wherein a material of the microstructure (e.g. of the handle) has a refractive index greater than or equal to 1.3;- the entrance is provided adjacent an inclined wall configured to lead the target into the chamber.

[0021] The present disclosure is further directed to an apparatus for in vitro fertilization, comprising an incubator configured to receive at least one microfluidic chip, at least one fluidic feed and at least one fluidic discharge configured to be connected to the at least one microfluidic chip, and a remote controller for remotely controlling a microstructure provided in the at least one microfluidic chip.

[0022] The incubator may be configured to receive the abovedescribed microfluidic chip. The remote controller may be configured to remotely control the above-described microstructure. The remote controller may be configured to remotely control the microstructure while the microfluidic chip is in the incubator.

[0023] The incubator may be configured to control the environment of the microfluidic chip, e.g. in terms of temperature (e.g. about 37°C), gas concentrations, humidity, etc.

[0024] Optionally, the apparatus further comprises at least one of: an imaging system configured to capture at least one image of the microfluidic chip; a pump configured to supply fluid to the fluidic feed or to suck fluid from the fluidic discharge; a first laser beam source.

[0025] The imaging system may be provided for monitoring purposes. The imaging system may comprise one or more microscopeobjectives, and / or one or more cameras, e.g. directed to the microfluidic chip.

[0026] The pump may be controlled to provoke the desired circulation of fluid within the microcircuit.

[0027] The first laser beam source may be provided for surgical operations on the cells (e.g. incision, ablation, etc. as will be described below). In the present disclosure, unless otherwise indicated, the mention of a "first" element, such as a first laser beam source, does not necessarily imply the existence of a "second" element, nor, where applicable, any relationship of order between the first and second elements. Ordinal qualifiers are, in this context, used solely for purposes of clarity and identification, without prejudging any particular characteristics. Similarly, and conversely, the mention of a higher-ranking element (third, etc.) in no way implies that lower-ranking elements, such as a possible second element, exist and / or possess the features that may have been presented elsewhere. In addition, an element (e.g. laser beam source, etc.) mentioned without "first" or "second" may indicate either one or both of the first element and the second element.

[0028] Optionally, the apparatus comprises a second laser beam source. The second laser beam source may be part of an optical remote controller.

[0029] Optionally, the first laser beam source and the second laser beam source are directed to a common focusing system. The focusing system may comprise a 2D or 3D scanning system for controlling the position of the laser focal point. The scanning system typically uses steering mirrors. Since the first laser beam source and the second laser beam source have different applications, they are generally not used simultaneously. In addition, both laser beam sources are used to perform operations on the microfluidic chip. This embodiment leverages these observations by providing a common focusing system for the first laserbeam source and the second laser beam source, which saves space and costs.

[0030] Independently but likewise, the same microscope objective may be used for the focalization of the laser beam sources and for the imaging system, which saves space and costs.

[0031] The present disclosure is further directed to a method for in vitro fertilization, comprising providing the microfluidic chip as described above, and remotely controlling the at least one microstructure to move a spermatozoon from the spermatozoon feed branch to the fertilization area, to move an oocyte from the oocyte feed branch to the fertilization area, and / or to move an embryo from the fertilization area to the embryo processing branch. Accordingly, IVF can be carried out with less or even no contact-requiring manipulations (e.g. micropipetting) as described above.

[0032] Optionally, the method further comprises placing the microfluidic chip within the incubator of the above-described apparatus, wherein the remote controlling is carried out while the microfluidic chip is in the incubator. Therefore, as described above, the microfluidic chip environment can be appropriately controlled during the IVF process.

[0033] Optionally, the method comprises circulating a fluid in the spermatozoon feed branch to separate motile spermatozoa from non- motile spermatozoa and less motile (e.g. poorly motile) spermatozoa. This feature relies on the motile sperm's ability to swim against the current and enables to efficiently select the most promising spermatozoa. The non- motile sperm and / or poorly motile sperm may be evacuated with the circulating fluid.

[0034] Optionally, the method comprises growing an embryo in at least one of the fertilization area and the embryo processing branch, optionally for a plurality of days.

[0035] Optionally, the remotely controlling the at least one microstructure to move a target cell comprises:- capturing the target cell in a chamber of the microstructure;- moving the microstructure enclosing the target cell;- releasing the target cell by moving the microstructure around the target so as to extract the target cell from the chamber.

