TWI656214B - Method for cleaning the surface of eggs or fertilized eggs - Google Patents

Abstract

The invention relates to a method for cleaning the surface of an egg or a fertilized egg. Specifically, the disclosed method includes step (1) placing untreated eggs or untreated fertilized eggs in a first culture dish, wherein the first culture dish is surface treated with a standard cell culture and contains A first culture liquid containing protein; and then adding hyaluronidase to treat the eggs or fertilized eggs to obtain the first treated eggs or fertilized eggs; (2) the first treatment Eggs or fertilized eggs are transferred to a second culture dish, wherein the second culture dish is not surface-treated and contains a second culture liquid, and the second culture liquid contains no protein; The rod-shaped tool is used to pluck the egg or fertilized egg, wherein the third culture liquid contains protein; the granular cells and / or excess sperm are detached from the surface of the egg or fertilized egg, thereby being completely removed.

Description

   Method for cleaning the surface of eggs or fertilized eggs   

The invention belongs to the field of reproductive medicine, and in particular, the invention relates to a method for cleaning the surface of an egg or a fertilized egg.

IVF technology is a powerful technique to fight infertility. But for various reasons, the success rate from embryo implantation to uterus to fetal birth is not high (usually only about 40%). In addition to maternal health reasons, fertilized egg quality is one of the important causes of embryo development failure.

Early in the process of IVF technology, only morphological observation under a microscope was used to select 2-3 relatively "normal" ones from multiple embryos for implantation into the mother's uterus. The normal morphology under the microscope does not reflect whether the chromosomes are normal. Improper selection of embryos with normal morphology and abnormal chromosomes into the mother's uterus has resulted in the failure of many IVF conceptions.

In recent years, some techniques (collectively called Preimplantation Genetic Screen (PGS)) have been established to detect the chromosomal status of in vitro cultured embryos in order to screen embryos with normal chromosomes and implant them into the mother's uterus, thereby improving the success rate of conception. The biological samples required for various PGS methods are one to several cells obtained from in vitro cultured embryos. The detection of these written cells can reflect whether the chromosomes of the entire embryo are normal. Embryo biopsy includes polar body biopsy, blastomere biopsy, and blastocyst trophoblast cell biopsy. The PGS test is usually a biopsy of blastomere or blastocyst trophoblast cells. However, more and more studies show that the safety of cleavage stage biopsy is questioned, so blastocyst trophoblast cell biopsy has gradually become the mainstream of PGS embryo biopsy.

When the fertilized egg grows and develops in culture for 5 days, the embryo is a sac-like structure composed of about 80-100 cells, called a blastocyst. At this time, one to several trophoblast cells can be detected with a capillary glass tube under a microscope. Previous studies have shown that a small amount of DNA is released into the culture medium (blastocyst culture medium) during the early in vitro development of the embryo (day 1-5). Although the mechanism is unknown, the presence of embryo-derived DNA in the blastocyst culture fluid provides a material basis for non-destructive testing of embryo chromosomal status. Our earlier invention "A Method for Detecting Embryo Chromosomal Abnormalities Using Blastocyst Culture Medium" (CN105368936A) is exactly a set of technical methods (NICS) to achieve this goal. However, the NICS method is susceptible to interference from factors that have been ignored during in vitro embryo culture of conventional IVFs.

The first generation of IVFs used large numbers of sperm to fertilize eggs in vitro. However, for each egg, only one sperm can fertilize effectively, making the egg a fertilized egg, and other unwanted sperm will adhere to the surface of the fertilized egg, and they will be released into the in vitro culture fluid after death. Therefore, the parental DNA will Subsequent NICS detection forms interference. In addition, a large number of maternal-derived granulocytes are unavoidably attached to the surface of the egg cells extracted from the mother, and these granulocytes are released into the culture medium. Therefore, maternal DNA will also interfere with the detection of NICS.

Although the second-generation IVF uses the intracytoplasmic sperm injection method (ICSI) to fertilize the eggs in vitro, the problem of contamination caused by excess sperm is avoided. In addition, before in vitro fertilization, the granular cells attached to the surface of the egg are routinely cleaned to partially expose the egg surface and provide a channel for the sperm injection needle. However, after cleaning the eggs, there will still be more particles attached to the surface of the egg. Detection forms interference.

