Culture solution for improving embryo health degree and embryo culture method

Abstract

The invention relates to the technical field of embryonic cell culture, in particular to a culture solution for improving embryo health degree, which is prepared by adding antioxidant active matter haematococcus pluvialis with specific concentration and cistanche deserticola extract on the basis of the existing embryo basic culture solution, when the culture solution is used for culturing embryonic cells, the blastocyst formation rate of 1-cell embryos can be increased, and a better in-vitro research model is provided for subsequently transplanting fertilized ova back to maternal uterus for development.

Description

Technical Field

[0001] The present invention relates to the technical field of embryo cell culture, and more specifically, to a culture medium and an embryo culture method for improving embryo health. Background Art

[0002] At present, embryo culture technology has developed rapidly and achieved remarkable results. At the technical level, it has developed from the initial simple in vitro fertilization technology to today's microdroplet culture technology, embryo freezing technology, single gene disease screening technology, etc. The technical level of some large hospitals and reproductive centers has reached an advanced level, and even leads the world in preimplantation genetic diagnosis technology. At the same time, blastocyst culture technology is also becoming more and more mature. Through advanced methods such as time-lapse photography to monitor embryo development, embryos with the greatest developmental potential can be better selected for transplantation, which improves the survival rate and implantation rate of embryos.

[0003] However, at present, the survival rate of embryo culture system is still not high enough. Relevant studies have shown that the survival rate of blastocysts cultured from IVF is usually between 30-40%, and the survival rate of primary embryos is generally between 60-70%. Therefore, it is necessary to optimize the embryo culture system. Summary of the invention

[0004] In order to solve the technical problem that the survival rate of the existing embryo culture system is not high enough, the purpose of the present invention is to provide a culture medium that improves the health of the embryo.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] A culture solution for improving embryo health comprises a basic culture solution and an antioxidant active extract; the antioxidant active extract is a Haematococcus pluvialis and Cistanche deserticola extract.

[0007] Studies have shown that Haematococcus pluvialis extract is rich in astaxanthin, which has a strong antioxidant capacity. Its antioxidant activity is 550 times that of vitamin E and 10 times that of β-carotene. It can protect cells from oxidative damage by quenching singlet oxygen and scavenging free radicals, and prevent a variety of diseases related to oxidative stress, such as cardiovascular disease, cancer, and neurodegenerative diseases. Cistanche deserticola extract can increase the body's antioxidant enzyme activity, such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), while reducing the content of lipid peroxidation product malondialdehyde (MDA), thereby reducing oxidative stress damage to cells and tissues, and playing an antioxidant and anti-aging role.

[0008] The inventor's research shows that adding Haematococcus pluvialis extract or Cistanche deserticola extract to the existing embryo basal culture medium can improve the formation rate of embryo blastocysts. However, adding Houttuynia cordata extract, which also has antioxidant activity, to the existing embryo basal culture medium has a counter-effect at certain concentrations.

[0009] The Haematococcus pluvialis extract of the invention is prepared from Haematococcus pluvialis. The specific extraction method comprises the following steps: weighing 100g of Haematococcus pluvialis powder, adding 1L of 95% food-grade alcohol, fully mixing, placing the mixture as a reaction system in a dark place at room temperature (25-30 DEG C) for extraction for 1 hour, and mixing the reaction system once every 15 minutes; after the extraction is completed, centrifuging at 2000rpm for 15min, discarding the residue and obtaining the supernatant; vacuum decompressing the supernatant at room temperature to recover the alcohol, and drying to obtain the Haematococcus pluvialis extract.

[0010] The Cistanche deserticola extract of the present invention is prepared from the stem of Cistanche deserticola. The specific extraction method comprises the following steps: weighing 1 kg of 60-mesh Cistanche deserticola stem powder, adding 10 times the weight of 60% ethanol solution to mix, heating and refluxing, each time for 2 hours, extracting twice in total, sieving through 100 mesh, combining the filtrate, and concentrating the filtrate under reduced pressure to 1.02 g / cm 3 , and obtain the Cistanche deserticola alcohol extract concentrate; then centrifuge at 4000rpm for 15min, discard the residue and take the supernatant; soak the D101 macroporous resin in 95% ethanol for 24h, and after sufficient swelling, rinse with 95% ethanol until the effluent mixed with water is not white turbid, wash with ultrapure water until there is no alcohol taste, and then pass the supernatant through 100g D101 macroporous resin, and elute with one volume of water and two volumes of 60% ethanol solution of the macroporous resin column in turn, collect the ethanol solution eluate, and perform vacuum decompression on the supernatant at room temperature to recover the alcohol concentration, and obtain the Cistanche deserticola extract after drying.

