CN111235092B - Blastocyst culture solution and preparation method thereof - Google Patents

Blastocyst culture solution and preparation method thereof Download PDF

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CN111235092B
CN111235092B CN202010151317.0A CN202010151317A CN111235092B CN 111235092 B CN111235092 B CN 111235092B CN 202010151317 A CN202010151317 A CN 202010151317A CN 111235092 B CN111235092 B CN 111235092B
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胡威
姜永存
曾玉洁
赵衡斌
陈烨
周星宇
吴秀芝
吴文林
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Abstract

The invention relates to a cyst embryo culture solution and a preparation method thereof. The blastocyst culture solution comprises a basic culture solution and additives; the additives comprise the following components in terms of addition amount in the basic culture solution: melatonin 0.01-100 nmol/L, coenzyme Q100.5mg/L-2.0 mg/L, IGF-20.1 nmol/L-300 nmol/L; the basic culture solution comprises inorganic salt buffer solution, glucose, sodium lactate, sodium pyruvate, amino acid, alanyl-glutamine, choline chloride, folic acid, inositol, nicotinamide, pantothenic acid, pyridoxine, riboflavin and thiamine. The blastocyst culture solution can effectively improve the blastocyst formation rate and the blastocyst quality.

Description

Blastocyst culture solution and preparation method thereof
Technical Field
The invention relates to the field of assisted reproduction, in particular to a cyst embryo culture solution and a preparation method thereof.
Background
Assisted Reproduction Technology (ART) is a short term for human Assisted reproduction Technology, and refers to a Technology for making a sterile couple pregnant by medical assistance, and includes two major types of technologies, namely Artificial Insemination (AI), In Vitro Fertilization and Embryo Transfer (IVF-ET), and derivatives thereof. In the in vitro fertilization-embryo transfer (IVF-ET), the ova and sperms of both patients are taken out, fertilized in vitro, and the fertilized ova or embryos are transferred back to the mother uterus to develop into fetuses. The aim of in vitro culture of human embryos is to keep the embryos continuously alive and enable the embryos to reach the optimal development stage within a certain time, and an optimized culture system can support the normal division of the viable embryos in vitro and develop the viable embryos into high-quality cleavage-stage embryos and blastulas.
IVF in vitro cultures are generally divided into two culture fluid series: single culture broth and sequential culture broth. The development of the single culture solution is based on the concept of 'allowing the embryo to select by itself', so that the embryo can automatically pick up and discard the required nutrients from the culture solution in the growth and development process. The research and development of the sequential culture solution are completed by researching different requirements of different development stages on nutrient substances and natural physiological environments of the nutrient substances in the reproductive tract based on the concept of 'approaching nature'. Since the culture from fertilized egg to blastocyst is a dynamic process, not only the division and development of fertilized egg is dynamic, but also the displacement of embryo from oviduct to uterus is a dynamic process, the in vitro culture of IVF should follow the concept of "close to nature", and different components of embryo culture solution are adopted at different embryo development stages.
The media used in IVF has undergone some evolution over the last 30 years. However, most are still aimed at improving the embryo culture medium's constituent-carbohydrate energy source, amino acids and salts/buffers etc., which make up the core or bulk of the medium. Over the last 10 to 15 years, there has been increasing interest in incorporating different biologically active substances and growth factors into the culture medium.
In vitro fertilization-embryo transfer (IVF-ET), the quality of the oocyte is critical for obtaining good offspring embryos and mitochondria are one of the core factors that determine the quality of the oocyte. The number, distribution and mtDNA copy number of mitochondria play a decisive role in the developmental maturity of oocytes and the development potential of embryos after fertilization. Mitochondria are organelles for producing energy in human cells, and are continuously damaged by oxygen toxicity while utilizing oxygen molecules, and when mitochondria generate energy for dividing oocytes, chromosomes are equally separated, and simultaneously a large amount of free radicals are released, and when the damage of mitochondria exceeds a certain limit, cells can be aged and die. At present, more and more researches show that the coenzyme Q10 and the melatonin have the effects of resisting oxidation, resisting aging, eliminating free radicals and the like, and play an important role in the process of oocyte generation and embryo development.
At present, most of the conventional blastocyst culture solutions clinically used are modified human oviduct salt solutions (HTF) which take sodium bicarbonate as a buffering agent, cannot effectively ensure the blastocyst formation of the embryo, and particularly have poor effect on the blastocyst development of the early embryo.
