Use of perfluorooctane for piezoelectric-mediated intracytoplasmic sperm injection

Abstract

The present invention relates to an operating fluid for piezoelectric-mediated microinjection. In particular, the present invention relates to the use of purified perfluorooctane or a composition comprising purified perfluorooctane as an operating fluid for piezoelectric-mediated intracytoplasmic sperm injection (Piezo-ICSI). The present invention also relates to a method for fertilizing an oocyte in vitro by Piezo-ICSI using purified perfluorooctane or a composition comprising purified perfluorooctane as an operating fluid and an assisted reproduction method using the method of the present invention.

Description

Field of the Invention

[0001] The present invention relates to an operating liquid for piezoelectrically mediated microinjection. In particular, the present invention relates to the use of purified perfluorooctane or a composition comprising purified perfluorooctane as an operating liquid for piezoelectrically mediated intracytoplasmic sperm injection (Piezo-ICSI). The present invention also relates to a method for fertilizing an oocyte in vitro by Piezo-ICSI using purified perfluorooctane or a composition comprising purified perfluorooctane as an operating liquid and an assisted reproduction method using the method of the present invention. Background Art

[0002] Male factor infertility is responsible for the inability of approximately 50% of couples to conceive. A number of procedures were developed in the 1980s to address fertilization failure due to male gamete dysfunction, including the injection of a single sperm cell (spermatozoa) into the oocyte, but with very limited success. However, it was during the oocyte subzonal injection procedure that the oocyte membrane was accidentally disrupted and the sperm was delivered into the ooplasm, which subsequently initiated the development of intracytoplasmic sperm injection, which is still performed in humans today.

[0003] Intracytoplasmic sperm injection (ICSI) is an in vitro fertilization procedure in which sperm cells are injected directly into the cytoplasm of the oocyte. The first human pregnancy resulting from an ICSI-derived embryo was reported in 1992. ICSI is performed by a human operator in a dish under a microscope using a micromanipulator and micropipettes to manipulate the oocyte and sperm. A fine glass micropipette (holding pipette) stabilizes the mature oocyte by gentle suction applied by the microinjector, while a single sperm is collected from the other side using a thin, spiked glass micropipette (injection micropipette), which is immobilized by cutting its tail with the tip of the micropipette. The mechanical force applied by the operator through the micromanipulator is used to pierce the zona pellucida and plasma membrane (oolemma) of the oocyte with the injection micropipette. The sperm is then injected into the cytoplasm of the oocyte.

[0004] To date, ICSI has been responsible for more than 2 million babies worldwide. But ICSI is a labor-intensive technique that requires highly skilled operators, and there is still room for improvement in terms of technical standardization and success rates. An alternative oocyte microinjection method called "piezo-mediated" ICSI (Piezo-ICSI) was developed for mouse oocytes in 1995 because conventional ICSI could not resolve problems associated with fertilization in mouse IVF.

[0005] Piezo-ICSI is similar to conventional ICSI (c-ICSI) in that a micropipette containing a single sperm pierces the oocyte to deliver the sperm to the oocyte's cytoplasm. However, whereas in c-ICSI the zona pellucida and tunica oolemma are pierced by mechanical forces applied to the micropipette by the operator via a micromanipulator, in piezo-ICSI the piezoelectric effect is used to pierce the oocyte. The piezoelectric effect is the phenomenon in which certain materials accumulate electrical charge in response to mechanical pressure, which in turn can be induced by the application of electrical force. The piezoelectric effect has many technological applications. In the case of piezo-ICSI, a brief "piezoelectric pulse" is applied to the micropipette containing the sperm to generate an ultrafast, submicron forward momentum of the micropipette. This precise and rapid motion is used to pierce the zona pellucida and tunica oolemma in a manner that places less pressure on the oocyte than the mechanical forces used in c-ICSI.

[0006] The success rate of piezo-ICSI was initially lower than that of c-ICSI. However, it was found that by adding a small amount of mercury to the injection micropipette, the success rate could be increased to above that of c-ICSI.

[0007] Therefore, piezo-ICSI requires the presence of an “operating fluid” in the injection micropipette. Currently, there is no complete scientific explanation for the physical characteristics of the puncture process in piezo-ICSI. Some have proposed that the axial movement of the micropipette, caused by the piezoelectricity, punctures the zona pellucida. Others have hypothesized that the puncture is produced by a pressure burst caused by the sudden forward movement of the operating fluid. Still others have hypothesized that when the piezoelectric pulse is applied, the micropipette moves forward, but the operating fluid does not move due to the law of inertia and the high specific gravity of the liquid. Some have suggested that the fast-moving micropipette and the stationary operating fluid generate a negative pressure at the micropipette tip, which pulls the zona pellucida apart. Recent studies have pointed out that with the introduction of a sequence of piezoelectric pulses, considerable lateral tip oscillations of the injection pipette occur. They claim that the lateral dynamics play an important role in the puncture and that this process is also mediated by the operating fluid. Most likely, all of the listed characteristics participate, to some extent, in the effective operation of piezo-mediated ICSI.

[0008] Although mercury is toxic and its use is extremely limited, piezo-ICSI is now used for in vitro fertilization of animals. More recently, fluorinated compounds have been used as operating fluids.

[0009] Piezo-ICSI was first successfully used in mice in 1995 and was later applied to microinjection in pigs and cattle. Piezo-ICSI achieves higher fertilization and survival rates than conventional ICSI (c-ICSI) and has therefore become the standard method in the field for microinjection of sperm into animal oocytes.

[0010] The first successful human pregnancy produced using piezoelectric ICSI technology was reported in 1996, and improved fertilization and survival rates were reported in 1999. However, piezoelectric-ICSI has not been approved for clinical use in humans.

[0011] Further development of piezo-ICSI for approval for clinical use has the potential to improve the success rate of assisted reproduction in humans. Summary of the invention

[0012] The present invention relates to an operating fluid for piezoelectrically mediated microinjection.