[0011] Preferably, the basal culture medium comprises sodium chloride at a final concentration of 95.45 mmol / L, potassium chloride at a final concentration of 5.55 mmol / L, magnesium sulfate at a final concentration of 0.92 mmol / L, glucose at a final concentration of 1.25 mmol / L, ethylenediaminetetraacetic acid at a final concentration of 0.02 mmol / L, sodium pyruvate at a final concentration of 9.52 mmol / L, alanylglutamine at a final concentration of 28.56 mmol / L, taurine at a final concentration of 0.52 mmol / L, D-sorbitol at a final concentration of 0.25 mmol / L, potassium dihydrogen phosphate at a final concentration of 0.25 mmol / L, sodium dihydrogen phosphate at a final concentration of 0.82 mmol / L, N-acetyl-L-cysteine ​​at a final concentration of 7.25 mmol / L, 4-hydroxyethylpiperazineethanesulfonic acid HEPES at a final concentration of 7.25 mmol / L. l / L 3-(N-morpholino)propanesulfonic acid sodium salt MOPS-Na, 1.0mmol / L amino acid complex, 6.2mg / L gentamicin and 0.52g / L serum albumin.

[0012] Preferably, the amino acid complex consists of L-valine, L-leucine, L-isoleucine, L-phenylalanine, L-tryptophan, L-methionine, L-lysine, L-threonine, L-alanine, L-proline, L-serine, L-tyrosine, L-cysteine, L-asparagine, L-glutamine, L-aspartic acid, L-glutamic acid, L-arginine and L-histidine.

[0013] Preferably, 1.0 mmol / L of the amino acid complex consists of 0.12 mmol / L of L-valine, 0.05 mmol / L of L-leucine, 0.11 mmol / L of L-isoleucine, 0.07 mmol / L of L-phenylalanine, 0.12 mmol / L of L-tryptophan, 0.07 mmol / L of L-methionine, 0.08 mmol / L of L-lysine, 0.15 mmol / L of L-threonine, 0.12 mmol / L of L-alanine, 0.03 mmol / L of L-proline, 0.01 mmol / L of L-serine, 0.03 mmol / L of L-tyrosine, 0.01 mmol / L of L-cysteine, 0.04 mmol / L of L-asparagine, 0.01 mmol / L of l / L L-glutamine, 0.02mmo l / L L-aspartic acid, 0.01mmo l / L L-glutamic acid, 0.02mmo l / L L-arginine and 0.03mmol / L L-histidine.

[0014] Preferably, the antioxidant active extract is added to the basal culture medium at a final concentration of 15-30 mg / L.

[0015] Preferably, Haematococcus pluvialis is added to the basic culture solution at a final concentration of 5-10 mg / L, and the Cistanche deserticola extract is added to the basic culture solution at a final concentration of 10-20 mg / L.

[0016] Preferably, Haematococcus pluvialis is added to the basic culture solution at a final concentration of 5 mg / L, and the Cistanche deserticola extract is added to the basic culture solution at a final concentration of 15 mg / L.

[0017] Another object of the present invention is to provide an embryo culture method, which utilizes the aforementioned culture medium for improving embryo health to culture embryos in vitro.

[0018] The present invention has the following beneficial effects: by adding Haematococcus pluvialis and Cistanche deserticola extracts with antioxidant activity to the existing embryo basic culture medium, the in vitro culture conditions of the embryo are optimized, so that the embryo blastocyst has a higher formation rate. DETAILED DESCRIPTION

[0019] The present invention is further described below in conjunction with embodiments, but the scope of protection claimed by the present invention is not limited to the scope described in the embodiments.

[0020] Example 1

[0021] A culture medium for improving embryo health, comprising a basic culture medium and an antioxidant active extract; wherein the antioxidant active extract is a Haematococcus pluvialis extract.

