阅读说明：本技术 一种特定位点辅助囊胚孵化的方法 (Method for assisting blastocyst incubation at specific site ) 是由 杜福良 安礼友 于 2020-12-17 设计创作，主要内容包括：本发明公开了一种特定位点辅助囊胚孵化的方法,通过显微操作使囊胚透明带融通形成通孔,使胚胎细胞从通孔出来完成胚胎孵化,孵化胚胎经胚胎移植后,得到出生个体。采用本方法得到的孵化胚胎经胚胎移植后,其胚胎发育到期的出生率比未做处理的正常胚胎显著提高,出生率高达77％。(The invention discloses a method for assisting hatching of blastocysts at specific sites, which is characterized in that a blastocyst zona pellucida is fused to form a through hole through micromanipulation, embryo cells come out from the through hole to complete embryo hatching, and a born individual is obtained after the hatched embryos are transplanted. After the hatching embryo obtained by the method is transplanted, the birth rate of the mature embryo is obviously improved compared with the normal embryo which is not processed, and the birth rate is up to 77%.)

1. A method for assisting blastocyst hatching at a specific site is characterized by comprising the following steps: the blastocyst zona pellucida is fused through a microscopic operation to form a through hole, embryo cells come out from the through hole to complete embryo incubation, and the incubated embryo is transplanted to obtain a birth individual.

2. The method for site-specific assisted blastocyst incubation according to claim 1, wherein: the position of the through hole is as follows: the Inner Cell Mass (ICM) of the blastocyst is defined as the clock 12 o 'clock position and the through hole position is between 2 and 4 o' clock or between 4 and 8 o 'clock or between 8 and 10 o' clock.

3. The method for assisting blastocyst hatching at a specific site according to claim 2, wherein: the position of the through hole is as follows: a 3-point location or a 6-point location.

4. The method for site-specific assisted blastocyst incubation according to claim 1, wherein: the micromanipulation comprises modified acidic working solutions, proteases, a piezo-electroporator, or a laser.

5. The method for assisting blastocyst hatching at a specific site according to claim 4, wherein: the improved acidic operating solution is obtained by modifying a standard acidic Tschmann solution, namely, adjusting the pH of the standard acidic Tschmann solution to be in a range of 1-2 by using 1-4 mol (M) of hydrochloric acid (HCl).

6. The method for site-specific assisted blastocyst incubation according to claim 1, wherein: the diameter of the through hole is 20-50 μm.

Technical Field

The invention relates to biotechnology, in particular to a method for assisting blastocyst incubation at a specific site.

Background

Embryo implantation refers to a complex process of close connection between blastocyst and uterus in accepting state, mainly including positioning, adhesion and invasion of free blastocyst and formation of placenta, and is a continuous dynamic development process. The hatching of the blastocyst is a prerequisite for establishing the relationship between the mother and the fetus, and the blastocyst must be hatched from the zona pellucida to be implanted, so that the blastocyst normally develops to the birth. Even embryos with good developmental potential will fail pregnancy due to blastocyst hatching failure. Assisted hatching technology (assisted hatching) is currently applied to human clinical application, but the effect of the assisted hatching technology is still controversial.

Assisted hatching technology (assisted hatching) is an assisted reproduction technology in animal and human reproduction technology, and is a technology for enabling embryo cells to hatch from zona pellucida by artificial means so as to improve the implantation and birth rate of animal embryos. In the past, the laser or the piezoelectric perforator is mainly used for assisting hatching of blastocysts, and the laser or the piezoelectric perforator is applied to assisting reproduction of human beings, mice, rats, goats, sheep, pigs and cows, but no clear technical effectiveness theory exists.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a method for assisting blastocyst hatching at a specific site capable of improving birth rate.

The technical scheme is as follows: the invention provides a method for assisting hatching of a blastocyst at a specific site.

Further, the position of the through hole: the Inner Cell Mass (ICM) of the blastocyst is defined as the clock 12 o 'clock position and the through hole position is between 2 and 4 o' clock or between 4 and 8 o 'clock or between 8 and 10 o' clock.

Further, the position of the through hole: a 3-point location or a 6-point location.

Further, the micromanipulation comprises modifying an acidic working solution, a protease, a piezo-electroporator, or a laser.

Further, the modified acidic working solution is modified based on a standard acidic tai solution by adjusting the PH of the standard acidic tai solution to a range of 1 to 2 using 1 to 4 moles (M) of hydrochloric acid (HCl).

Further, the diameter of the through hole is 20-50 μm.

Has the advantages that: the invention melts, thins and even melts the zona pellucida at the special position of the zona pellucida of the blastocyst by a micromanipulation method to form a small hole, embryo cells can come out from the zona pellucida melting through hole to finish embryo incubation, and the birth rate of the incubated embryo obtained by the method after embryo transplantation is obviously improved compared with the normal embryo without treatment, and the birth rate is up to 77 percent.

Drawings

FIG. 1 is an acid operating solution at a position between 2 and 4 points to assist in blastocyst incubation;

FIG. 2 shows the comparison of the birth rate of embryos incubated with acid operating solution in an assisted manner with that of embryos not incubated with the acid operating solution in an assisted manner;

FIG. 3 is a schematic representation of the use of a piezo-electroporator to assist in hatching blastocyst in a 2 to 4 o' clock position;

FIG. 4 is a comparison of the birth rate of blastocysts incubated with the aid of a piezoelectric electroporator and embryos not incubated with the aid of incubation.