Disclosure of Invention
Based on the above, one of the objectives of the present invention is to provide a blastocyst culture solution, which can effectively improve the blastocyst formation rate and the quality of blastocysts.
The specific technical scheme is as follows:
a blastocyst culture solution comprising a basal medium and additives; the additives comprise the following components in terms of addition amount in the basic culture solution:
melatonin 0.01-100 nmol/L
Coenzyme Q100.5mg/L-2.0 mg/L
IGF-2 0.1nmol/L～300nmol/L；
The basic culture solution comprises inorganic salt buffer solution, glucose, sodium lactate, sodium pyruvate, amino acid, alanyl-glutamine, choline chloride, folic acid, inositol, nicotinamide, pantothenic acid, pyridoxine, riboflavin and thiamine;
the molar ratio of glucose, sodium lactate, sodium pyruvate, amino acid, alanyl-glutamine, choline chloride, folic acid, inositol, nicotinamide, pantothenic acid, pyridoxine, riboflavin and thiamine is 0.1-6: 1-22: 0.01-1.1: 0.19 to 7.2: 0.1-2.2: 0.001 to 0.014: 0.001 to 0.005: 0.001-0.02: 0.001-0.015: 0.001-0.009: 0.001-0.009: 0.0001 to 0.0006: 0.001 to 0.006.
Compared with the prior art, the invention has the following beneficial effects:
the blastocyst culture solution of the invention adds melatonin, coenzyme Q10 and IGF-2 into the basic culture solution, can effectively promote mitosis and cell development of embryo cells in the cleavage stage, and improves the blastocyst formation rate and the quality of blastocysts.
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FIG. 1 shows four-stage and five-stage blastocysts of mice cultured with the blastocyst medium of the present invention.
Detailed Description
In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It is to be understood that the experimental procedures in the following examples, where specific conditions are not noted, are generally in accordance with conventional conditions, or with conditions recommended by the manufacturer. The various reagents used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The embodiment provides a blastocyst culture solution, which comprises a basic culture solution and additives; the additives comprise the following components in terms of addition amount in the basic culture solution:
melatonin 0.01-100 nmol/L
Coenzyme Q100.5mg/L-2.0 mg/L
IGF-2 0.1nmol/L～300nmol/L；
The basic culture solution comprises inorganic salt buffer solution, glucose, sodium lactate, sodium pyruvate, amino acids, alanyl-glutamine, choline chloride, folic acid, inositol, nicotinamide, pantothenic acid, pyridoxine (pyridoxine), riboflavin, and thiamine (thiamine);
the molar ratio of glucose, sodium lactate, sodium pyruvate, amino acid, alanyl-glutamine, choline chloride, folic acid, inositol, nicotinamide, pantothenic acid, pyridoxine, riboflavin and thiamine is 0.1-6: 1-22: 0.01-1.1: 0.19 to 7.2: 0.1-2.2: 0.001 to 0.014: 0.001 to 0.005: 0.001-0.02: 0.001-0.015: 0.001-0.009: 0.001-0.009: 0.0001 to 0.0006: 0.001 to 0.006.
In some embodiments, the additives include the following components in terms of the amount added in the basic culture solution:
0.1 nmol/L-10 nmol/L of melatonin;
coenzyme Q100.75mg/L-1.5 mg/L;
IGF-2 1nmol/L～100nmol/L。
in some embodiments, the additives include the following components in terms of the amount added in the basic culture solution:
melatonin is 0.1 nmol/L-5 nmol/L;
coenzyme Q100.75mg/L-1.5 mg/L;
IGF-2 20nmol/L～80nmol/L。
the addition amount of the additive is preferably as follows:
melatonin is 0.5 nmol/L-1.5 nmol/L;
coenzyme Q100.85mg/L-1.15 mg/L;
IGF-2 40nmol/L～60nmol/L。
the addition amount of the additive is preferably as follows:
melatonin is 0.8 nmol/L-1.2 nmol/L;
coenzyme Q100.95mg/L-1.05 mg/L;
IGF-2 45nmol/L～55nmol/L。
further preferably, the additive is added in an amount of:
melatonin 1 nmol/L;
coenzyme Q101 mg/L;
IGF-2 50nmol/L。
in some of these embodiments, the molar ratio of glucose, sodium lactate, sodium pyruvate, amino acids, alanyl-glutamine, choline chloride, folic acid, inositol, nicotinamide, pantothenic acid, pyridoxine, riboflavin, and thiamine is 2.5-3.5: 5-7: 0.05-0.12: 3.5-5: 0.3-0.8: 0.005-0.008: 0.0015 to 0.003: 0.008-0.013: 0.007-0.009: 0.003 to 0.005: 0.004-0.006: 0.0002 to 0.0003: 0.002-0.004.