Therefore, the present invention establishes a technical method for completely removing granular cells and / or excess sperm on the surface of an egg (for ICSI) or a fertilized egg (for IVF) to avoid interference with NICS detection.

The purpose of the present invention is to establish a technical method for completely removing granular cells and / or excess sperm from the surface of an egg during the process of IVF and ICSI, so that the subsequent embryo culture solution is suitable for NICS detection.

In a first aspect of the present invention, a method for cleaning the surface of an egg or a fertilized egg is provided, which comprises the steps of: (1) placing an untreated egg or an untreated fertilized egg in a first culture dish Wherein, the first culture dish is surface-treated with a standard cell culture and contains a first culture liquid, and the first culture liquid contains protein; and then hyaluronidase is added to treat the eggs or fertilized eggs to obtain a cultured first culture dish. Once-treated eggs or fertilized eggs; (2) moving the first-treated eggs or fertilized eggs into a second culture dish, wherein the second culture dish is not surface-treated and contains a second culture liquid, so Said second culture medium does not contain protein; then the egg or fertilized egg is plucked with a rod-shaped tool with a third culture medium, wherein said third culture medium contains protein; and granulocytes and / or excess sperm are detached The surface of the egg or fertilized egg is completely removed.

In another preferred example, the rod-shaped tool with a third culture solution means that the rod-shaped tool is dipped with a third culture solution.

In another preferred example, the rod-shaped tool is a stick or a stick.

In another preferred example, the rod-shaped tool is made of any suitable material such as glass, plastic, and the like.

In another preferred example, the protein is a serum protein.

In another preferred example, the untreated egg is an egg that is removed from the mother for 2-3 hours.

In another preferred example, the untreated fertilized eggs are fertilized eggs that are fertilized in vitro for 4 hours or 16-20 hours.

In another preferred example, the first culture solution simulates a component of human fallopian tube fluid, and uses HEPES as a buffer component.

In another preferred example, the first culture liquid can refer to the literature published by Quinn et al. In 1984 ("Fertil Steril." 1984; 41: 202 or 1985; 44: 493.) preparation.

The culture medium uses HEPES as a buffer component, and can maintain pH stability in the air for a long time. No carbon dioxide gas environment is required for cultivation.

The culture solution can be purchased from various commercial companies (for example, SAGE media) or can be prepared by itself.

In another preferred example, the first culture solution is an IM culture solution.

In another preferred example, the first petri dish is a Falcon 353037 petri dish, a Corning 430166 petri dish, or a Thermomer 150288 petri dish.

In another preferred example, the first culture dish is any commercially available cell culture dish treated with a standard cell culture surface. It can be sold by any other company and can be of any shape or size.

In another preferred example, the hyaluronidase can be purchased from any cell culture reagent supplier.

In another preferred example, in step (1), the processing time is 10-60 seconds; preferably, 20-40 seconds.

In another preferred example, the composition of the second culture solution is the same as that of the first culture solution, except that the second culture solution does not contain protein.

In another preferred example, the second culture solution is an M-IM culture solution.

In another preferred example, the second petri dish is a Falcon 351006 petri dish, a Corning 430589 petri dish, or a Thermo Fisher 150340 petri dish.

In another preferred example, the second culture dish is any commercially available cell surface membrane without surface treatment. It can be sold by any other company and can be of any shape or size.

In another preferred example, the composition of the third culture solution is the same as that of the first culture solution, except that the third culture solution does not contain the buffer component HEPES.

In another preferred example, the third culture liquid can refer to the literature published by Quinn et al. ("Fertil Steril." 1984; 41: 202 or 1985; 44: 493.) In 1984. preparation.

The third culture medium differs from the first culture medium in that it does not contain a buffer component (HEPES), so it must be used in a 5% carbon dioxide environment (in a carbon dioxide incubator).

The culture solution can be purchased from various commercial companies (for example, SAGE media) or can be prepared by itself.

In another preferred example, the third culture solution is a GM culture solution.

In another preferred example, the granular cells and / or excess sperm are detached from the surface of the egg or fertilized egg and attached to the bottom surface of the culture dish.

It should be understood that, within the scope of the present invention, the above technical features of the present invention and the technical features specifically described in the following (such as the embodiments) may be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them here.