[0013] The present invention provides use of purified perfluoro-n-octane or a composition comprising purified perfluoro-n-octane as an operating fluid for piezoelectric-mediated intracytoplasmic sperm injection (piezo-ICSI).

[0014] The present invention also provides a method for fertilizing an oocyte in vitro, the method comprising injecting a sperm into the oocyte by piezoelectric-ICSI, wherein purified perfluoro-n-octane or a composition comprising purified perfluoro-n-octane is used as an operating fluid for piezoelectric-ICSI.

[0015] The present invention also provides an assisted reproductive method, which comprises

[0016] (a) fertilizing an oocyte in vitro to form an embryo using a method according to the invention;

[0017] (b) culturing the embryos; and

[0018] (c) implanting the embryo into a subject.

[0019] Suitably, the use of purified perfluorooctane as an operating fluid according to the present invention can provide advantages for assisted reproductive methods. These advantages may include a higher proportion of successful fertilization, improved embryo development rates (e.g., a higher proportion of embryos reaching the blastocyst stage), and a higher proportion of successful implantation. The use of perfluorooctane as an operating fluid for piezo-ICSI may be approved for human clinical use, thereby providing the possibility of standardization of ICSI procedures. The use of piezo-ICSI may be the first step in achieving full automation of assisted reproduction. Piezo-ICSI is also a technology that is easier to learn than c-ICSI, which requires highly skilled operators. Therefore, the use of perfluorooctane as an operating fluid for piezo-ICSI has the potential to simplify and develop more effective human assisted reproductive methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 - Position of the operating fluid in the injection micropipette

[0021] Figure 1Depicted is an injection micropipette (1) that has been loaded with a working fluid (2). The working fluid forms a column about 10-15 mm long in the middle of the micropipette.

[0022] Figure 2 -Connection between micropipette and syringe

[0023] Figure 2 A depicts the insertion of an injection micropipette (1) into a micropipette holder (3).

[0024] Figure 2 B depicts the insertion of the micropipette holder (3) into the syringe (4).

[0025] Figure 3 -Piezoelectric Micromanipulator (PMM) / Piezoelectric Actuator

[0026] Figure 3 A depicts a piezoelectric actuator (5) connected to a micropipette holder (3).

[0027] Figure 3 B is a photograph of the piezoelectric actuator (5).

[0028] Figure 4 - Injection micropipettes and culture medium

[0029] Figure 4 A depicts culture medium, such as PVP, being taken up by an injection micropipette.

[0030] Figure 4 B is a photograph of an injection micropipette used to absorb PVP medium.

[0031] Figure 5 -Schematic diagram of Piezo-ICSI

[0032] Figure 5 Depicted are the steps for injecting sperm into an oocyte using piezo-ICSI. The tip of the injection micropipette is gently placed against the zona pellucida, without deformation of the oocyte (a). A piezo pulse is applied to the micropipette (b), advancing the micropipette through the zona pellucida puncture hole (c). The injection micropipette is removed from the zona pellucida, and the hollowed-out zona pellucida portion is expelled by discharging the injection micropipette. This discharging serves to mobilize sperm to the tip of the injection micropipette (d). The injection micropipette is advanced through the zona pellucida (e) to approximately 80-90% of the diameter of the oocyte, pushing and stretching the oolemma (f). A piezo pulse is applied to disrupt the oolemma, allowing the cytoplasm to surround the injection micropipette (g). Sperm is injected (h). DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention provides use of purified perfluoro-n-octane or a composition comprising purified perfluoro-n-octane as an operating fluid for piezoelectric-mediated intracytoplasmic sperm injection (piezo-ICSI).

[0035] The present invention also provides a method for in vitro fertilization of an oocyte, the method comprising injecting a sperm into the oocyte by piezoelectric-ICSI, wherein purified perfluoro-n-octane or a composition comprising purified perfluoro-n-octane is used as an operating fluid for piezoelectric-ICSI.

[0036] Perfluorooctane

[0037] Perfluorooctane consists of an unbranched chain of eight carbon atoms, each of which is bonded to the maximum number of fluorine atoms (i.e., three fluorine atoms are bonded to the terminal carbon and two fluorine atoms are bonded to the other carbons). The lUPAC name of perfluorooctane is 1,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-octafluorooctane. The CAS registry number for perfluorooctane is 307-34-6. The chemical formula for perfluorooctane is C8F 18 The structural formula of perfluorooctane is as follows:

[0038]

[0039] At room temperature, perfluorooctane is an odorless, colorless liquid. The physical properties of perfluorooctane are shown in Table 1 below (temperature-dependent properties are quoted at 25°C).

[0040] Table 1 - Physical properties of perfluorooctane

[0041] Boiling Point 103℃ Pour Point -27℃ Molecular weight 438 density 1.755g / L Dynamic viscosity <![CDATA[1.03mm 2 / s(1.8mPa.s)]]> Vapor Pressure 3.4mbar Surface tension 16mN / m

[0042] Perfluorooctane is chemically inert. In other words, it is stable and does not react easily with other compounds. This stability is due to the high strength of the carbon-fluorine bonds that dominate its structure.

[0043] Perfluorooctane may be referred to as a perfluorocarbon, a compound consisting only of carbon and fluorine atoms. Perfluorooctane may be referred to as a perfluorocarbon liquid (PFCL). Perfluorooctane may be referred to as a perfluoroalkane, a compound consisting only of carbon and fluorine atoms connected by single bonds.

[0044] Methods for synthesizing perfluorooctane are known in the art.

[0045] For example, perfluoro-n-octane can be synthesized by electrochemical fluorination, in which electrolysis is used to replace hydrogen atoms on organic compounds with fluorine atoms from donor compounds. Two exemplary forms of electrochemical fluorination that can be used are the Simons process and the Phillips petroleum process. In the Simons process (Simons and Harland 1949 Journal of the Electrochemical Society 95: 47-66), organic compounds are electrolyzed in a hydrogen fluoride solution. The Phillips petroleum process is similar to the Simons process, but uses potassium fluoride in hydrogen fluoride as a fluorine source (Banks et al. (eds.). Organofluorine Chemistry. Boston, MA: Springer. Alsmeyer et al. 1994 Organofluorine Chemistry: Principles and Commercial Applications in pages 121–143).