[0036] Optionally, the releasing comprises moving the microstructure with respect to the target cell according to a relative movement substantially reversed as compared to a capturing movement.

[0037] Optionally, the method further comprises:- making an incision in an oocyte, optionally with a laser beam;- remotely controlling the at least one microstructure to move a spermatozoon closer to the incision and releasing the spermatozoon;- immobilizing the spermatozoon, optionally with a laser beam.

[0038] The incision in the oocyte is intended to enable sperm to go through, as known per se in the art.

[0039] The sperm is moved closer to the incision than it previously was, and is immobilized. Note that immobilization may occur before, during or after the remote controlling. More generally, the above-recited steps may be performed in the above-recited order or any other suitable order; for instance, making an incision in the oocyte may be performed after controlling the microstructure.

[0040] Thanks to these features, IVF is facilitated.

[0041] Optionally, the spermatozoon is released at a distance of the incision less than twice a diameter of the oocyte, optionally less than the diameter of the oocyte. Therefore, the spermatozoon is ready for fertilization.

[0042] Optionally, the method further comprises remotely controlling the at least one microstructure to push the released and immobilized spermatozoon through the incision.

[0043] Instead of conventional intracytoplasmic sperm injection (ICSI), in which sperm is injected by a micropipette inserted inside the oocyte, the present method offers a contactless alternative using the microstructure to push the (possibly immobilized) sperm inside the oocyte. Therefore, the above-mentioned advantages of remote control are maintained throughout the whole IVF cycle, including this so-called contactless ICSI. In addition, this alternative may be less aggressive for the oocyte.

[0044] Optionally, the immobilizing of the spermatozoon comprises ablating a flagella thereof.

[0045] The present disclosure is further directed to a method for contactless intracytoplasmic injection, comprising:- making an incision in an oocyte, optionally with a laser beam;- remotely controlling at least one microstructure to move a spermatozoon closer to the incision and releasing the spermatozoon;- immobilizing the spermatozoon;- remotely controlling at least one microstructure to push the released and immobilized spermatozoon through the incision.

[0046] The above comments apply mutatis mutandis to that method. In particular, the steps may be performed in any suitable order, including the above order.BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The invention and advantages thereof will be better understood upon reading the detailed description which follows, of embodiments given as non-limiting examples. This description refers to the appended drawings, wherein:- Fig. 1 is a diagram illustrating a microfluidic chip for IVF according to an embodiment;- Fig. 2 is a cross-sectional view of a microstructure according to an embodiment;- Fig. 3 is a diagram illustrating an apparatus for IVF according to an embodiment;- Fig. 4 depicts steps of an IVF method according to an embodiment.DETAILED DESCRIPTION OF EMBODIMENTS

[0048] A microfluidic chip 100 for in vitro fertilization according to an embodiment is diagrammatically illustrated in Fig. 1. The microfluidic chip 100 comprises a microfluidic circuit 102 provided on a support 104, as known per se in the art.

[0049] The microfluidic circuit 102 comprises an oocyte feed branch 110, a spermatozoon feed branch 120, a fertilization area 130 and an embryo processing branch 140. The microfluidic circuit 102 fluidically connects the oocyte feed branch 110, the spermatozoon feed branch 120, the fertilization area 130 and the embryo processing branch 140 to one another, directly or indirectly, in series or in parallel. For instance, in this embodiment, the fertilization area 130 is downstream of the oocyte feed branch 110 and of the spermatozoon feed branch 120, and upstream of the embryo processing branch 130. In this context, upstream and downstream are defined with respect to the normal direction of the gametes in view of the fertilization process. Thus, an oocyte 112 present in the oocyte feed branch 110 and a sperm 122 present in the spermatozoon feed branch 120 can be brought together to the fertilization area 130, where fertilization occurs. The resulting embryo may then be brought to the embryo processing branch 140.

[0050] At least one fluidic inlet 150 and at least one fluidic outlet 152 may be provided to enable fluid to enter and exit the microfluidic circuit 102. For instance, respective fluidic inlets 150 open out into the oocyte feed branch 110 and the spermatozoon feed branch 120, and afluidic outlet 152 exits from the embryo processing branch 140. More fluidic inlets and outlets may be provided depending on the desired design, e.g. an additional fluidic outlet for evacuating fluid without passing through the embryo processing branch 140. Besides, dedicated fluidic inlets 150 and / or fluidic outlets 152 may be provided for any each of the oocyte feed branch 110, the spermatozoon feed branch 120 and the embryo processing branch 140. Although the microfluidic circuit 102 has been illustrated in a simplified manner, other microfluidic components may be provided besides fluidic inlets and outlets, as known per se in the art, e.g. microvalves and the like, to direct the fluid as desired.