Figure 1 shows untreated eggs freshly removed from the mother, with a large number of granular cells attached to the surface.

Figure 2 shows that after the egg was washed in step 3, the granulocyte attachment on the surface was significantly reduced, but granulocyte attachment was still observed.

Figure 3 shows the granule cells detached from the egg during the operation and attached to the bottom of the culture dish.

Figure 4 shows that the granule cells on the surface of the eggs treated in steps 3 and 4 were completely removed.

Figure 5 shows that the granular cells attached to the zona pellucida of both eggs were completely removed.

Figure 6 shows that granulated cells remained on the surface of the two eggs after treatment.

After extensive and intensive research, the present inventor unexpectedly discovered a method for thoroughly cleaning the surface of an egg or a fertilized egg. On this basis, the inventors have completed the present invention.

The proteins mentioned herein are serum proteins.

Method for cleaning the surface of eggs or fertilized eggs

The invention provides a method for cleaning the surface of an egg or a fertilized egg, the method comprising the steps of: (1) placing an untreated egg or an untreated fertilized egg in a first culture dish, wherein the first The petri dish is surface-treated with a standard cell culture and contains a first medium containing protein; then hyaluronidase (available from any cell culture reagent supplier) is added to treat the eggs or fertilized eggs for a period Time (such as 10-60 seconds; preferably, 20-40 seconds), so as to obtain the first-treated eggs or fertilized eggs; (2) move the first-treated eggs or fertilized eggs to a second culture In a dish, wherein the second culture dish is not surface-treated and contains a second culture fluid, the second culture fluid contains no protein; and then the egg or The fertilized egg, wherein the third culture liquid contains protein; the granular cells and / or excess sperm are detached from the surface of the egg or the fertilized egg, thereby being completely removed.

Wherein, the granular cells and / or excess sperm are detached from the surface of the egg or fertilized egg and attached to the bottom surface of the culture dish.

The rod-shaped tool is a rod or stick-like tool. The rod-shaped tool may be made of any suitable material such as glass, plastic, and the like. The rod-shaped tool with a third culture solution means that the rod-shaped tool is dipped with a third culture solution.

Untreated eggs or unfertilized eggs

The untreated egg may be an egg that has been removed from the mother for 2-3 hours.

The untreated fertilized egg may be a fertilized egg after 4 hours or 16-20 hours of in vitro fertilization.

First culture broth

The first culture solution simulates the components of human fallopian tube fluid, and uses HEPES as a buffer component. The culture medium uses HEPES as a buffer component, and can maintain pH stability in the air for a long time. No carbon dioxide gas environment is required for cultivation.

The first culture liquid can be prepared by referring to the literature published by Quinn et al. In 1984 ("Fertil Steril." 1984; 41: 202 or 1985; 44: 493.). It can also be purchased from various commercial companies (eg, SAGE media).

In another preferred example, the first culture solution is an IM culture solution.

First petri dish

The first culture dish is any commercially available cell culture dish treated with a standard cell culture surface. It can be sold by any other company and can be of any shape or size.

In another preferred example, the first petri dish is a Falcon 353037 petri dish, a Corning 430166 petri dish, or a Thermomer 150288 petri dish.

Second culture broth

The composition of the second culture solution is the same as that of the first culture solution, except that the second culture solution does not contain protein.

In another preferred example, the second culture solution is an M-IM culture solution.

Second petri dish

The second culture dish is any commercially available unsurfaced cell surface membrane. It can be sold by any other company and can be of any shape or size.

In another preferred example, the second petri dish is a Falcon 351006 petri dish, a Corning 430589 petri dish, or a Thermo Fisher 150340 petri dish.

Third culture broth

The composition of the third culture solution is the same as that of the first culture solution, except that the third culture solution does not contain the buffer component HEPES. The third culture medium differs from the first culture medium in that it does not contain a buffer component (HEPES), so it must be used in a 5% carbon dioxide environment (in a carbon dioxide incubator).

In another preferred example, the third culture liquid can refer to the literature published by Quinn et al. (Fertil Steril. 1984; 41: 202 or 1985; 44: 493.) In 1984. Prepared or available from various commercial companies (eg, SAGE media).

In another preferred example, the third culture solution is a GM culture solution.