[0046] Perfluorooctane can also be synthesized by the Fowler method (Fowler et al. 1947 Ind. Eng. Chem. 39: 292-298), in which hydrocarbons are fluorinated using cobalt (III) fluoride in the gas phase. Specifically, in the first step, cobalt (II) fluoride is fluorinated to cobalt (III) fluoride by exposure to fluorine gas at high temperature. In the second step, cobalt (III) fluoride is exposed to the hydrocarbon to be fluorinated (e.g. octane, in the case of perfluorooctane) at high temperature. The hydrogen atoms of the hydrocarbon are replaced by the fluorine atoms in cobalt (III) fluoride, which is converted back to cobalt (II) fluoride.

[0047] Perfluorooctane is readily available from commercial sources.

[0048] Purified perfluorooctane

[0049] The term "purified perfluoro-n-octane" refers to perfluoro-n-octane containing low levels of under-fluorinated impurities.

[0050] Underfluorinated impurities are byproducts of the synthesis of perfluorocarbon compounds. Underfluorinated impurities contain fewer fluorine atoms in their molecular structure than the intended perfluorocarbon product of the synthesis.

[0051] Specifically, perfluorooctane is fully fluorinated, with each carbon atom in its molecular structure bonded to the maximum number of fluorine atoms. However, during the synthesis of perfluorooctane, other molecules may be produced that are not fully fluorinated, i.e., they still contain hydrogen and / or carbon multiple bonds and therefore contain fewer fluorine atoms than perfluorooctane. These molecules are underfluorinated impurities.

[0052] The underfluorinated impurities of perfluoro-n-octane may contain carbon-carbon double bonds. The underfluorinated impurities of perfluoro-n-octane may additionally or alternatively contain carbon-carbon triple bonds. The underfluorinated impurities of perfluoro-n-octane may additionally or alternatively contain one or more hydrogen atoms in place of the fluorine atoms present in the perfluoro-n-octane.

[0053] The under-fluorinated impurity may be referred to as an under-fluorinated compound, an under-fluorinated by-product, an incompletely fluorinated impurity, an incompletely fluorinated compound, or an incompletely fluorinated by-product.

[0054] Compared to the chemically inert perfluorooctane, the underfluorinated impurities are reactive. In other words, the underfluorinated impurities can react with compounds that do not react with perfluorooctane.

[0055] Underfluorinated impurities may be removed from the synthesized perfluoro-n-octane by purification. In some embodiments of the present invention, the purified perfluoro-n-octane comprises perfluoro-n-octane that has been subjected to purification.

[0056] Purification methods for perfluorocarbon compounds applicable to perfluorooctane are known in the art (see, e.g., U.S. Pat. Nos. 3,696,156, 3,887,629, and 5,563,306, all incorporated by reference). Such methods generally utilize the reactivity of underfluorinated impurities to distinguish them from the inert perfluorooctane. In an exemplary purification method for perfluorooctane, the underfluorinated impurities are purified in the presence of Ca 2+ Or Ba 2+ It reacts with amines or strong bases such as potassium hydroxide in the presence of ions.

[0057] The amount of under-fluorinated impurities in the preparation of perfluoro-n-octane can be expressed as the number of CH bonds equivalent to those impurities. This value is called the H value and is expressed in parts per million (ppm). For example, the amount of under-fluorinated impurities in a preparation of perfluoro-n-octane can be measured using fluoride selective ion analysis.

[0058] The use of fluoride selective ion analysis to quantify under-fluorinated impurities is known in the art. The principle of the method is to react perfluorooctane with a strong base that cleaves fluorine atoms from the molecule during the preparation process, producing fluoride ions. The fluoride ions are then quantified, from which the amount of under-fluorinated impurities can be calculated. An illustrative method for fluoride selective ion analysis is listed below.

[0059] Step 1: Chemical transformation of under-fluorinated impurities.

[0060] 10 ml of perfluorocarbon liquid (PFCL) was mixed with 3.4 g of 1,6-diaminohexane and 15 mL of nonane. The mixture was heated with stirring for 8 hours to a temperature of 120° C. in a 100 mL glass flask equipped with a reflux condenser. After cooling to room temperature, the solution was vigorously mixed with 30 mL of hydrochloric acid (1.3 moles) and the aqueous phase was separated.

[0061] Subsequently, 15 mL of the aqueous phase was neutralized with 1.3 molar aqueous hydrochloric acid using phenolphthalein as an indicator and diluted to 25 mL with deionized water. 10 mL of this neutralized, diluted solution was transferred to a 25 mL glass beaker and 1 mL of TISAB-III was added under stirring.

[0062] Step 2: Fluoride Selective Potentiometry

[0063] Ion selective potentiometry is used to quantify fluoride ions in sample solutions. Prior to sample measurement, a sodium fluoride solution with a fluoride ion concentration between 0.005 mmol / L and 0.05 mmol / L must be calibrated as a reference standard. In addition, a blank value is recorded. The sample solution is then analyzed. The amount of reactive underfluorinated impurities is expressed as the number of CH bonds equivalent to those in these impurities, i.e., the H value. The H value (c F-C-H ) is calculated using the following formula:

[0064]

[0065] in:

[0066] c F-C-H is the concentration of incompletely fluorinated contaminants in the sample (i.e., H value);

[0067] c F - is the measured concentration of fluoride ions in the sample;

[0068] is the molecular weight of the measured PFCL;

[0069] ρ PFCL is the density of the measured PFCL;

[0070] bv is the recorded blank value (reagent and flask blank);

[0071] 5 is a factor to compensate for the dilution step; and

[0072] 1 / 3 is the stoichiometric factor (counting the number of CH bonds).