[0051] The fluidic inlets 150 and the fluidic outlet 152 may be the only points of access to the inside of the microfluidic circuit 102. In other words, apart from the fluidic inlets 150 and the fluidic outlet 152, the microfluidic circuit 102 may be hermetically enclosed from an outside of the microfluidic chip 100. More generally, the fertilization area 130 may be hermetically enclosed from an outside of the microfluidic chip 100. More generally, the microfluidic circuit 102 may be designed so that it is not possible to access the fertilization area 130 without going through another component or branch of the microfluidic circuit 102. In particular, the fertilization area 130 may be inaccessible to instruments external to the microfluidic chip 100, e.g. micropipettes and the like.

[0052] Respective gametes may be provided to the oocyte feed branch 110 and the spermatozoon feed branch 120 via conventional manipulations, e.g. introduced through the fluidic inlets 150.

[0053] The oocyte feed branch 110 may have any desired feature to process one or more oocyte 112 for IVF. In particular, as mentioned above, the oocyte feed branch 110 may comprise an oocyte decoronization conduit 114. Decoronization conduits are known per se in the art and undesired parts may be evacuated fluidically, e.g. through a dedicated evacuation conduit.

[0054] Other oocyte processing may occur in the oocyte feed branch 110, e.g. the visualization and estimation of oocyte maturity using images from an imaging system such as that described below; and / or the delivery of different liquids at different flowrates, such as for oocyte washing, refreshing of the culture medium, or introducing other solutions like hyaluronidase to facilitate decoronization.

[0055] The spermatozoon feed branch 120 may have any desired feature to process one or more sperm 122 for IVF. In particular, as mentioned above, the spermatozoon feed branch 120 may be suitable for circulating a fluid therein to separate motile spermatozoa from non-motile spermatozoa. Non-motile sperm may be evacuated with the circulating fluid while motile sperm resist the flow of fluid and remain in the spermatozoon feed branch 120. During this processing, the communication between the spermatozoon feed branch 120 and the fertilization area 130 may be closed, and circulating fluid may be directed to an evacuation conduit after flowing through part or all of the spermatozoon feed branch 120. As mentioned above, the spermatozoon feed branch 120 may include a complex pathway, e.g. have mazes and / or obstacles which are suitable to separate motile spermatozoa from non- motile spermatozoa and / or poorly motile spermatozoa. The motile sperms will swim through the obstacles and / or mazes, separating them from non- motile sperm and poor motile sperm.

[0056] Other sperm processing may occur in the spermatozoon feed branch 120, e.g. the visualization and estimation of spermatozoa motility using images from an imaging system such as that described below; and / or the delivery of different liquids and solutions at different flowrates, such as for sperm washing, refreshing of the culture medium, or for sperm activation.

[0057] The embryo processing branch 140 may have any desired feature to process one or more embryo after fertilization has occurred.The embryo may grow in the embryo processing branch 140. Alternatively or in addition, the embryo may grow directly in the fertilization area 130. The growing embryo may be kept in the microfluidic chip 100 for a desired time, e.g. a plurality of days, e.g. from 2 to 7 days. In the fertilization area 130 and / or the embryo processing branch 140 may occur others processing, e.g. the visualization and estimation of cells (zygote and embryo) maturity using images from an imaging system such as that described below; and / or the delivery of different liquids and solutions at different flowrates, such as for cells washing or refreshing of the culture medium.

[0058] In order to efficiently perform IVF, a plurality of fertilizations may occur in the fertilization area 130, resulting in a plurality of embryos. For appropriate growth of the embryos, the embryo processing branch 140 may comprise a plurality of chambers 142, each chamber being configured to receive an embryo or a plurality of embryos. The chambers 142 may be connected in parallel to each other by a main conduit. Controlling the flow rate within the main conduit of embryo processing branch 140, allows controlling the flow rate within the chambers 142. Flow rate of chambers 142 may be an order of magnitude lower that flow rate in the main conduit. This provides a distribution of medium flow in a low shear stress environment, which imparts a positive impact on in vitro embryo development.