The beneficial effects of the technical solution of the present invention are:

1. Thoroughly remove the granular cells on the surface of the egg (or fertilized egg), avoiding the particulate cells and / or excess sperm contamination that may be encountered during the test of IVF and ICSI.

2. Avoiding the operation of embryo zona pellucida stripping, simplifying the technical operation steps, and avoiding the risk of embryo damage that may be caused by zona pellucida stripping operation.

The present invention will be further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are generally performed according to conventional conditions, such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing conditions. Conditions recommended by the manufacturer. Unless stated otherwise, percentages and parts are by weight.

Example 1.     I. Reagents and Consumables         Reagent    

1.1 HEPES-HTF culture solution containing 80U / ml hyaluronidase (Hyaluronidase 80U / ml in HEPES-HTF) (Specification: SAGE, product number: ART4007-A)

1.2 M-IM medium: Quinn's Advantage with HEPES medium (Quinn's Advantage Medium with HEPES) (Specification: SAGE, Product No .: ART-1023)

1.3 IM medium: Quinn's Advantage plus protein fertilization medium (Quinn's AdvantageTM Protein Plus Fertilization (TIF) Medium) (Specification: SAGE, product number: ART-1520)

1.4 GM medium: Quinn's Advantage plus protein cleavage medium (Quinn's Advantage Protein Plus Cleavage Medium) (Specification: SAGE, product number: ART-1526)

    2. Consumables    

2.1 Falcon 351006 Petri Dish

2.2 Falcon 363037 Petri Dish

2.3 Pasteur straws

    3. Operating timing    

ICSI's egg clearance granule cell treatment is 2-3 hours after egg retrieval.

Untreated eggs just removed from the mother have a large number of granular cells attached to the surface, as shown in Figure 1.

    4. Operating procedures:    

4.1 Before the eggs are cleaned, prepare a straw (100-120 μm) for egg peeling, a straw (180-200 μm) for egg removal, and a small glass rod with a rounded end.

4.2 Add 1 ml IM culture fluid (ART-1520) to a Falcon 353037 (polystyrene material, standard cell culture surface treatment) culture dish; add 2 ml to a Falcon 351006 (polystyrene material, no treatment) culture dish Protein-free M-IM broth (ART-1023).

4.3 Place the untreated eggs from the mother's body into a Falcon 353037 dish containing IM medium (ART-1520), then add 1 ml of 80 U / ml of hyaluronidase-containing HEPES-HTF culture medium, and repeat Inhale for 30 seconds. In this step, because the culture medium contains negatively charged proteins, the positive charge on the surface of the granular cells is neutralized. At the same time, the surface of the polystyrene culture dish was treated with cell culture, and its surface became hydrophilic and neutral. Therefore, the granule cells attached to the egg and its surface will be suspended in the liquid culture medium. The digestion of hyaluronidase weakens the adhesion between granulocytes and eggs. Under the action of flushing, part of the granular cells can be detached from the egg.

After the eggs washed and washed in step 3 above, the granule cell adhesion on the surface is significantly reduced, but the granule cells still adhere. as shown in picture 2.

4.4 Transfer the eggs with residual granulocytes adhered to the surface into a Falcon 351006 dish containing serum-free M-IM broth (ART-1023). At this time, granule cells are positively charged because there is no negative neutralization of proteins in the culture medium. At the same time, Falcon 351006 petri dish is made of untreated polystyrene, and its surface is hydrophobic and negatively charged. Therefore, the granule cells attached to the egg and its surface will adhere to the bottom surface of the culture dish (as shown in Figure 3). At this time, use a glass rod dipped in protein-containing GM culture medium (ART-1526) (the role of the protein-containing culture medium to neutralize the negative charge on the surface of the glass rod and prevent the cells from sticking to the glass rod) to constantly stir the eggs Roll the eggs on the bottom surface of the petri dish. During the rolling process, the remaining granular cells on the surface of the egg will adhere to the bottom surface of the petri dish and detach from the egg, and continue to push the egg to roll until the granular cells on the surface are completely removed.

The granule cells on the surface of the eggs treated after the above steps 3 and 4 are completely removed, as shown in FIG. 4.