[0073] Suitably, the purified perfluoro-n-octane may have an H value of about 1000 parts per million (ppm) or less. The purified perfluoro-n-octane may have an H value of about 500 ppm or less. The purified perfluoro-n-octane may have an H value of about 100 ppm or less. The purified perfluoro-n-octane may have an H value of about 50 ppm or less. The purified perfluoro-n-octane may have an H value of about 40 ppm or less. The purified perfluoro-n-octane may have an H value of about 30 ppm or less. The purified perfluoro-n-octane may have an H value of about 20 ppm or less. The purified perfluoro-n-octane may have an H value of about 10 ppm or less. The purified perfluoro-n-octane may have an H value of about 5 ppm or less. The purified perfluoro-n-octane may have an H value of about 1 ppm or less. In a preferred embodiment, the purified perfluoro-n-octane has an H value of about 10 ppm or less.

[0074] Suitably, the operating fluid may have an H value of about 1000 parts per million (ppm) or less. The operating fluid may have an H value of about 500 ppm or less. The operating fluid may have an H value of about 100 ppm or less. The operating fluid may have an H value of about 50 ppm or less. The operating fluid may have an H value of about 40 ppm or less. The operating fluid may have an H value of about 30 ppm or less. The operating fluid may have an H value of about 20 ppm or less. The operating fluid may have an H value of about 10 ppm or less. The operating fluid may have an H value of about 5 ppm or less. The operating fluid may have an H value of about 1 ppm or less. In a preferred embodiment, the operating fluid has an H value of about 10 ppm or less.

[0075] Purified perfluorooctane is available from commercial sources. In particular, purified perfluorooctane is sold for use in vitreoretinal surgery. Examples of commercially available purified perfluorooctane include BIO OCTANE sold by Liquid and Biotech TM OPTISOL sold by PFS and Moss Vision Inc.

[0076] Piezo-ICSI

[0077] Piezoelectric-mediated intracytoplasmic sperm injection (piezo-ICSI) is a technique known in the art for injecting sperm into the cytoplasm of an oocyte in vitro. Piezoelectric-ICSI can be used for in vitro fertilization procedures in assisted reproduction. Piezoelectric-mediated ICSI may also be referred to as "piezo-driven" ICSI or "piezo-actuated" ICSI in the art. Kimura and Yanagimachi 1995 Biol. Reprod. 52 (4): 709-720 and Nagy et al. (ed.) In Vitro Fertilization, Springer, Cham. Hiraoka et al. 2019 Piezo-ICSI Chapter 39, pp. 481-489 provide examples of using piezoelectric-ICSI. Each of these documents is incorporated herein by reference.

[0078] Piezo-ICSI is performed on oocytes and sperm in a culture dish under a microscope. Human operators use micromanipulators and micropipettes to handle oocytes and sperm. A single oocyte is held in place by a holding micropipette through gentle suction, while a single sperm is captured in an opposing micropipette (called an "injection micropipette" to distinguish it from the micropipette that holds the oocyte in place). The injection micropipette is used to pierce the zona pellucida and then the oolemma. The piezoelectric effect is used to pierce the zona pellucida and oolemma. The piezoelectric effect is the phenomenon by which certain materials accumulate electrical charge in response to mechanical pressure, which in turn can be induced by the application of electrical force. In piezo-ICSI, a brief "piezoelectric pulse" is applied to the injection micropipette containing the sperm to generate an ultrafast, submicron forward momentum of the injection micropipette. This precise and rapid motion is used to pierce the zona pellucida and oolemma. The sperm is then injected into the cytoplasm of the oocyte and the injection micropipette is removed from the oocyte.

[0079] The injection micropipette contains the operating fluid, which is necessary to safely penetrate the oocyte using the piezoelectric effect.

[0080] Thus, in some embodiments of the invention, piezo-ICSI comprises the following steps:

[0081] (a) providing an injection micropipette containing sperm and operating fluid;

[0082] (b) Piercing the oocyte with an injection micropipette using a piezoelectric pulse;

[0083] (c) Injection of sperm into the oocyte.

[0084] In some embodiments of the invention, step (b) comprises:

[0085] (i) puncturing the zona pellucida of an oocyte using a piezoelectric pulse;

[0086] (ii) moving the injection micropipette through the zona pellucida to push against the oolemma of the oocyte;

[0087] (iii) A piezoelectric pulse is used to pierce the oocyte membrane, thereby moving the injection micropipette into the cytoplasm of the oocyte.

[0088] Thus, in some embodiments of the invention, piezoelectric-ICSI comprises the following steps:

[0089] (a) providing an injection micropipette containing sperm and operating fluid;

[0090] (b) Impale the oocyte with an injection micropipette using a piezoelectric pulse by following these steps:

[0091] (i) puncturing the zona pellucida of an oocyte using a piezoelectric pulse;

[0092] (ii) moving the injection micropipette through the zona pellucida to push against the oolemma of the oocyte;

[0093] (iii) using a piezoelectric pulse to pierce the oocyte membrane, thereby moving the injection micropipette into the cytoplasm of the oocyte; and

[0094] (c) Injection of sperm into the oocyte.

[0095] The "piezoelectric pulse" is an electric current applied to the injection micropipette to induce ultrafast submicron forward momentum in the micropipette. This momentum is caused by deformation of the crystal in response to an externally applied voltage. In addition to propelling the injection micropipette forward (axial momentum), lateral momentum is also generated. Both axial and lateral dynamics are thought to play an important role in piercing the oocyte.

[0096] Equipment for performing piezo-ICSI may include an inverted microscope (e.g., IX73) for viewing oocytes, sperm, and micropipettes, a three-axis micromanipulator (e.g., XenoWorksPT) for handling oocytes, sperm, and micropipettes, a syringe (e.g., a pneumatic microinjector such as PNJ-T2) for controlling injections through the injection micropipette, and a piezoelectric micromanipulator (PMM), also known as a "piezo driver," (e.g., Piezo PMM4GD) for applying piezoelectric pulses to the injection micropipette. Consumables required for piezo-ICSI may include a micropipette for injecting sperm (e.g., ultra-thin PINU06-20FT), a micropipette for holding an oocyte, an operating fluid, and a device for loading the operating fluid into the micropipette.

