Electro-fusion microscopic insemination method

Abstract

The invention discloses an electrofusion microscopic insemination method, and relates to the technical field of human assisted reproduction. The method comprises the following steps: step 1, braking sperms, and sucking the braked sperms through a puncture needle; 2, puncturing the zona pellucida by using the puncture needle, and injecting sperms under the zona pellucida; 3, sucking out the pole body by using the puncture needle, and removing the needle; and 4, releasing cell nucleuses of sperms into oocytoplasm in an electric fusion mode to realize fertilization. According to the method, mechanical membrane rupture is replaced with electric fusion, cell membrane puncture is not needed, mechanical damage to cell membranes, cytoskeletons and imitation hammer bodies of egg cells is directly avoided, damage to oocytes is reduced to the maximum extent, and therefore the clinical success rate is increased, and health of offspring is better facilitated; in the aspect of cell screening, the better the egg cell quality is, the more failed the traditional method is during wall breaking, while the method provided by the invention can select the egg cells with better quality for operation, so that the clinical success rate is greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of human assisted reproduction, and in particular to an electrofusion micro-insemination method. Background Art

[0002] Cell fusion is the normal physiological way for sperm and egg to combine, that is, the sperm passes through the zona pellucida of the egg and contacts the cell membrane of the oocyte. The cell membranes of the oocyte and sperm will fuse, and after fusion, the sperm will release its nucleus into the cytoplasm of the egg, completing the fertilization process. A considerable number of couples (about 30%) who receive IVF-ET treatment need microinsemination, that is, intracytoplasmic sperm injection (ICSI) to achieve fertilization.

[0003] The currently commonly used microinsemination method is to inject the immobilized sperm into the cytoplasm with a fine needle. This method was first used in clinical practice in 1992 and was successful. It is still used today, but it also has obvious disadvantages. An important step in this method is mechanical membrane rupture. The most commonly used method is re-absorption rupture, and a few use piezoelectric rupture. Both methods require the destruction of the integrity of the cell membrane. In particular, re-absorption rupture will also damage the cytoskeleton, causing a certain proportion of oocyte degeneration. In a certain proportion of cases, the mitochondria will be punctured. Once the mitochondria are damaged, the oocyte loses its core function as a gamete and cannot continue to form a zygote. The above disadvantages directly affect the clinical outcome.

[0004] Based on the above problems, we proposed an electrofusion microinsemination method to solve the above problems.

[0005] The above information disclosed in this background technology is only used to increase the understanding of the background technology of the present invention and therefore, it may include information that does not constitute the prior art known to ordinary technicians in this field. Summary of the invention

[0006] The object of the present invention is to provide an electrofusion micro-insemination method to solve the problem that both the aspiration membrane rupture and the piezoelectric membrane rupture proposed in the above-mentioned background technology require the destruction of the integrity of the cell membrane, especially the aspiration membrane rupture will also cause damage to the cytoskeleton, causing a certain proportion of oocyte degeneration, and in a certain proportion of cases the cysticercus will be punctured. Once the cysticercus is damaged, the oocyte loses its core function as a gamete and cannot continue to form a zygote, which directly affects the clinical outcome.

[0007] To achieve the above object, the present invention provides the following technical solution: an electrofusion microinsemination method, comprising the following steps:

[0008] Step 1: Sperm braking and aspiration of the braked sperm through the puncture needle;

[0009] Step 2: Use the puncture needle to puncture the zona pellucida and inject the sperm under the zona pellucida;

[0010] Step 3: Use the puncture needle again to aspirate the polar body and remove the needle;

[0011] Step 4: Use electrofusion to release the sperm nucleus into the egg cytoplasm to achieve fertilization.

[0012] Preferably, in step 1, sperm braking is performed by mechanical braking, laser braking or piezoelectric braking.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] (1) The present invention establishes a new microscopic insemination method that is closer to the natural fertilization process, using electrofusion instead of mechanical membrane rupture, without puncturing the cell membrane, directly avoiding mechanical damage to the cell membrane, cytoskeleton and mitochondria of the oocyte, minimizing damage to the oocyte, thereby improving the clinical success rate and being more beneficial to the health of future generations; in terms of cell screening, the better the quality of the oocyte, the more likely it is to fail during cell rupture by the traditional method, while the method of the present invention can select oocytes of better quality for operation, greatly improving the clinical success rate.