[0059] The microfluidic chip 100 further comprises at least one remotely controllable microstructure 10, which is depicted diagrammatically in Fig. 1, e.g. in the fertilization area 130. The microstructure 10 is provided inside the microfluidic circuit 102 and is movable within the microfluidic circuit 102. In particular, the microstructure 10 is mobile between the fertilization area 130 and at least one of the oocyte feed branch 110, a spermatozoon feed branch 120 and the embryo processing branch 140.

[0060] The microstructure 10 assists IVF by helping the cells to move to desired locations. As the microstructure 10 can be remotely controlled, it is sometimes referred to as a microrobot although it may not have the features that macroscopic robots usually have (e.g. processorbased control, articulated parts, etc.). As its name suggests, the microstructure may have dimensions less than 1 millimeter (mm), preferably less than 0.5 mm, preferably less than 300 micrometer (pm). For instance, a microstructure 10 may range from 10 to 100 pm (e.g. for sperm), or from 50pm to 500pm (e.g. for oocytes and embryos).

[0061] When a plurality of microstructures 10 are provided, the microstructures 10 may differ from one another, e.g. based on their desired function. For instance, an end-effector of the microstructure 10 may vary. For instance, a microstructure 10 for capturing and manipulating an embryo may be greater than a microstructure 10 for capturing and manipulating sperm. Besides, a microstructure 10 for capturing and manipulating a motile cell (e.g. motile sperm) may have different features than a microstructure 10 for capturing and manipulating a non-motile cell (e.g. immobilized sperm).

[0062] A microstructure 10 according to an embodiment is illustrated in more detail, in cross-section, in Fig. 2. As shown in Fig. 2, the microstructure 10 may comprise a chamber 12 for housing at least one of a spermatozoon 122, an oocyte 112 and an embryo. The chamber 12 may be sized to receive one target cell at a time. The chamber 12 may be enclosed by a wall 16 having an entrance 14 and an optional opposite vent 20.

[0063] The microstructure 10 may further comprise at least one handle 22 for manipulation by a remote controller. The handles 22 are connected to the wall 16 and movement of the handles 22 provokes movement of the microstructure 10, and thus manipulation of the captured sperm 122.

[0064] The remote controller may be an optical controller, a magnetic controller, an acoustic controller or the like. As regards optical remote control, reference is made to the Applicant's international publications WO 2022 / 090760 Al and WO 2022 / 090761 Al. Respective optical tweezers may be used to control respective handles 22 of the microstructure 10. The handles 22, or more generally the microstructure 10, may have appropriate features to be controlled by the remote controller, e.g. a refractive index greater than the surrounding fluid refractive index.

[0065] Conversely, the type of remote controller and the material of the microfluidic chip 100 may be chosen so that they do not interfere with each other, thus enabling a reliable control of the microstructure 10. In an example, the microfluidic chip 100 may be made of polydimethylsiloxane (PDMS) or another polymer, e.g. photoresins.

[0066] An apparatus 200 for IVF is depicted in Fig. 3. The apparatus comprises an incubator 202 configured to receive at least one microfluidic chip, e.g. ten or more microfluidic chips. The microfluidic chips may be the same as the above-described microfluidic chip 100.

[0067] The incubator 202 may be configured to keep the microfluidic chips under a controlled environment. The controlled parameters may be temperature, humidity, gas concentrations, etc. In this respect, sensors 204 may be provided to facilitate monitoring and controlling the incubator parameters. The incubator 202 may comprise a hermetic housing for enclosing the microfluidic chips 100. At least part of the housing may be transparent to enable observation therein; alternatively, observation devices may be provided directly inside the incubator. In this embodiment, the apparatus 200 comprises an imaging system 210 (e.g. comprising objective microscope and a camera) configured to capture at least one image of the microfluidic chip 100. Thecamera may be a framerate camera, and may use CCD or CMOS image sensors, or may be a hyperspectral camera or an event camera.

[0068] The apparatus 200 further comprises at least one fluidic feed 250 and at least one fluidic discharge 252 configured to be connected to the at least one microfluidic chip 100, in particular to the above-described fluidic inlets 150 and fluidic outlets 152.

[0069] At least one pump 254 may be configured to control the flow rate in the fluidic feed 250 and / or the fluidic discharge 252, e.g. to supply fluid to the fluidic feed 250 or to suck fluid from the fluidic discharge 252. Such a pump is known per se in the art. The pump 254 may be a peristaltic pump, syringe pump, pressure-driven pump or other.