Detailed Description

The embryo develops into a blastocyst in vivo or in vitro, the blastocyst is transferred into a micromanipulation liquid drop, the blastocyst is fixed by an egg holding needle, and the assisted hatching of a specific site is completed by adopting micromanipulation. The micromanipulation consists of using a Modified Acidic reagent's solution (see example 1) and a piezoelectric electroporator (Piezo) in example 2. After the blastocysts which are subjected to the auxiliary hatching operation are cleaned by the culture solution, the blastocysts are transferred into the culture solution for culture, and the blastocysts are transplanted to a receptor in estrus at the same period after the transient culture.

Example 1

(1) Improved preparation of acidic operating solution

The modified acidic working solution is modified based on a standard acidic Tschel solution by adjusting the pH of the standard acidic Tschel solution to a range of 1 to 2 using 1 to 4 moles (M) of hydrochloric acid (HCl).

(2) Acid operating solution assisted mouse blastocyst incubation

Mouse embryo develops into expanded blastocyst in vivo or in vitro, the blastocyst is transferred into M2 micromanipulation liquid drop, the blastocyst is fixed by egg holding needle (figure 1A), the operating needle sucks acid operating liquid in the liquid drop prepared separately, and then the opening of the operating needle is aligned with the zona pellucida of the target hatching position, for example, the ICM position is defined as the clock 12 point position, and the target hatching position can be 2-4 (or 8-10 on the opposite side) and 4-8 point positions; and (3) slowly discharging the acidic operating solution from the opening of the operating needle by applying pressure to form an acidic operating solution flow, dissolving the zona pellucida in the target area by the acidic operating solution, gradually thinning the zona pellucida in the target area, melting the zona pellucida to form open holes (positions between arrows in the figure 1B), cleaning the blastocysts which finish the auxiliary hatching operation by the KSOM culture solution, transferring the blastocysts into the KSOM culture solution for culturing for 30 minutes, and observing that the embryonic cells begin to hatch from the auxiliary hatching position (figure 1C).

(3) Comparison of birth rates of mice with blastocysts incubated with acidic operating solution and without assisted incubation treatment

After the blastocysts which finish the auxiliary hatching operation are cleaned by KSOM culture solution, the blastocysts are transferred into the KSOM culture solution for culturing for 30 minutes, and then embryo transplantation is carried out. The blastocysts incubated in the auxiliary hatches were microsurgical implanted into the uterus of a recipient mouse in estrus. The contemporaneous blastocyst without auxiliary hatching treatment was used as a transplantation control and transplanted into the uterus of a recipient mouse in contemporaneous estrus. By day 19 of gestation, mice pups were removed by cesarean section and the rate of birth recorded. Statistics shows that the birth rate of the untreated Blastocyst (4.0dpc blast) is 36%, the birth rate of the random locus (AH random) assisted hatching Blastocyst is 59%, the birth rate of the 3-site assisted hatching Blastocyst (3-site) is 77%, the birth rate of the 6-site assisted hatching Blastocyst (6-site) is 55%, the birth rates of the random locus, the 3-site assisted hatching Blastocyst and the 6-site assisted hatching Blastocyst are significantly higher (P < 0.05) than those of the untreated Blastocyst, and the birth rate of the 3-site assisted hatching Blastocyst (P < 0.05) is the highest (P < 0.05), as shown in FIG. 2.

Example 2

(1) Mouse blastocyst incubation assisted by piezoelectric perforator

Mouse embryo develops into expanded blastocyst in vivo or in vitro, the blastocyst is transferred into M2 micromanipulation liquid drop, the blastocyst is fixed by an ovum holding needle (figure 3A), then the opening of the operation needle is aligned with a zona pellucida of a target hatching position, for example, the ICM position is defined as the 12 o 'clock position, and the target hatching position can be 2-4 (or 8-10 on the opposite side) and 4-8 o' clock position; triggering a piezoelectric electroporator (Peizo) to act to form a small hole (the position between arrows in figure 3B) in the zona pellucida of the target area, cleaning the blastocyst which finishes the auxiliary hatching operation by KSOM culture solution, and then transferring the blastocyst into the KSOM culture solution for culturing for 30 minutes.

(2) Comparison of birth rates of blastocysts incubated by a piezoelectric electroporator and mice without assisted incubation treatment of blastocysts

The blastocysts incubated in the auxiliary hatches were microsurgical implanted into the uterus of a recipient mouse in estrus. The contemporaneous blastocyst without auxiliary hatching treatment was used as a transplantation control and transplanted into the uterus of a recipient mouse in contemporaneous estrus. By day 19 of gestation, mice pups were removed by cesarean section and the rate of birth recorded. The birth rate of the untreated Blastocyst (4.0dpc blast) was counted as 32%, the birth rate of random locus (AH random) assisted hatching Blastocyst was counted as 43%, the birth rate of 3-position (3-position) assisted hatching Blastocyst was counted as 49%, and the birth rate of 6-position (6-position) assisted hatching Blastocyst was counted as 42% (fig. 4).