CN115011552A - Whole-process culture solution for prenatal embryo development and preparation method thereof - Google Patents

Whole-process culture solution for prenatal embryo development and preparation method thereof Download PDF

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CN115011552A
CN115011552A CN202210768289.6A CN202210768289A CN115011552A CN 115011552 A CN115011552 A CN 115011552A CN 202210768289 A CN202210768289 A CN 202210768289A CN 115011552 A CN115011552 A CN 115011552A
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冯怀亮
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Reprobiotech Corp China Ltd
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Abstract

The invention provides a whole-course culture solution for prenatal embryo development and a preparation method thereof, belonging to the technical field of assisted reproduction. The whole-course culture solution comprises the following effective components: glycine, threonine, leucine, isoleucine, lysine, alanine, aspartic acid, asparagine, glutamic acid, glutamine, tyrosine, methionine, histidine, phenylalanine, tryptophan, valine; vitamin B 1 Vitamin B 12 Folic acid, panthenol, inositol; taurine, hyaluronic acid, EDTA. The whole-process culture solution for the pre-implantation embryo development provided by the invention is used for culturing sperms, ova and fertilized eggs, and can improve the blastocyst formation rate, the high-quality blastocyst rate and the pregnancy rate.

Description

Whole-process culture solution for prenatal embryo development and preparation method thereof
Technical Field
The invention relates to the technical field of assisted reproduction, in particular to a whole-course culture solution for prenatal embryo development and a preparation method thereof.
Background
The assisted reproduction technology refers to a technology for making sterile couples pregnant by adopting medical auxiliary means, and comprises two major technologies of artificial insemination, in vitro fertilization-embryo transplantation and derivation technology. Artificial insemination refers to a method of artificially injecting semen into a female to make it pregnant instead of the sexual intercourse route. The in vitro fertilization-embryo transfer technology and various derivative technologies thereof refer to a technology that an ovum is taken out from a female body, cultured in a vessel, added with sperm processed by the technology, continuously cultured after the ovum is fertilized, transferred to a uterus for implantation when an early embryo is formed, and developed into a fetus until delivery. Thus, the ivf-embryo transfer technique may involve the in vitro pre-implantation embryo development culture of fertilized eggs.
The pre-implantation embryo development process mainly refers to the development process of 1-6 days after the combination of sperms and ova. This process, which is a dynamic process, is a fertilized egg cell that develops into a blastocyst. During the pre-implantation embryo development culture process, culture components required by different stages of fertilized egg development are different. Therefore, the culture solution used in this stage can significantly affect the incidence of blastocysts and the quality of blastocyst development.
Disclosure of Invention
The invention aims to provide a whole-course culture solution for prenatal embryo development and a preparation method thereof, which are used for improving the blastocyst formation rate and the blastocyst quality.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a whole-process culture solution for pre-implantation embryo development, which comprises the following effective components: glycine, threonine, leucine, isoleucine, lysine, alanine, aspartic acid, asparagine, glutamic acid, glutamine, tyrosine, methionine, histidine, phenylalanine, tryptophan, valine; vitamin B 1 Vitamin B 12 Folic acid, panthenol, inositol; taurine, hyaluronic acid, EDTA.
Preferably, the concentration of glycine, threonine, leucine, isoleucine, lysine, alanine, aspartic acid, asparagine, glutamic acid, glutamine, tyrosine, methionine, histidine, phenylalanine, tryptophan and valine in the whole culture solution is independently 0.1-0.7 mmol/L.
Preferably, the vitamin B 1 Vitamin B 12 The concentrations of folic acid, panthenol and inositol in the whole culture solution are independently 0.001-0.02 mmol/L.
Preferably, the concentration of the taurine in the whole culture solution is 0.01-0.05 mmol/L; the concentration of the hyaluronic acid in the whole culture solution is 0.01-0.1 mmol/L; the concentration of the EDTA in the whole culture solution is 0.01-0.1 mmol/L.