4.5 After removing the granulocytes, transfer the eggs to the GM broth with a pipette (aspirate a little protein-containing broth first) and put them in a 37 ° C, 5% CO ₂ , 5% O ₂ incubator, and then you can Into the routine single sperm injection (ICSI), embryo in vitro culture (IVF and ICSI) and NICS detection procedures.

Example 2

The method of Example 1 was used, except that the eggs were replaced with fertilized eggs. The treatment time for IVF to remove granular cells and excess sperm from the surface of fertilized eggs is 4 hours (short fertilization) or 16-20 hours (conventional fertilization) after fertilization.

It was found that granular cells and excess sperm on the surface of IVF fertilized eggs could be completely removed.

Example 3

The method of Example 1 was used, except that a Petri dish of Corning was used, and the culture solution was made according to the formula in the literature (Fertil Steril. 1984; 41: 202, 1985; 44: 493.).

Replace 1ml IM culture medium (ART-1520) with HTF culture medium containing human serum albumin and HEPES (self-made according to the literature) and petri dishes with standard cell culture surface treatment (purchased from: Corning; product number: 430166) Falcon 353037 (polystyrene, standard cell culture surface treatment) culture dishes; use protein-free HTF culture medium containing HEPES (made according to the literature) and non-surface-treated culture dishes (purchased from: Corning; (Product No. 430589) instead of a Falcon 351006 (polystyrene, untreated) culture dish containing 2 ml of protein-free M-IM culture medium (ART-1023).

The results showed that the granular cells attached to the zona pellucida of the two eggs were completely removed, as shown in Figure 5.

Comparative Example 1

The method of Example 1 was used, except that the protein-free M-IM medium was replaced with the IM medium in step 4.4.

As a result, the granulocytes could not adhere to the bottom of the petri dish and could not be detached from the egg surface. Granular cells remained on the surface of the two eggs after treatment, as shown in Figure 6.

The advantage of the invention is that it does not cause any damage to the eggs, fertilized eggs or embryos, and does not touch the zona pellucida, and provides a method for thoroughly removing granular cells and / or excess sperm on the surface of the egg. The main point is to use the charge relationship between the cell surface, the culture medium component (protein) and the bottom surface of the cell culture dish to remove granular cells and / or excess sperm from the surface of the egg (or fertilized egg).

All documents mentioned in the present invention are incorporated by reference in this application, as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the scope of the patents attached to this application.

Claims (10)

A method for cleaning the surface of an egg or a fertilized egg, comprising the steps of: (1) placing an untreated egg or an untreated fertilized egg in a first culture dish, wherein the first culture dish is cultured with standard cells; Surface treated and containing a first culture solution, said first culture solution contains protein; and then the hyaluronidase is added to treat the eggs or fertilized eggs to obtain the first treated eggs or fertilized eggs; (2) The eggs or fertilized eggs treated for the first time are transferred to a second culture dish, wherein the second culture dish is not surface-treated and contains a second culture fluid, and the second culture fluid contains no protein; The rod-shaped tool of three culture fluids stirs the egg or fertilized egg, wherein the third culture fluid contains protein; the granular cells and / or excess sperm are detached from the surface of the egg or fertilized egg, thereby being completely removed. The method according to claim 1, wherein the untreated eggs are eggs 2-3 hours after removal from the mother. The method according to claim 1, wherein the untreated fertilized egg is a fertilized egg 4 hours or 16-20 hours after in vitro fertilization. The method according to claim 1, wherein the first culture fluid mimics a component of human fallopian tube fluid and uses HEPES as a buffer component. The method of claim 1, wherein the first petri dish is a Falcon 353037 petri dish, a Corning 430166 petri dish, or a Thermo Fisher 150288 petri dish. The method according to claim 1, wherein in step (1), the processing time is 10-60 seconds. The method according to claim 1, wherein the composition of the second culture liquid is different from that of the first culture liquid only in that the second culture liquid does not contain protein. The method of claim 1, wherein the second petri dish is a Falcon 351006 petri dish, a Corning 430589 petri dish, or a Thermo Fisher 150340 petri dish. The method according to claim 1, wherein the third culture medium is different from the first culture medium only in that the third culture medium does not contain the buffer component HEPES. The method according to claim 1, wherein the granular cells and / or excess sperm are detached from the surface of the egg or fertilized egg and attached to the bottom surface of the culture dish.