[0022] The basal culture medium uses ultrapure water as a solvent, which includes 95.45 mmol / L sodium chloride, 5.55 mmol / L potassium chloride, 0.92 mmol / L magnesium sulfate, 1.25 mmol / L glucose, 0.02 mmol / L ethylenediaminetetraacetic acid, 9.52 mmol / L sodium pyruvate, 28.56 mmol / L alanyl-glutamine, 0.52 mmol / L taurine, 0.25 mmol / L D-sorbitol, 0.25 mmol / L potassium dihydrogen phosphate, 0.25 mmol / L sodium dihydrogen phosphate, 0.82 mmol / L N-acetyl-L-cysteine, 7.25 mmol / L l / L 4-hydroxyethylpiperazineethanesulfonic acid HEPES, 7.25mmol / L 3-(N-morpholino)propanesulfonic acid sodium salt MOPS-Na, 1.0mmo l / L amino acid complex, 6.2mg / L gentamicin and 0.52g / L serum albumin.

[0023] The 1.0 mmol / L amino acid complex in this example consists of 0.12 mmol / L L-valine, 0.05 mmol / L L-leucine, 0.11 mmol / L L-isoleucine, 0.07 mmol / L L-phenylalanine, 0.12 mmol / L L-tryptophan, 0.07 mmol / L L-methionine, 0.08 mmol / L L-lysine, 0.15 mmol / L L-threonine, 0.12 mmol / L L-alanine, 0.03 mmol / L L-proline, 0.01 mmol / L L-serine, 0.03 mmol / L L-tyrosine, 0.01 mmol / L L-cysteine, 0.04 mmol / L L-asparagine, 0.01 mmol / L L-glutamine ... The composition of the present invention is 0.1mmo l / L L-glutamine, 0.02mmo l / L L-aspartic acid, 0.01mmo l / L L-glutamic acid, 0.02mmo l / L L-arginine and 0.03mmo l / L L-histidine.

[0024] The Haematococcus pluvialis extract in this embodiment is prepared from Haematococcus pluvialis. The specific extraction method comprises the following steps: weighing 100 g of Haematococcus pluvialis powder, adding 1 L of 95% food-grade alcohol, mixing thoroughly, placing the mixture in a dark place as a reaction system, extracting at room temperature (25-30° C.) for 1 hour, and mixing the reaction system once every 15 minutes; centrifuging at 2000 rpm for 15 minutes after the extraction, discarding the residue and obtaining the supernatant; vacuum-decompressing the supernatant at room temperature to recover the alcohol concentration, and drying to obtain the Haematococcus pluvialis extract.

[0025] The antioxidant active extract was added to the basal culture medium at a final concentration of 5 ml / L.

[0026] Example 2

[0027] A culture medium for improving embryo health, which is different from Example 1 only in that the antioxidant active extract is added to the basic culture medium at a final concentration of 10 ml / L.

[0028] Example 3

[0029] A culture medium for improving embryo health, which is different from Example 1 only in that the antioxidant active extract is added to the basic culture medium at a final concentration of 15 ml / L.

[0030] Example 4

[0031] A culture medium for improving embryo health, which is different from Example 1 only in that the antioxidant active extract is added to the basic culture medium at a final concentration of 15 ml / L.

[0032] Example 5

[0033] A culture medium for improving embryo health, which is different from Example 1 only in that the antioxidant active extract is added to the basic culture medium at a final concentration of 20 ml / L.

[0034] Example 6

[0035] A culture medium for improving embryo health, which is different from Example 1 only in that the antioxidant active extract is a Cistanche deserticola extract.