The invention also provides a preparation method of the whole process culture solution for the pre-implantation embryo development, which comprises the following steps:
(1) mixing vitamin B 1 Vitamin B 12 Folic acid, panthenol, inositol; dissolving taurine, hyaluronic acid and EDTA in a salt solution, and adjusting the pH value to 7.1-7.8 to obtain a weakly alkaline culture solution;
(2) mixing the alkalescent culture solution with glycine, threonine, leucine, isoleucine, lysine, alanine, aspartic acid, asparagine, glutamic acid, glutamine, tyrosine, methionine, histidine, phenylalanine, tryptophan and valine to obtain a whole-course culture solution.
Preferably, the salt solution comprises the following components: 90-98 mmol/L of sodium chloride, 3-5 mmol/L of potassium chloride, 0.1-0.2 mmol/L of magnesium sulfate, 0.5-0.7 mmol/L of monopotassium phosphate, 0.1-0.5 mmol/L of sodium dihydrogen phosphate, 1.5-2.5 mmol/L of calcium chloride, 15-25 mmol/L of sodium bicarbonate, 0.3-0.9 mmol/L of glucose, 0.1-0.2 mmol/L of sodium pyruvate, 1-3 g/L of sodium lactate, 0.1-0.2 mmol/L of monopotassium phosphate, 3.25-4.75 mg/L of phenol red, 8-10 mg/L of gentamycin, 8-12 v/L of human serum albumin and the balance of water.
The whole-process culture solution for the pre-implantation embryo development is used for culturing sperms, ova and fertilized ova, can ensure higher fertilization rate and cleavage rate, and improve the blastocyst formation rate, the high-quality blastocyst rate and the pregnancy rate.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparing a salt solution: the following materials were weighed at a concentration of 95mmol/L sodium chloride, 4mmol/L potassium chloride, 0.15mmol/L magnesium sulfate, 0.6mmol/L potassium dihydrogen phosphate, 0.3mmol/L sodium dihydrogen phosphate, 2.0mmol/L calcium chloride, 20mmol/L sodium bicarbonate, 0.6mmol/L glucose, 0.15mmol/L sodium pyruvate, 2g/L sodium lactate, 0.15mmol/L potassium dihydrogen phosphate, 4.0mg/L phenol red, 9mg/L gentamicin, 10v/v human serum albumin, and the balance water. Dissolving the materials except phenol red, gentamicin and human serum albumin in water (ultrapure water), adding phenol red and gentamicin, and adding 5% CO at 37 deg.C 2 The incubator (2) was equilibrated for 12 hours, and the pH was adjusted to 7.2. Human serum albumin was then added to give a salt solution.
Preparing a whole-course culture solution: the active ingredients were precisely weighed at concentrations of 0.4mmol/L for each of glycine, threonine, leucine, isoleucine, lysine, alanine, aspartic acid, asparagine, glutamic acid, glutamine, tyrosine, methionine, histidine, phenylalanine, tryptophan, and valine. The concentrations of vitamin B1, vitamin B12, folic acid, panthenol and inositol are all 0.01 mmol/L. Taurine 0.03mmol/L, hyaluronic acid 0.05mmol/L, EDTA 0.05 mmol/L.
Mixing vitamin B 1 Vitamin B 12 Dissolving folic acid, panthenol, inositol, taurine, hyaluronic acid, and EDTA in the above well-matched salt solution, and adding 5% CO at 37 deg.C 2 After 3 hours of equilibration in the incubator, the solution pH was measured and adjusted to 7.5 to obtain a weakly alkaline culture solution. Adding glycine, threonine, leucine, isoleucine, lysine, alanine, aspartic acid, asparagine, glutamic acid, glutamine, tyrosine, methionine, histidine, phenylalanine, tryptophan and valine into a weakly alkaline culture solution, and uniformly mixing to obtain a whole-course culture solution. The whole process culture solution is filtered through a 0.2 micron filter membrane, and sampling detection is carried out.
A.pH is 7.5 at 20-30 ℃, and is 7.3-7.8;
B. the osmotic pressure is 260-280 mOsm/Kg;
C. endotoxin is less than 0.15 EU/mL.
A culture test was carried out using the whole-course culture solution prepared in example 1 and a conventional culture solution (CooperSurgical one step culture medium).
The results are shown in Table 1.
TABLE 1 Effect of different whole-course culture solutions on fertilization and embryo development
Figure BDA0003726445620000031
Figure BDA0003726445620000041
Note: b. indicating significant difference (p < 0.5); a. indicating that the difference was not significant (p > 0.5).
The test results show that the fertilization rate and the cleavage rate difference of the whole process culture solution provided by the invention are not obvious compared with other cultures, but the whole process culture solution provided by the invention obviously improves the blastocyst formation rate, the high-quality blastocyst rate and the fresh cycle clinical pregnancy rate. Meanwhile, the whole-course culture solution containing amino acids with different concentrations also has obvious differences in blastocyst formation rate, high-quality blastocyst rate and fresh cycle clinical pregnancy rate, and the concentration of the amino acids optimized by the method has better culture effect.