In some of these embodiments, the inorganic salt buffer comprises sodium chloride, potassium chloride, sodium dihydrogen phosphate, magnesium sulfate, sodium bicarbonate, and calcium chloride.
In some embodiments, the molar ratio of the sodium chloride, the potassium chloride, the sodium dihydrogen phosphate, the magnesium sulfate, the sodium bicarbonate and the calcium chloride is 80-110: 3.5-8: 0.05-1.2: 0.2-3.2: 10-35: 0.5 to 3; preferably, the molar ratio of the sodium chloride to the potassium chloride to the sodium dihydrogen phosphate to the magnesium sulfate to the sodium bicarbonate to the calcium chloride is 85-95: 5-6: 0.2-0.4: 0.8-1.2: 20-30: 1.5 to 2.
In some embodiments, the amino acids include non-essential amino acids and essential amino acids;
in some embodiments, the basic culture solution further comprises, based on the amount of the additive in the basic culture solution:
1-10 g/L of human serum albumin; and/or the presence of a gas in the gas,
20-100 mg/L of bactericide.
In some of these embodiments, the antimicrobial agent is at least one of penicillin, streptomycin, and gentamicin; the addition amount of the bactericide in the basic culture solution is 40-60 mg/L; the addition amount of the human serum albumin is 4.5-5.5 g/L.
In some of these embodiments, the nonessential amino acids include alanine, aspartic acid, asparagine, glutamic acid, glycine, proline and serine; the mol ratio of the alanine to the aspartic acid to the asparagine to the glutamic acid to the glycine to the proline to the serine is 0.01-0.16: 0.01-0.16: 0.01-0.16: 0.01-0.16: 0.01-0.16: 0.01-0.16: 0.01-0.16, and the mol ratio of alanine, aspartic acid, asparagine, glutamic acid, glycine, proline and serine is preferably 0.09-0.11: 0.09-0.11: 0.09-0.11: 0.09-0.11: 0.09-0.11: 0.09-0.11: 0.09 to 0.11.
In some of these embodiments, the essential amino acids include arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine;
the mol ratio of arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine and valine is 0.1-1: 0.01-0.15: 0.05-0.6: 0.1-1: 0.1-1: 0.1-1: 0.01-0.15: 0.05-0.6: 0.1-1: 0.01-0.1: 0.05-0.6: 0.1 to 1;
preferably, the molar ratio of arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine and valine is 0.5-0.7: 0.09-0.11: 0.1-0.3: 0.3-0.5: 0.3-0.5: 0.3-0.5: 0.09-0.11: 0.1-0.3: 0.3-0.5: 0.04-0.06: 0.1-0.3: 0.3 to 0.5.
The embodiment also provides a preparation method of the cyst embryo culture solution, which comprises the following steps:
melatonin, coenzyme Q10, IGF-2 and the components of the basic culture solution were added to water and sterile filtered.
Preferably, the water is ultrapure water.
When the blastocyst culture solution further comprises human serum albumin, the human serum albumin can be added into water together with other raw materials; or the human serum albumin can be added into the prepared culture solution according to the corresponding proportion when the human serum albumin is ready to be used.
The present invention will be described in further detail with reference to specific examples.
Example 1A culture solution for blastocysts
Figure BDA0002402535720000061
Figure BDA0002402535720000071
The preparation method comprises the following steps:
1) the preparation method comprises the steps of preparing the raw materials under hundred-grade dust-free workshop conditions, accurately weighing the raw materials in sequence according to the respective proportions, adding the raw materials into ultrapure water, and mixing to obtain a solution I.
2) Filtering and sterilizing the solution I prepared in the step 1) by using a 0.2um filter membrane, and performing aseptic subpackage to obtain a cyst embryonic stage culture solution. Wherein, Human Serum Albumin (HSA) can be added into the solution I in advance, or HSA is not added, and HSA is added and mixed according to a corresponding proportion when the solution is used.
Example 2A culture solution for blastocysts
Figure BDA0002402535720000072
Figure BDA0002402535720000081
The preparation method comprises the following steps:
1) the preparation method comprises the steps of preparing the raw materials under hundred-grade dust-free workshop conditions, accurately weighing the raw materials in sequence according to the respective proportions, adding the raw materials into ultrapure water, and mixing to obtain a solution I.