[0015] (2) The present invention is simpler to operate and easier to master. Since mechanical membrane rupture is a relatively complex operation step and is also the step that is least likely to be standardized, eliminating this step makes the entire operation process more repeatable, thereby simplifying the methodology for implementing automated microinsemination.

[0016] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of the process of the electrofusion microinsemination method of the present invention;

[0018] Figure 2 Schematic diagram of the process of traditional microinsemination method. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] Example

[0021] See also Figure 1 , an electrofusion microinsemination method, comprising the following steps:

[0022] Step 1: Sperm braking and aspirating the braked sperm through a puncture needle. The sperm braking is performed by mechanical braking, laser or piezoelectric braking. The puncture needle is a needle with an inner diameter of 15-20 μm and an eagle's beak.

[0023] Step 2: Use the puncture needle to puncture the zona pellucida and inject the sperm under the zona pellucida;

[0024] Step 3: Use the puncture needle again to aspirate the polar body and remove the needle;

[0025] Step 4: Use electrofusion to release the sperm nucleus into the egg cytoplasm to achieve fertilization.

[0026] This process is similar to the process of natural fertilization. In natural fertilization, after the sperm passes through the zona pellucida, the cell membranes of the sperm and egg fuse, and the sperm nucleus enters the cytoplasm of the egg. The biggest advantage of this method is that it uses electrical fusion instead of mechanical membrane rupture, avoiding the damage of mechanical membrane rupture to the oocyte.

[0027] Comparative Example

[0028] like Figure 2 As shown, the traditional microinsemination method used today is as follows:

[0029] Step 1: Sperm braking and aspirating the braked sperm through the puncture needle. Sperm braking is usually done by mechanical compression braking.

[0030] Step 2: After puncturing the zona pellucida with the needle, continue to insert the needle into the center of the oocyte;

[0031] Step 3: Create a rupture in the oocyte by suction (this step is the most difficult to control and is the main step that causes cell damage);

[0032] Step 4: Inject the sperm into the cytoplasm of the oocyte, withdraw the needle, and end the operation.

[0033] The biggest disadvantage is the need to suck back to break the membrane. The better the quality of the egg, the more resilient the membrane will be, and the less likely it is to break back. The easier it is to use excessive force during operation and damage the egg, leading to degeneration.

[0034] In summary, the present invention has established a new microscopic insemination method that is closer to the natural fertilization process. It uses electrical fusion instead of mechanical membrane rupture, and there is no need to puncture the cell membrane. It directly avoids mechanical damage to the cell membrane, cytoskeleton and cytokines of the oocyte, minimizes damage to the oocyte, thereby improving the clinical success rate and being more beneficial to the health of future generations. In terms of cell screening, the better the quality of the oocyte, the more likely it is to fail during cell cell rupture in traditional methods. The method of the present invention can select oocytes with better quality for operation, greatly improving the clinical success rate.

[0035] At the same time, the present invention is simpler to operate and easier to master; since mechanical membrane rupture is a relatively complex operation step and is also the step that is least likely to be standardized, this step is eliminated, so that the entire operation process has better repeatability, and the methodology is simplified for realizing the implementation of automated microinsemination.

[0036] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0037] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An electrofusion microinsemination method, characterized in that: The steps include: Step 1: Sperm braking and aspiration of the braked sperm through the puncture needle; Step 2: Use the puncture needle to puncture the zona pellucida and inject the sperm under the zona pellucida; Step 3: Use the puncture needle again to aspirate the polar body and remove the needle; Step 4: Use electrofusion to release the sperm nucleus into the egg cytoplasm to achieve fertilization.

2. The electrofusion microinsemination method according to claim 1, characterized in that: In step 1, sperm braking is performed by mechanical braking, laser braking or piezoelectric braking.

Images