[0070] The apparatus 200 may further comprise a surgical system 220, comprising a first laser beam source 222 for carrying out laser surgical operations on the cells in the microfluidic chips 100. Such surgical operations are known per se in the art. The first laser beam source 222 may be a pulsed laser beam, conveniently, a femtosecond laser, i.e. a laser configured to emit ultra-short pulses lasting from a few femtoseconds to a few hundred femtoseconds (1 femtosecond (fs) = 1015seconds). The first laser beam may have a near infrared wavelength as biological objects and water have low absorption coefficient at this wavelength. The first laser beam source 222 may be directed to a focusing system 224. The focusing system 224 may be designed by the skilled person according to its needs based on their general knowledge. The first laser beam emitted by the first laser beam source may be used as a scalpel. The surgical system 220 may be used e.g. for polar globule biopsy, pre-implantation genetic test, etc.

[0071] The apparatus 200 further comprises a remote controller 230 for remotely controlling a microstructure 10 provided in the at least one microfluidic chip 100. The example of Fig. 3 depicts an optical remotecontroller 230 (e.g. based on optical tweezers), however other embodiments are contemplated, as discussed above.

[0072] The optical remote controller 230 may comprise a second laser beam source 232. The second laser beam may be a continuous laser beam, e.g. an Nd:YAG laser. The second laser beam may have a near infrared wavelength as biological objects and water have low absorption coefficient at this wavelength. The second laser beam source 232 may have its own focusing system. However, as illustrated, the first laser beam source 222 and the second laser beam source 232 may be directed to a common focusing system, namely the focusing system 224. The focusing system 224 may be designed according to the Applicant's above- mentioned international publications WO 2022 / 090760 Al and WO 2022 / 090761 Al.

[0073] The apparatus 200 may comprise further components as appropriate, e.g. a control processor, interface, heater, etc. to carry out tasks which are known per se in the art.

[0074] With reference back to Fig. 1, a method for in vitro fertilization, comprises providing the microfluidic chip 100 as described above, and remotely controlling the at least one microstructure 10 to move a spermatozoon 122 from the spermatozoon feed branch 120 to the fertilization area 130, to move an oocyte 112 from the oocyte feed branch 110 to the fertilization area 130, and / or to move an embryo from the fertilization area 130 to the embryo processing branch 140, as detailed above, for instance using optical remote manipulation.

[0075] The method may further comprise placing the microfluidic chip 100 within the incubator 202 of the above-described IVF apparatus 200. The remote controlling may be carried while the microfluidic chip 100 is in the incubator 202. In this way, the environment of the cells is controlled as much as possible during the IVF cycle.

[0076] Figure 4 illustrates more specifically a possible embodiment for the fertilization step per se. The fertilization step may comprise contactless intracytoplasmic sperm injection (contactless ICSI). While represented in a certain order, the steps of Figs. 4A-4D may be performed in any desired technically feasible order.

[0077] Figure 4A shows making an incision in an oocyte 112, in view of facilitating injection of sperm therein. The oocyte 112 may be incised by any desired surgical technique, e.g. contactless techniques such as by a laser beam. The surgical system 220 may be used in this respect. As Fig. 4A shows, a laser beam may be focused by the focusing system 224 to make the incision.

[0078] Figure 4B illustrates remotely controlling a microstructure 10 to spermatozoon 122 closer to the incision and releasing the spermatozoon 122. The microstructure 10 may be controlled as detailed above, e.g. through the handles 22. To facilitate injection, the sperm 122 may be released at a distance of the incision less than a diameter of the oocyte 112.

[0079] Figure 4C illustrates immobilizing the sperm 122, which may comprise ablating a flagella thereof. For doing so, the surgical system 220 may be used. The above comments relating to the incision of the oocyte 112 apply mutatis mutandis to ablating the flagella. As mentioned above, the sperm 122 may be immobilized at any desired step of the IFV cycle.

[0080] Finally, Fig. 4D illustrates remotely controlling a microstructure 10' to push the released and immobilized spermatozoon through the incision of the oocyte 112. Note that the microstructure 10' may differ from the above-described microstructure 10 e.g. in its shape, in order to be more adapted to pushing the immobilized sperm 122 (e.g. with a more opened chamber) as opposed to capturing a motile sperm. However, alternatively, the same microstructure 10 may be used for capturing, manipulating and pushing the sperm 122.

[0081] In this way, contactless ICSI can be perform without any mechanical contact between an operator and the gametes, thus leaving the cells in the controlled environment of the microfluidic chip 100.