[0036] The Cistanche deserticola extract in this embodiment is prepared from the stem of Cistanche deserticola. The specific extraction method comprises the following steps: weighing 1 kg of 60-mesh Cistanche deserticola stem powder, adding 10 times the weight of 60% ethanol solution, mixing, heating and refluxing, each time for 2 hours, extracting twice in total, sieving through 100 mesh, combining the filtrate, and concentrating the filtrate under reduced pressure to 1.02 g / cm 3 , and obtain the Cistanche deserticola alcohol extract concentrate; then centrifuge at 4000rpm for 15min, discard the residue and take the supernatant; soak the D101 macroporous resin in 95% ethanol for 24h, and after sufficient swelling, rinse with 95% ethanol until the effluent mixed with water is not white turbid, wash with ultrapure water until there is no alcohol taste, and then pass the supernatant through 100g D101 macroporous resin, and elute with one volume of water and two volumes of 60% ethanol solution of the macroporous resin column in turn, collect the ethanol solution eluate, and perform vacuum decompression on the supernatant at room temperature to recover the alcohol concentration, and obtain the Cistanche deserticola extract after drying.

[0037] The antioxidant active extract was added to the basal culture medium at a final concentration of 5 mg / L.

[0038] Example 7

[0039] A culture medium for improving embryo health, which is different from Example 6 only in that the antioxidant active extract is added to the basic culture medium at a final concentration of 10 ml / L.

[0040] Example 8

[0041] A culture medium for improving embryo health, which is different from Example 6 only in that the antioxidant active extract is added to the basic culture medium at a final concentration of 15 ml / L.

[0042] Example 9

[0043] A culture medium for improving embryo health, which is different from Example 6 only in that the antioxidant active extract is added to the basic culture medium at a final concentration of 15 ml / L.

[0044] Example 10

[0045] A culture medium for improving embryo health, which is different from Example 6 only in that the antioxidant active extract is added to the basic culture medium at a final concentration of 20 ml / L.

[0046] Embodiment 11

[0047] A culture medium for improving embryo health, which is different from Example 1 only in that the antioxidant active extract is an alcohol extract of Houttuynia cordata.

[0048] The antioxidant active extract was added to the basal culture medium at a final concentration of 5 mg / L.

[0049] Example 12

[0050] A culture medium for improving embryo health, which differs from Example 11 only in that an antioxidant active extract is added to the basal culture medium at a final concentration of 10 ml / L.

[0051] Embodiment 13

[0052] A culture medium for improving embryo health, which differs from Example 11 only in that the antioxidant active extract is added to the basic culture medium at a final concentration of 15 ml / L.

[0053] Embodiment 14

[0054] A culture medium for improving embryo health, which differs from Example 11 only in that the antioxidant active extract is added to the basic culture medium at a final concentration of 15 ml / L.

[0055] Embodiment 15

[0056] A culture medium for improving embryo health, which differs from Example 11 only in that an antioxidant active extract is added to the basal culture medium at a final concentration of 20 ml / L.

[0057] Example 16

[0058] A culture medium for improving embryo health, comprising a basic culture medium and an antioxidant active extract; wherein the antioxidant active extract comprises a Haematococcus pluvialis extract and a Cistanche deserticola extract. The extraction method of the Haematococcus pluvialis extract is the same as in Example 1, and the extraction method of the Cistanche deserticola extract is the same as in Example 6.

[0059] The Haematococcus pluvialis extract was added to the basic culture solution at a final concentration of 5 ml / L, and the Cistanche deserticola extract was added to the basic culture solution at a final concentration of 5 ml / L.

[0060] Embodiment 17

[0061] A culture medium for improving embryo health, comprising a basic culture medium and an antioxidant active extract; wherein the antioxidant active extract comprises a Haematococcus pluvialis extract and a Cistanche deserticola extract. The extraction method of the Haematococcus pluvialis extract is the same as in Example 1, and the extraction method of the Cistanche deserticola extract is the same as in Example 6.

[0062] The Haematococcus pluvialis extract was added to the basic culture solution at a final concentration of 5 ml / L, and the Cistanche deserticola extract was added to the basic culture solution at a final concentration of 10 ml / L.

[0063] Embodiment 18

[0064] A culture medium for improving embryo health, comprising a basic culture medium and an antioxidant active extract; wherein the antioxidant active extract comprises a Haematococcus pluvialis extract and a Cistanche deserticola extract. The extraction method of the Haematococcus pluvialis extract is the same as in Example 1, and the extraction method of the Cistanche deserticola extract is the same as in Example 6.

[0065] The Haematococcus pluvialis extract was added to the basic culture solution at a final concentration of 10 ml / L, and the Cistanche deserticola extract was added to the basic culture solution at a final concentration of 5 ml / L.