[0097] The present invention provides a device for piezoelectric-ICSI, which comprises an injection micropipette containing purified perfluoro-n-octane as an operating fluid.

[0098] In some embodiments, a device for piezoelectric-ICSI according to the present invention comprises an inverted microscope, a micromanipulator, a syringe, an injection micropipette, and a piezoelectric micromanipulator.

[0099] The present invention also provides a consumable kit for piezoelectric-ICSI, which comprises purified perfluorooctane as an operating fluid and one or more components selected from the following: one or more injection micropipettes, one or more holding pipettes, and a device for loading the operating fluid into the one or more injection micropipettes.

[0100] Suitably, the consumable kit according to the present invention comprises two or more components selected from: one or more injection micropipettes, one or more holding pipettes, and means for loading operating fluid into the one or more injection micropipettes.

[0101] Suitably, the consumable kit according to the present invention comprises purified perfluorooctane as a working fluid, one or more injection micropipettes, one or more holding pipettes and means for loading the working fluid into the one or more injection micropipettes.

[0102] Piezo-ICSI can be performed as follows:

[0103] (a) using a loading device to fill an injection micropipette to a length of about 2-25 mm, e.g., about 10-15 mm, within the micropipette (see Figure 1 ).

[0104] (b) Insert the injection micropipette into the micropipette holder (see Figure 2 A) and connect it to the syringe (see Figure 2 B), connected to a piezoelectric micromanipulator (PMM) (see Figure 3 ).

[0105] (c) Place the injection micropipette under the microscope.

[0106] (d) Use a syringe to push the operating solution into the tip of the injection micropipette so that no air is trapped.

[0107] (e) The tip of the micropipette is placed into a drop of culture medium containing sperm. The culture medium may be a nutrient medium or a medium such as polyvinylpyrrolidone (PVP).

[0108] (f) Aspirate and then expel a small amount of medium from the droplet into the injection micropipette, making sure not to completely aspirate or expel the working solution. Repeat this operation until the interface between the medium and the working solution in the injection micropipette slides smoothly (see Figure 4 ).

[0109] (g) Immobilizing the sperm cell and drawing it into an injection micropipette. For example, the sperm can be immobilized by crushing the tail with the tip of the micropipette. The sperm can be positioned within the injection micropipette, approximately one oocyte diameter from the tip of the injection micropipette.

[0110] (h) Position the oocyte using a holding micropipette. The oocyte should be positioned so that it can be penetrated in a wide area of ​​the perivitelline space, avoiding the spindle.

[0111] (i) Gently place the tip of the injection micropipette against the zona pellucida (see Figure 5 a), without oocyte deformation, the PMM is then turned on to apply a piezoelectric pulse to the micropipette (see Figure 5 b). The injection micropipette is propelled through the zona pellucida to pierce a hole by ultrafast submicrometer motion of the injection micropipette (see Figure 5 c).

[0112] (j) Remove the injection micropipette from the zona pellucida and dislodge the hollowed-out zona pellucida portion remaining within the injection micropipette by expelling the injection micropipette. This expulsion of the injection micropipette will move the spermatozoa to the tip of the injection micropipette (see Figure 5 d).

[0113] (k) Set the PMM to the single pulse setting. Only a single piezoelectric pulse is needed to disrupt the egg membrane.

[0114] (l) Push the injection micropipette through the zona pellucida (see Figure 5 e) to penetrate approximately 80-90% of the oocyte diameter, pushing and stretching the oocyte membrane (see Figure 5 f).

[0115] (m) Activate the PMM to disrupt the egg membrane with a piezoelectric pulse. Allow the cytoplasm to surround the injection micropipette (see Figure 5 g).

[0116] (n) Inject sperm, taking care not to discharge too much fluid into the cytoplasm (cf. Figure 5 h).

[0117] (o) Removal of the injection micropipette from the oocyte.

[0118] In c-ICSI, an injection micropipette with a spike or beveled tip is used to pierce the oocyte with mechanical force. In piezo-ICSI, because no mechanical force is used, a flat-headed injection micropipette can be used to pierce the oocyte (see Nagy et al. (eds.) In Vitro Fertilization, Springer, Cham., Fliraoka et al. 2019 Piezo-ICSI Chapter 39, pp. 481-489). Therefore, in one embodiment, the injection micropipette is flat-headed.

[0119] Injection micropipettes of the same wall thickness have been used for piezoelectric-ICSI (Hiraoka and Kitamura 2015 J. Assist. Reprod. Genet. 32: 1827-1833). Any injection micropipettes of appropriate wall thickness can be used in the method of the present invention. Suitably, the injection micropipettes may have a wall thickness between 0.5 μm and 1 μm. The injection micropipettes may have a wall thickness of 0.925 μm. The injection micropipettes may have a wall thickness of 0.625 μm.

[0120] The injection micropipette that holds sperm for Piezo-ICSI may also be referred to as a capillary or microcapillary.

[0121] In some embodiments of the invention, piezo-ICSI comprises injecting mammalian sperm into a mammalian oocyte. Thus, the invention encompasses all applications of piezo-ICSI performed on mammals where the operating fluid is purified perfluorooctane. In some embodiments, the sperm and oocyte are human. In some embodiments, the sperm and oocyte are murine. In some embodiments, the sperm and oocyte are bovine. In some embodiments, the sperm and oocyte are porcine. In some embodiments, the sperm and oocyte are equine.

[0122] Preferably, the sperm and oocytes may be human.