[0082] Although the present disclosure refers to specific exemplary embodiments, modifications may be provided to these examples without departing from the general scope of the invention as defined by the claims. In particular, individual characteristics of the different illustrated / mentioned embodiments may be combined in additional embodiments. Therefore, the description and the drawings should be considered in an illustrative rather than in a restrictive sense.

Claims

CLAIMS1. A microfluidic chip (100) for in vitro fertilization, comprising a microfluidic circuit (102) having an oocyte feed branch (110), a spermatozoon feed branch (120), a fertilization area (130) and an embryo processing branch (140), the microfluidic chip (100) further comprising at least one remotely controllable microstructure (10) that is mobile between the fertilization area (130) and at least one of the oocyte feed branch (110), a spermatozoon feed branch (120) and the embryo processing branch (140).

2. The microfluidic chip of claim 1, wherein the fertilization area (130) is hermetically enclosed from an outside of the microfluidic chip.

3. The microfluidic chip of claim 1 or 2, wherein the embryo processing branch (140) comprises a plurality of chambers (142), each chamber being configured to receive one or more embryos.

4. The microfluidic chip of any one of claims 1 to 3, wherein the fertilization area (130) is downstream of the oocyte feed branch (110) and of the spermatozoon feed branch (120), and upstream of the embryo processing branch (140).

5. The microfluidic chip of any one of claims 1 to 4, wherein the oocyte feed branch (110) comprises an oocyte decoronization conduit (114).

6. The microfluidic chip of any one of claims 1 to 5, wherein the spermatozoon feed branch (120) comprises a complex pathway, optionally including one or more obstacle or one or more maze.

7. The microfluidic chip of any one of claims 1 to 6, wherein the microstructure (10) comprises a chamber (12) for housing at least one of a spermatozoon (122), an oocyte (112) and an embryo, and further comprises at least one handle (22) for manipulation by a remote controller (230), optionally an optical controller, a magnetic controller or an acoustic controller.

8. An apparatus (200) for in vitro fertilization, comprising an incubator (202) configured to receive at least one microfluidic chip (100), at least one fluidic feed (250) and at least one fluidic discharge (252) configured to be connected to the at least one microfluidic chip (100), and a remote controller (230) for remotely controlling a microstructure (10) provided in the at least one microfluidic chip (100).

9. The apparatus of claim 8, further comprising at least one of: an imaging system (210) configured to capture at least one image of the microfluidic chip (100); a pump (254) configured to supply fluid to the fluidic feed (250) or to suck fluid from the fluidic discharge (252); a first laser beam source (222).

10. The apparatus of claim 9, comprising a second laser beam source (232), optionally wherein the first laser beam source (222) and the second laser beam source (232) are directed to a common focusing system (224).

11. A method for in vitro fertilization, comprising providing the microfluidic chip (100) of any one of claims 1 to 7, and remotely controlling the at least one microstructure (10) to move a spermatozoon (122) from the spermatozoon feed branch (120) to the fertilization area (130), to move an oocyte (112) from the oocyte feed branch (110) to thefertilization area (130), and / or to move an embryo from the fertilization area (130) to the embryo processing branch (140).

12. The method of claim 11, further comprising placing the microfluidic chip (100) within the incubator (202) of the apparatus (200) of any one of claims 8 to 10, wherein the controlling is carried while the microfluidic chip (100) is in the incubator (202).

13. The method of claim 11 or 12, comprising circulating a fluid in the spermatozoon feed branch (120) to separate motile spermatozoa from non-motile or less motile spermatozoa.

14. The method of any one of claims 11 to 13, comprising growing an embryo in at least one of the fertilization area (130) and the embryo processing branch (140), optionally for a plurality of days.

15. The method of any one of claims 11 to 14, further comprising:- making an incision in an oocyte (112), optionally with a laser beam;- remotely controlling the at least one microstructure (10) to move a spermatozoon (122) closer to the incision and releasing the spermatozoon (122);- immobilizing the spermatozoon (122).

16. The method of claim 15, wherein the spermatozoon (122) is released at a distance of the incision less than twice a diameter of the oocyte (112), optionally less than the diameter of the oocyte (112).

17. The method of claim 15 or 16, further comprising remotely controlling the at least one microstructure (10') to push the released and immobilized spermatozoon (122) through the incision.

18. The method of any one of claims 15 to 17, wherein the immobilizing the spermatozoon (122) comprises ablating a flagella thereof.

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