[0066] Embodiment 19

[0067] A culture medium for improving embryo health, comprising a basic culture medium and an antioxidant active extract; wherein the antioxidant active extract comprises a Haematococcus pluvialis extract and a Cistanche deserticola extract. The extraction method of the Haematococcus pluvialis extract is the same as in Example 1, and the extraction method of the Cistanche deserticola extract is the same as in Example 6.

[0068] The Haematococcus pluvialis extract was added to the basic culture solution at a final concentration of 5 ml / L, and the Cistanche deserticola extract was added to the basic culture solution at a final concentration of 15 ml / L.

[0069] Embodiment 20

[0070] A culture medium for improving embryo health, comprising a basic culture medium and an antioxidant active extract; wherein the antioxidant active extract comprises a Haematococcus pluvialis extract and a Cistanche deserticola extract. The extraction method of the Haematococcus pluvialis extract is the same as in Example 1, and the extraction method of the Cistanche deserticola extract is the same as in Example 6.

[0071] The Haematococcus pluvialis extract was added to the basic culture solution at a final concentration of 15 ml / L, and the Cistanche deserticola extract was added to the basic culture solution at a final concentration of 5 ml / L.

[0072] Comparative Example 1

[0073] An embryo culture medium comprising only the basal culture medium in Example 1.

[0074] The specific compositions of the culture medium for improving embryo health prepared in Examples 1-20 and the embryo culture medium prepared in Comparative Example 1 are shown in Table 1.

[0075] Table 1 Composition of embryo culture medium obtained in Examples and Comparative Examples

[0076]

[0077]

[0078] The culture medium for improving embryo health prepared in Examples 1-20 and the embryo culture medium prepared in Comparative Example 1 were subjected to a mouse embryo test, and the specific steps were as follows: female mice with thrombosis aged 6-8 weeks were prepared, and the female mice with thrombosis were killed 24 hours after hCG injection, and the fertilized egg mass was collected from the oviduct. The collected flocculent fertilized egg mass was placed in hyaluronidase preheated in advance at 37°C, and after the cumulus and granulosa cells around the embryo were digested and separated, the normal morphological fertilized 1-cell embryos were immediately transferred and washed, and the collected fertilized 1-cell embryos were randomly divided into 21 groups, each with 100; the microdroplet method was used for culture, and each group of fertilized 1-cell embryos was placed in the culture medium of the embodiment and the comparative example, respectively, and cultured in an incubator at 37°C, 5% CO2, 5% O2, and saturated humidity, and the number of blastocysts was recorded by observing the blastocyst morphology after 96 hours of culture. The results are shown in Table 2.

[0079] Table 2 Blastocyst formation rate of embryos cultured in culture medium obtained in Examples and Comparative Examples

[0080]

[0081]

[0082] As shown in Table 2, adding a specific concentration of Haematococcus pluvialis extract or Cistanche deserticola extract to the existing embryo basal culture medium can promote the formation of blastocysts from 1-cell embryos, while adding Houttuynia cordata extract, which also has an antioxidant effect, has a counter-effect. Among them, when the concentration of Haematococcus pluvialis extract is 5-15 mg / L, the blastocyst formation rate can be increased from 75% to between 81-87%, but when its concentration is further increased, the blastocyst formation rate decreases instead. The possible reason is that the active free radicals (as signal molecules) are excessively removed, affecting the normal signal transduction pathway, resulting in abnormal processes such as proliferation, differentiation and apoptosis of embryonic cells, thereby inhibiting the development of the embryo and reducing the blastocyst formation rate. When the concentration of Cistanche deserticola Haematococcus extract is 10-20 mg / L, the blastocyst formation rate can be increased from 75% to between 82-87%. However, when the concentration is too high or too low, the blastocyst formation rate cannot be effectively improved. The possible reasons are: when the concentration is too low, the content of active ingredients such as phenylethanoid glycosides, polysaccharides, and echinacoside in the Cistanche deserticola extract is also insufficient, and cannot meet the demand for these ingredients during embryonic development, thereby resulting in restricted embryonic development and the inability to effectively improve the blastocyst formation rate; when the concentration is too high, it may cause the osmotic pressure of the culture medium to increase, thereby destroying the osmotic pressure balance inside and outside the embryonic cells.