Example 2
Preparing a salt solution: the following were weighed as follows, sodium chloride 90mmol/L, potassium chloride 3mmol/L, magnesium sulfate 0.2mmol/L, potassium dihydrogen phosphate 0.5mmol/L, sodium dihydrogen phosphate 0.5mmol/L, calcium chloride 1.5mmol/L, sodium bicarbonate 25mmol/L, glucose 0.3mmol/L, sodium pyruvate 0.1mmol/L, sodium lactate 1g/L, potassium dihydrogen phosphate 0.2mmol/L, phenol red 4.0mg/L, gentamicin 9mg/L, human serum albumin 10 v/v%, and balance water. Dissolving the materials except phenol red, gentamicin and human serum albumin in water (ultrapure water), adding phenol red and gentamicin, and adding 5% CO at 37 deg.C 2 The incubator (2) was equilibrated for 12 hours, and the pH was adjusted to 7.2. Then human serum albumin was added to obtain a salt solution.
Preparing a whole-course culture solution: the active ingredients were precisely weighed at concentrations of 0.1mmol/L for each of glycine, threonine, leucine, isoleucine, lysine, alanine, aspartic acid, asparagine, glutamic acid, glutamine, tyrosine, methionine, histidine, phenylalanine, tryptophan, and valine. Vitamin B 1 Vitamin B 12 The concentrations of folic acid, panthenol and inositol were all 0.005 mmol/L. Taurine 0.05mmol/L andthe concentration of the hyaluronic acid and the concentration of EDTA were respectively 0.01mmol/L and 0.05 mmol/L.
Mixing vitamin B 1 Vitamin B 12 Dissolving folic acid, panthenol, inositol, taurine, hyaluronic acid, and EDTA in the above well-matched salt solution, and adding 5% CO at 37 deg.C 2 The incubator (2) was equilibrated for 3 hours, and then the solution pH was measured and adjusted to 7.3 to obtain a weakly alkaline culture solution. Adding glycine, threonine, leucine, isoleucine, lysine, alanine, aspartic acid, asparagine, glutamic acid, glutamine, tyrosine, methionine, histidine, phenylalanine, tryptophan and valine into a weakly alkaline culture solution, and uniformly mixing to obtain a whole-course culture solution. The whole process culture solution is filtered through a 0.2 micron filter membrane, and sampling detection is carried out.
A.pH is 7.4 at 20-30 ℃, and is 7.3-7.8;
D. the osmotic pressure is 260-280 mOsm/Kg;
E. endotoxin is less than 0.15 EU/mL.
Example 3
Preparing a salt solution: the following materials, sodium chloride 98mmol/L, potassium chloride 5mmol/L, magnesium sulfate 0.1mmol/L, potassium dihydrogen phosphate 0.7mmol/L, sodium dihydrogen phosphate 0.1mmol/L, calcium chloride 2.5mmol/L, sodium bicarbonate 15mmol/L, glucose 0.9mmol/L, sodium pyruvate 0.2mmol/L, sodium lactate 3g/L, potassium dihydrogen phosphate 0.1mmol/L, phenol red 4.0mg/L, gentamicin 10mg/L, human serum albumin 12 v/v%, and water in balance were weighed. Dissolving the materials except phenol red, gentamicin, and human serum albumin in water (ultrapure water), adding phenol red and gentamicin, and adding 5% CO at 37 deg.C 2 The incubator (2) was equilibrated for 12 hours, and the pH was adjusted to 7.2. Human serum albumin was then added to give a salt solution.
Preparing a whole-course culture solution: the active ingredients were precisely weighed at concentrations of 0.7mmol/L for each of glycine, threonine, leucine, isoleucine, lysine, alanine, aspartic acid, asparagine, glutamic acid, glutamine, tyrosine, methionine, histidine, phenylalanine, tryptophan, and valine. Vitamin B 1 Vitamin B 12 Folic acid, panthenol and inositolThe concentration of (A) was 0.02 mmol/L. Taurine 0.01mmol/L, hyaluronic acid 0.1mmol/L, EDTA 0.05 mmol/L.
Mixing vitamin B 1 Vitamin B 12 Dissolving folic acid, panthenol, inositol, taurine, hyaluronic acid, and EDTA in the above well-matched salt solution, and adding 5% CO at 37 deg.C 2 After equilibrating in the incubator for 3 hours, the pH of the solution was measured and adjusted to 7.8, yielding a weakly alkaline culture solution. Adding glycine, threonine, leucine, isoleucine, lysine, alanine, aspartic acid, asparagine, glutamic acid, glutamine, tyrosine, methionine, histidine, phenylalanine, tryptophan and valine into a weakly alkaline culture solution, and uniformly mixing to obtain a whole-course culture solution. The whole process culture solution is filtered through a 0.2 micron filter membrane, and sampling detection is carried out.
A.pH is 7.6 at 20-30 ℃, and is 7.3-7.8;
F. the osmotic pressure is 260-280 mOsm/Kg;
G. endotoxin is less than 0.15 EU/mL.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (6)