[0123] Operating fluid

[0124] The term "operating fluid" refers to the liquid in the injection micropipette that assists in piercing the oocyte during piezo-ICSI. If the micropipette does not contain an operating fluid, but only contains culture medium or oil, piezo-mediated oocyte piercing is less efficient and results in a higher proportion of oocyte degeneration. Therefore, it is recommended to use an operating fluid in the injection micropipette to provide efficient and safe oocyte piercing.

[0125] There is currently no scientific consensus on the precise physical effects of the operating fluid that improve piezo-ICSI outcomes. The operating fluid has a relatively high specific gravity. It is believed that this high specific gravity means that the operating fluid remains in place due to inertia as the injection micropipette moves. The stationary operating fluid may stabilize and dampen unwanted lateral vibrations while reinforcing the axial (i.e., piercing) movement of the micropipette tip. The stationary operating fluid may also create a slight vacuum as the micropipette moves axially, thereby creating a slight suction force that helps open the zona pellucida or tunica ovale.

[0126] The first operating fluid used was mercury. However, the toxicity of mercury requires that it be handled with care in the laboratory, and it may not be used for piezo-ICSI in humans.

[0127] Less toxic alternatives to mercury for use as operating fluids are fluorocarbons and fluoroethers.

[0128] In particular, fluorocarbon liquids, such as perfluoro-n-alkylmorpholines (see Hiraoka et al. 2019 Piezo-ICSI Chapter 39, pp. 481-489 in Nagy et al. (eds.) In Vitro Fertilization, Springer, Cham.) and perfluorocarbon mixtures have been used as operating fluids in piezo-ICSI. Fluorocarbon liquids are clear, colorless, odorless, non-flammable liquids with a high specific gravity but a viscosity similar to that of water.

[0129] Commercially available hydrofluoroethers have also been used as operating fluids in piezo-ICSI.

[0130] Although fluorocarbon fluids and hydrofluoroethers are used for piezo-ICSI in animals, these compounds are not approved for use in piezo-ICSI in humans. In addition, these fluids are not as effective as mercury for piezo-ICSI.

[0131] The present invention relates to the use of purified perfluoro-n-octane or a composition comprising purified perfluoro-n-octane as an operating fluid in piezoelectric ICSI.

[0132] As used herein, the term "purified perfluoro-n-octane" means that the perfluoro-n-octane contains low levels of reactive under-fluorinated impurities. Thus, the process fluid may contain other compounds, as long as these other compounds are not substantially under-fluorinated impurities (i.e., under-fluorinated compounds). In other words, in addition to perfluoro-n-octane, the process fluid may also include other perfluorinated compounds.

[0133] Therefore, the process liquid used in the present invention can be "purified perfluoro-n-octane" while containing compounds other than perfluoro-n-octane, because "purified" refers to the proportion of under-fluorinated compounds in the process liquid. As described herein, the proportion of under-fluorinated impurities is represented by H value (ppm).

[0134] The proportion of purified perfluorooctane in the process fluid can be expressed as a percentage. Similarly, the proportion of any other compound in the process fluid can be expressed as a percentage. The percentage can refer to the weight percentage (wt.%) of the process fluid containing the purified perfluorooctane or other compound or the volume percentage (vol.%) of the process fluid containing the purified perfluorooctane or other compound.

[0135] Suitably, the present use of purified perfluoro-n-octane as an operating fluid for piezo-ICSI refers to an operating fluid comprising about 90% or more purified perfluoro-n-octane, such as about 95% or more purified perfluoro-n-octane, such as about 96% or more purified perfluoro-n-octane, such as about 97% or more purified perfluoro-n-octane, such as about 98% or more purified perfluoro-n-octane, such as about 99% or more purified perfluoro-n-octane, such as 100% purified perfluoro-n-octane. In some embodiments, the operating fluid comprises about 95% to about 100% purified perfluoro-n-octane.

[0136] Preferably, the process fluid comprises from about 95% to about 100% purified perfluorooctane.

[0137] In some embodiments, the process fluid comprises about 95% or more perfluoro-n-octane and has an H value of about 100 ppm or less. In some embodiments, the process fluid comprises about 95% or more perfluoro-n-octane and has an H value of about 10 ppm or less.

[0138] The present invention also encompasses the use of a composition comprising purified perfluoro-n-octane as an operating fluid for piezo-ICSI.

[0139] Suitably, the use of the composition comprising purified perfluorooctane as a process fluid means that the purified perfluorooctane should be the major component of the process fluid (ie the compound constituting the majority of the process fluid). The composition for use as a process fluid may contain other compounds in addition to purified perfluorooctane.

[0140] For example, in some embodiments, the composition comprises purified perfluorooctane as the predominant purified perfluorinated compound. In other words, no other purified perfluorinated compound is present in the composition in an amount equal to or greater than purified perfluorooctane.

[0141] Suitably, the operating fluid may be a composition comprising about 50% or more purified perfluorooctane, such as about 60% or more purified perfluorooctane, such as about 70% or more purified perfluorooctane, such as about 80% or more purified perfluorooctane, such as about 90% or more purified perfluorooctane.

[0142] In some embodiments, the process fluid comprises a compound other than perfluoro-n-octane. The other compound is not substantially an underfluorinated compound. The other compound is substantially a perfluorinated compound.

[0143] The term "other perfluorinated compounds" refers to perfluorinated compounds other than perfluorooctane.

[0144] In some embodiments, the operating fluid contains about 50% or less of other perfluorinated compounds, about 40% or less of other perfluorinated compounds, such as about 30% or less of other perfluorinated compounds, such as about 20% or less of other perfluorinated compounds, such as about 10% or less of other perfluorinated compounds, such as about 5% or less of other perfluorinated compounds, such as about 4% or less of other perfluorinated compounds, such as about 3% or less of other perfluorinated compounds, such as about 2% or less of other perfluorinated compounds, such as about 1% or less of other perfluorinated compounds, such as no other perfluorinated compounds.