2) Filtering and sterilizing the solution I prepared in the step 1) by using a 0.2um filter membrane, and performing aseptic subpackage to obtain a cyst embryonic stage culture solution. Wherein, Human Serum Albumin (HSA) can be added into the solution I in advance, or HSA is not added, and HSA is added and mixed according to a corresponding proportion when the solution is used.
Example 3A culture solution for blastocysts
Figure BDA0002402535720000091
Figure BDA0002402535720000101
Example 4A culture solution for blastocysts
Figure BDA0002402535720000102
Figure BDA0002402535720000111
The preparation method comprises the following steps:
1) the preparation method comprises the steps of preparing the raw materials under hundred-grade dust-free workshop conditions, accurately weighing the raw materials in sequence according to the respective proportions, adding the raw materials into ultrapure water, and mixing to obtain a solution I.
2) Filtering and sterilizing the solution I prepared in the step 1) by using a 0.2um filter membrane, and performing aseptic subpackage to obtain a cyst embryonic stage culture solution. Wherein, Human Serum Albumin (HSA) can be added into the solution I in advance, or HSA is not added, and HSA is added and mixed according to a corresponding proportion when the solution is used.
Comparative example 1A cyst embryo culture solution
Figure BDA0002402535720000112
Figure BDA0002402535720000121
The preparation method is the same as example 1.
Comparative example 2
Figure BDA0002402535720000122
Figure BDA0002402535720000131
The preparation method is the same as example 1.
Comparative example 3
Figure BDA0002402535720000141
Figure BDA0002402535720000151
The preparation method is the same as example 1.
Comparative example 4
The comparison example provides a blastocyst culture solution, which is prepared by mixing the existing commercial finished blastocyst basic culture solution (source: G-2.5, Denmark vitrolite company), coenzyme Q10(1mg/L), melatonin (1nmol/L) and Igf-2(50nmol/L) as raw materials.
Effect test
Selecting the blastocyst culture solutions of the examples and the comparative examples, selecting embryos of high-quality mice scored as embryos on the third day (the source is ICR strain SPF level mice purchased from Beijing Wintolite laboratory animal technology Co., Ltd.), respectively placing the embryos in the cleavage period into the blastocyst culture solutions, continuously culturing in an incubator with the carbon dioxide concentration of 6% -oxygen concentration of 5% at 37 ℃, observing the development condition of the embryos, and calculating the blastocyst formation rate and the high-quality blastocyst formation rate on the fifth day.
The grading standard of the embryo adopts a Garnder grading system, the excellent embryo standard of the fertilized eggs on the third day is that the number of cells is 7-10 blastomere cells, the shapes and the sizes of the blastomere cells are normal and uniform or slightly uneven, and the proportion of non-nuclear fragments or fragments is smaller than the volume of cell masses 1/3; and a blastocyst cavity is formed on the fifth day to be a blastocyst, and the standard of the high-quality blastocyst is that the blastocyst cavity is expanded to more than 3 and 4 stages.
The development of the blastocyst is divided into six stages.
Stage 1: the early stage is provided with a cavity blastula, and the cavity of the blastula is smaller than 1/2 of the total volume of the embryos;
stage 2: the blastocoel volume is greater than or equal to 1/2 of the total embryo volume;
stage 3: fully expanding the blastocyst, the blastocyst cavity fully occupying the total volume of the embryo;
stage 4: expanding the blastocyst, wherein the blastocyst cavity is completely filled with the embryo, the total volume of the embryo is increased, and the zona pellucida is thinned;
stage 5: a blastocyst that is hatching, a portion of which escapes from the zona pellucida;
stage 6: the blastocyst is completely escaped from the zona pellucida.