1. A whole-course culture solution for pre-implantation embryo development is characterized by comprising the following effective components: glycine, threonine, leucine, isoleucine, lysine, alanine, aspartic acid, asparagine, glutamic acid, glutamine, tyrosine, methionine, histidine, phenylalanine, tryptophan, valine; vitamin B 1 Vitamin B 12 Folic acid, panthenol, inositol; taurine, hyaluronic acid, EDTA.
2. The whole process culture solution of claim 1, wherein the concentrations of glycine, threonine, leucine, isoleucine, lysine, alanine, aspartic acid, asparagine, glutamic acid, glutamine, tyrosine, methionine, histidine, phenylalanine, tryptophan, and valine in the whole process culture solution are independently 0.1 to 0.7 mmol/L.
3. The whole process culture solution of claim 2, wherein the vitamin B is vitamin B 1 Vitamin B 12 The concentrations of folic acid, panthenol and inositol in the whole culture solution are independently 0.001-0.02 mmol/L.
4. The whole course culture solution of claim 3, wherein the concentration of taurine in the whole course culture solution is 0.01 to 0.05 mmol/L; the concentration of the hyaluronic acid in the whole culture solution is 0.01-0.1 mmol/L; the concentration of the EDTA in the whole culture solution is 0.01-0.1 mmol/L.
5. A method for preparing the whole-process culture solution for the development of the preimplantation embryo as claimed in any one of claims 1 to 4, which comprises the following steps:
(1) mixing vitamin B 1 Vitamin B 12 Folic acid, panthenol, inositol; dissolving taurine, hyaluronic acid and EDTA in a salt solution, and adjusting the pH value to 7.1-7.8 to obtain a weakly alkaline culture solution;
(2) mixing the alkalescent culture solution with glycine, threonine, leucine, isoleucine, lysine, alanine, aspartic acid, asparagine, glutamic acid, glutamine, tyrosine, methionine, histidine, phenylalanine, tryptophan and valine to obtain a whole-course culture solution.
6. The method of claim 5, wherein the salt solution comprises the following ingredients: 90-98 mmol/L of sodium chloride, 3-5 mmol/L of potassium chloride, 0.1-0.2 mmol/L of magnesium sulfate, 0.5-0.7 mmol/L of monopotassium phosphate, 0.1-0.5 mmol/L of sodium dihydrogen phosphate, 1.5-2.5 mmol/L of calcium chloride, 15-25 mmol/L of sodium bicarbonate, 0.3-0.9 mmol/L of glucose, 0.1-0.2 mmol/L of sodium pyruvate, 1-3 g/L of sodium lactate, 0.1-0.2 mmol/L of monopotassium phosphate, 3.25-4.75 mg/L of phenol red, 8-10 mg/L of gentamycin, 8-12 v/L of human serum albumin and the balance of water.
CN202210768289.6A 2022-07-01 2022-07-01 Whole-process culture solution for prenatal embryo development and preparation method thereof Pending CN115011552A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108624551A (en) * 2018-06-05 2018-10-09 瑞柏生物（中国）股份有限公司 A kind of fertilization culture solution and preparation method thereof
CN108753690A (en) * 2018-06-05 2018-11-06 瑞柏生物（中国）股份有限公司 One step culture solution of one kind and preparation method thereof
US20190194621A1 (en) * 2016-09-08 2019-06-27 Kyowa Hakko Bio Co., Ltd. Medium for culturing stem cells, method for culturing stem cells, growth promoter for stem cells, and cell composition and method for producing same
CN110669724A (en) * 2019-11-08 2020-01-10 广州达瑞生殖技术有限公司 Saccharum embryo culture solution and preparation method thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190194621A1 (en) * 2016-09-08 2019-06-27 Kyowa Hakko Bio Co., Ltd. Medium for culturing stem cells, method for culturing stem cells, growth promoter for stem cells, and cell composition and method for producing same
CN108624551A (en) * 2018-06-05 2018-10-09 瑞柏生物（中国）股份有限公司 A kind of fertilization culture solution and preparation method thereof
CN108753690A (en) * 2018-06-05 2018-11-06 瑞柏生物（中国）股份有限公司 One step culture solution of one kind and preparation method thereof
CN110669724A (en) * 2019-11-08 2020-01-10 广州达瑞生殖技术有限公司 Saccharum embryo culture solution and preparation method thereof

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Application publication date: 20220906

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