[0145] Perfluoro-n-heptane and perfluoro-n-nonane are examples of other perfluorinated compounds that may be included in the process fluid. In some embodiments, the process fluid comprises perfluoro-n-heptane. In some embodiments, the process fluid comprises perfluoro-n-nonane. In some embodiments, the process fluid comprises perfluoro-n-heptane and perfluoro-n-nonane. In some embodiments, the process fluid comprises perfluoro-n-heptane, perfluoro-n-nonane, and other compounds.

[0146] Perfluoro-n-heptane and perfluoro-n-nonane each have the same structure as perfluoro-n-octane, except that they contain 7 and 9 carbon atoms, respectively, instead of 8. In other words, perfluoro-n-heptane consists of a chain of 7 carbon atoms, with each carbon bonded to the maximum number of fluorine atoms, and perfluoro-n-heptane consists of a chain of 9 carbon atoms, with each carbon bonded to the maximum number of fluorine atoms.

[0147] The structural formula of perfluoro-n-heptane is as follows:

[0148]

[0149] The structural formula of perfluoro-n-nonane is as follows:

[0150]

[0151] In some embodiments, the process fluid comprises about 5% or less perfluoro-n-heptane, such as about 4% or less perfluoro-n-heptane, such as about 3% or less perfluoro-n-heptane, such as about 2% or less perfluoro-n-heptane, such as about 1% or less perfluoro-n-heptane, such as no perfluoro-n-heptane.

[0152] In some embodiments, the operating fluid comprises about 10% or less perfluoron-nonane, such as about 6% or less perfluoron-nonane, such as about 5% or less perfluoron-nonane, such as about 4% or less perfluoron-nonane, such as about 3% or less perfluoron-nonane, such as about 2% or less perfluoron-nonane, such as about 1% or less perfluoron-nonane, such as no perfluoron-nonane.

[0153] In some embodiments, the process fluid comprises about 5% or less perfluoro-n-heptane and about 5% or less perfluoro-n-nonane. In some embodiments, the process fluid comprises about 2% or less perfluoro-n-heptane and about 5% or less perfluoro-n-nonane.

[0154] In some embodiments, the process fluid comprises about 95% to about 100% perfluoro-n-octane, about 2% or less perfluoro-n-heptane, and about 5% or less perfluoro-n-nonane.

[0155] In some embodiments, the process fluid comprises about 95% to about 100% perfluoro-n-octane, about 2% or less perfluoro-n-heptane, and about 5% or less perfluoro-n-nonane, and has an H value of about 10 ppm or less.

[0156] Assisted Reproduction

[0157] The present invention provides a method for assisted reproduction, comprising

[0158] (a) fertilizing an oocyte in vitro to form an embryo using a method according to the invention;

[0159] (b) culturing the embryos; and

[0160] (c) implanting the embryo into a subject.

[0161] In some embodiments of the assisted reproductive methods of the present invention, the subject is a human.

[0162] In some embodiments, methods of assisted reproduction according to the present invention include retrieving an oocyte from a subject.

[0163] Technologies for assisted reproduction are known in the art. In particular, products and techniques for culturing embryos in vitro and then implanting them into a subject are known in the art. For example, Vitrolife produces needles for retrieval of oocytes (e.g., Sense retrieval needles), gradients for optimal sperm preparation (e.g., SpermGrad TM ), culture medium for culturing fertilized oocytes and culture medium for transferring viable and genetically competent embryos to subjects (e.g., EmbryoGlue).

[0164] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure.Numerical ranges are inclusive of the numbers defining the range.

[0165] Where a numerical range is provided, each smaller range between any stated value or intervening value in the stated range and any other stated value or intervening value in the stated range is included in the disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range in which either or both of the upper and lower limits are included or excluded in the smaller range is also included in the disclosure, subject to any explicitly excluded limits in the stated range. Where the stated range includes one or both of the upper and lower limits, ranges excluding one or both of those included upper and lower limits are also included in the disclosure.

[0166] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0167] As used herein, the terms "comprising," "including," and "consisting of" are synonymous with "including," "comprising," or "containing," and are closed or open-ended and do not exclude additional, unrecited members, elements, or method steps. The terms "comprising," "including," and "consisting of" also include the term "composed of."

[0168] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that such publications constitute prior art to the appended claims.

[0169] The present invention will now be further described by way of examples, which are intended to help those skilled in the art to practice the present invention and are not intended to limit the scope of the present invention in any way. Example

[0170] Example 1 - Suitability of fluorinated compounds as operating fluids

[0171] The suitability of three fluorinated test compounds for use as piezoelectric-ICSI operating fluids was tested. The three test compounds were perfluorodecalin, perfluoro-n-octane, and perfluoroperhydrophenanthrene. The test compounds were compared with commercially available hydrofluoroethers that were previously used as operating fluids as controls. The results of this comparison are listed in Table 2 below.

[0172] Table 2 - Suitability of fluorinated compounds for use as piezo-ICSI operating fluids

[0173]

[0174]

[0175] In conclusion, perfluorooctane was found to be as suitable as standard hydrofluoroether operating fluids for use as an operating fluid for piezo-ICSI. Other fluorinated compounds were either less suitable or not suitable at all.

[0176] Example 2 - Effects of fluorinated compounds on embryonic development

[0177] The effects of incubation in different fluorinated compounds on embryonic development were tested.

[0178] 223 2PN (two pronuclei) mouse embryos were randomly divided into four groups: A) stock perfluorooctane; B) a commercially available perfluorocarbon mixture previously used as a working fluid for piezo-ICSI; C) purified perfluorooctane; and D) untreated control.

[0179] Embryos of groups A, B and C were incubated in the respective compounds for 3 minutes and then transferred to culture medium. Embryos of all groups were then cultured in an incubator (37.5°C, 100% relative humidity, 5% CO2 in air) for 5 days.

[0180] The number of embryos showing signs of degeneration was counted in each group.

[0181] The number of embryos reaching the 2-cell, 3-cell, expanded blastocyst and hatching / hatched blastocyst developmental stages in 5 days of incubation were counted in each group.