[0083] By comparing Examples 16-20 with Examples 1-10, it can be seen that the addition of Haematococcus pluvialis extract and Cistanche deserticola extract to the basal culture medium can play a synergistic role in improving the blastocyst formation rate. On this basis, the addition of a relatively low concentration of Haematococcus pluvialis extract and a relatively high concentration of Cistanche deserticola extract can further improve the blastocyst formation rate, increasing the blastocyst formation rate from 75% to between 93-95%.

[0084] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A culture medium for improving embryo health, characterized in that: The invention comprises a basic culture solution and an extract with antioxidant activity; the extract with antioxidant activity is an extract of Haematococcus pluvialis and Cistanche deserticola.

2. The culture solution for improving embryo health according to claim 1, characterized in that: The basic culture medium comprises sodium chloride with a final concentration of 95.45 mmol / L, potassium chloride with a final concentration of 5.55 mmol / L, magnesium sulfate with a final concentration of 0.92 mmol / L, glucose with a final concentration of 1.25 mmol / L, ethylenediaminetetraacetic acid with a final concentration of 0.02 mmol / L, sodium pyruvate with a final concentration of 9.52 mmol / L, alanylglutamine with a final concentration of 28.56 mmol / L, taurine with a final concentration of 0.52 mmol / L, D-sorbitol with a final concentration of 0.25 mmol / L, potassium dihydrogen phosphate with a final concentration of 0.25 mmol / L, sodium dihydrogen phosphate with a final concentration of 0.82 mmol / L. l / L N-acetyl-L-cysteine, 7.25mmol / L 4-hydroxyethylpiperazineethanesulfonic acid HEPES, 7.25mmol / L 3-(N-morpholino)propanesulfonic acid sodium salt MOPS-Na, 1.0mmol / L amino acid complex, 6.2mg / L gentamicin and 0.52g / L serum albumin.

3. The culture solution for improving embryo health according to claim 2, characterized in that: The amino acid complex consists of L-valine, L-leucine, L-isoleucine, L-phenylalanine, L-tryptophan, L-methionine, L-lysine, L-threonine, L-alanine, L-proline, L-serine, L-tyrosine, L-cysteine, L-asparagine, L-glutamine, L-aspartic acid, L-glutamic acid, L-arginine and L-histidine.

4. The culture solution for improving embryo health according to claim 3, characterized in that: 1.0mmo l / L amino acid complex consists of 0.12mmo l / L L-valine, 0.05mmo l / L L-leucine, 0.11mmo l / L L-isoleucine, 0.07mmo l / L L-phenylalanine, 0.12mmo l / L L-tryptophan, 0.07mmo l / L L-methionine, 0.08mmol / L L-lysine, 0.15mmo l / L L-threonine, 0.12mmo l / L L-alanine, 0.03mmo l / L L-proline, 0.01mmo l / L L-serine, 0.03mmo l / L L-tyrosine, 0.01mmo l / L L-cysteine, 0.04mmo l / L L-asparagine, 0.01mmo l / L The composition of the present invention is 0.1mmo l / L L-glutamine, 0.02mmo l / L L-aspartic acid, 0.01mmo l / L L-glutamic acid, 0.02mmo l / L L-arginine and 0.03mmo l / L L-histidine.

5. The culture solution for improving embryo health according to claim 1, characterized in that: The antioxidant active extract was added to the basal culture medium at a final concentration of 15-30 mg / L.

6. The culture solution for improving embryo health according to claim 5, characterized in that: Haematococcus pluvialis is added to the basic culture solution at a final concentration of 5-10 mg / L, and the Cistanche deserticola extract is added to the basic culture solution at a final concentration of 10-20 mg / L.

7. The culture solution for improving embryo health according to claim 6, characterized in that: Haematococcus pluvialis was added to the basic culture solution at a final concentration of 5 mg / L, and the Cistanche deserticola extract was added to the basic culture solution at a final concentration of 15 mg / L.

8. An embryo culture method, characterized in that: The embryos are cultured in vitro using the culture medium for improving embryo health as described in any one of claims 1 to 7.

Images