The results are shown in Table 1 (FIG. 1 shows four-stage and five-stage blastocysts of mice cultured with the blastocyst culture medium of the present invention):
TABLE 1
Figure BDA0002402535720000161
The results in table 1 show that the blastocyst culture solution of the present invention, in which melatonin, coenzyme Q10 and IGF-2 are added to the basic culture solution of the present invention, has excellent effects of inhibiting and scavenging oxygen radicals in vivo, and simultaneously, can promote mitosis and cell development of blastocyst cells in the cleavage stage, and improve the blastocyst formation rate and blastocyst quality. From the results of comparative examples 1 to 4, it is clear that the above effects are achieved by the mutual cooperation and promotion between melatonin, coenzyme Q10, IGF-2 in the blastocyst culture solution of the present invention and the basic culture solution of the present invention.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (8)

1. A blastocyst culture solution, which comprises a basic culture solution and additives; the additives comprise the following components in terms of addition amount in the basic culture solution:
melatonin is 0.8 nmol/L-1.2 nmol/L;
100.95 mg/L-1.05 mg/L of coenzyme Q;
IGF-2 45 nmol/L~55nmol/L；
the basic culture solution comprises inorganic salt buffer solution, glucose, sodium lactate, sodium pyruvate, amino acid, alanyl-glutamine, choline chloride, folic acid, inositol, nicotinamide, pantothenic acid, pyridoxine, riboflavin and thiamine;
the inorganic salt buffer solution comprises the following components in a molar ratio of 80-110: 3.5-8: 0.05-1.2: 0.2-3.2: 10-35: 0.5-3 parts of sodium chloride, potassium chloride, sodium dihydrogen phosphate, magnesium sulfate, sodium bicarbonate and calcium chloride;
the amino acids include non-essential amino acids and essential amino acids;
the nonessential amino acid comprises the following components in a molar ratio of 0.01-0.16: 0.01-0.16: 0.01-0.16: 0.01-0.16: 0.01-0.16: 0.01-0.16: 0.01-0.16 of alanine, aspartic acid, asparagine, glutamic acid, glycine, proline and serine;
the essential amino acid comprises the following components in a molar ratio of 0.1-1: 0.01-0.15: 0.05-0.6: 0.1-1: 0.1-1: 0.1-1: 0.01-0.15: 0.05-0.6: 0.1-1: 0.01-0.1: 0.05-0.6: 0.1-1 of arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine;
the molar ratio of glucose, sodium lactate, sodium pyruvate, amino acid, alanyl-glutamine, choline chloride, folic acid, inositol, nicotinamide, pantothenic acid, pyridoxine, riboflavin and thiamine is 2.5-3.5: 5-7: 0.05-0.12: 3.5-5: 0.3-0.8: 0.005-0.008: 0.0015 to 0.003: 0.008-0.013: 0.007-0.009: 0.003 to 0.005: 0.004-0.006: 0.0002 to 0.0003: 0.002-0.004.
2. The blastocyst medium according to claim 1, wherein the additives include the following components in terms of the amount added to the basic medium:
melatonin 1 nmol/L;
coenzyme Q101 mg/L;
IGF-2 50nmol/L。
3. the blastocyst broth of claim 1, wherein the molar ratio of glucose, sodium lactate, sodium pyruvate, amino acids, alanyl-glutamine, choline chloride, folic acid, inositol, nicotinamide, pantothenic acid, pyridoxine, riboflavin, and thiamine is 3.15: 5.87: 0.1: 4.15: 0.5: 0.0072: 0.0023: 0.01: 0.0082: 0.0042: 0.0049: 0.00027: 0.00296.
4. the blastocyst culture solution of claim 1, wherein the molar ratio of sodium chloride, potassium chloride, sodium dihydrogen phosphate, magnesium sulfate, sodium bicarbonate and calcium chloride is 85-95: 5-6: 0.2-0.4: 0.8-1.2: 20-30: 1.5 to 2.
5. The blastocyst medium according to any one of claims 1 to 4, wherein the basic medium further comprises, based on the amount of the medium added to the basic medium:
1-10 g/L of human serum albumin; and/or the presence of a gas in the gas,
20-100 mg/L of bactericide.
6. The blastocyst medium according to claim 5, wherein the bactericide is at least one of penicillin, streptomycin and gentamicin; the addition amount of the bactericide in the basic culture solution is 40-60 mg/L; the addition amount of the human serum albumin is 4.5-5.5 g/L.
7. The blastocyst culture solution of claim 1, wherein the molar ratio of alanine, aspartic acid, asparagine, glutamic acid, glycine, proline and serine is 0.09-0.11: 0.09-0.11: 0.09-0.11: 0.09-0.11: 0.09-0.11: 0.09-0.11: 0.09 to 0.11.
8. The method for preparing a blastocyst culture solution according to any one of claims 1 to 7, comprising the steps of:
adding melatonin, coenzyme Q10, IGF-2 and the components in the basic culture solution into water according to the addition amount, and filtering and sterilizing.
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