[0182] The average time taken for those embryos to reach the 2-cell stage (T2) and the average time taken for those embryos to develop from the 2-cell stage to the 3-cell stage (T3) were measured.

[0183] The results are shown in Table 3 below.

[0184] Table 3 - Mouse embryonic development after incubation in fluorinated compounds

[0185]

[0186] A small percentage of embryos incubated in a commercially available perfluorocarbon mixture showed signs of degeneration, whereas none of the embryos incubated in perfluorooctane showed signs of degeneration.

[0187] Embryos incubated in the commercially available perfluorocarbon mixture working fluid (Group B) or raw material perfluorooctane (Group C) developed more slowly than control embryos (Group A). ​​This slowing was reflected in a lower proportion of embryos reaching the enlarged blastocyst and hatching / hatched blastocyst stages, and an increase in the time required to develop from the 2-cell stage to the 3-cell stage (average T2 to T3).

[0188] In contrast, embryos incubated in purified PFOS (Group D) developed at a rate comparable to that of untreated control embryos.

[0189] These data suggest that purified perfluorooctane does not negatively affect embryonic development compared with commercially available perfluorocarbon mixture operating fluids.

[0190] Example 3 - Assisted Reproduction Using Piezo-ICSI with Perfluorooctane Operating Fluid

[0191] The efficacy of c-ICSI and piezo-ICSI for assisted reproduction was compared. Purified perfluorooctane was used as the operating fluid for piezo-ICSI.

[0192] Specifically, 69 patients with at least 6 mature oocytes were randomly divided into two groups. The first group underwent assisted reproduction, in which sperm was introduced into the oocyte using c-ICSI, and the second group underwent assisted reproduction, in which sperm was introduced into the oocyte using piezo-ICSI.

[0193] c-ICSI was performed using an Eppendorf 2K control with an ICSI micropipette (TPC, Cooper Surgical). Piezo-ICSI was performed using a PMM and a piezo micropipette (Primetech). Purified perfluorooctane was used as the operating fluid in piezo-ICSI.

[0194] Patients who underwent assisted reproduction with piezo-ICSI had significantly higher fertilization rates compared to those who underwent c-ICSI (80.6% of oocytes were successfully fertilized using piezo-ICSI vs. 65.9% of oocytes were successfully fertilized using c-ICSI, p<0.05). The use of piezo-ICSI also reduced the rate of oocyte degeneration compared to c-ICSI (3.8% with piezo-ICSI vs. 10.2% with c-ICSI). The utilization rates of c-ICSI and piezo-ICSI were similar (45.6% with c-ICSI vs. 47.3% with piezo-ICSI). The clinical pregnancy rates of c-ICSI and piezo-ICSI were also comparable (50% with c-ICSI vs. 55% with piezo-ICSI).

[0195] On average, one more embryo was produced for vitrification using piezo-ICSI compared with c-ICSI (average 3.8 vitrified embryos from piezo-ICSI vs. 2.7 vitrified embryos from c-ICSI).

[0196] These data suggest that piezo-ICSI using purified PFOS as the operating fluid in assisted reproduction is more effective than c-ICSI in producing embryos for assisted reproduction.

[0197] All publications mentioned in the above description are incorporated herein by reference. Various modifications and variations of the method and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be overly limited to these specific embodiments. In fact, various modifications of the modes for implementing the present invention that are apparent to those skilled in the art of obstetrics and reproduction or assisted reproductive technology or molecular biology or related fields are intended to fall within the scope of the appended claims.

Claims

1. Use of purified perfluoro-n-octane or a composition comprising purified perfluoro-n-octane in the preparation of an operating solution for piezoelectric-mediated intracytoplasmic sperm injection (piezo-ICSI).

2. The use according to claim 1, wherein Piezo-ICSI comprises injecting mammalian sperm into a mammalian oocyte.

3. The use according to claim 2, wherein the sperm and oocytes are human.

4. The use according to claim 2, wherein the sperm and oocytes are murine.

5. The use according to any one of claims 1 to 4, wherein piezoelectric-ICSI comprises (a) providing an injection micropipette containing the sperm and the operating solution; (b) Piercing the oocyte with an injection micropipette using a piezoelectric pulse; and (c) injecting the sperm into the oocyte.

6. The use according to claim 5, wherein (b) comprises (i) puncturing the zona pellucida of the oocyte using a piezoelectric pulse; (ii) moving the injection micropipette through the zona pellucida to push against the tunica ovale of the oocyte; and (iii) Using a piezoelectric pulse to pierce the oocyte membrane, the injection micropipette is moved into the oocyte cytoplasm.

7. A device for piezoelectric-ICSI, comprising an injection micropipette filled with purified perfluoro-n-octane or a composition containing purified perfluoro-n-octane as an operating fluid.

8. The device of claim 7, wherein the device comprises an inverted microscope, a micromanipulator, a syringe and an injection micropipette.

9. The device of claim 8, wherein the device comprises a piezoelectric micromanipulator.

10. A kit for Piezo-ICSI comprising purified perfluoro-n-octane or a composition comprising purified perfluoro-n-octane as an operating fluid and one or more injection micropipettes.

11. A kit according to claim 10, comprising one or more holding pipettes, and / or a device for loading operating fluid into the one or more injection micropipettes.

12. The use according to any one of claims 1 to 6, the device according to any one of claims 7 to 9 or the kit according to claim 10 or claim 11, wherein the purified perfluoro-n-octane has an H value of 1000 ppm or less.

13. The use, device or kit according to claim 12, wherein the purified perfluorooctane has an H value of 10 ppm or less.

14. The use according to any one of claims 1 to 6, 12 or 13, the device according to any one of claims 7 to 9, 12 or 13 or the kit according to any one of claims 10 to 13, wherein the composition comprises 90% or more purified perfluoro-n-octane.

15. The use, device or kit according to claim 14, wherein the composition comprises 95% or more purified perfluoro-n-octane.

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