Culture solution and method for improving in-vitro maturation and quality of remaining GV-stage ova after ICSI

Abstract

The invention discloses a culture solution and a method for improving in-vitro maturation and quality of remaining GV stage ova after ICSI, and relates to the field of assisted reproduction. The invention provides a culture solution for improving in-vitro maturation and quality of remaining GV stage ova after ICSI, the culture solution is a BM-CoE finished product culture solution composed of 50 [mu] M of coenzyme Q10, 10 ng / ml of epidermal growth factors and a BM basic culture solution, and the BM basic culture solution is composed of a finished product blastocyst culture solution 1029, a 10% serum protein substitute, 29 [mu] M of sodium pyruvate and 1.5 IU / ml of human menopausal gonadotropin. By jointly adding the coenzyme Q10 and the epidermal growth factor with certain concentration into the BM basic culture solution, compared with a commercially available finished product oocyte in-vitro maturation culture solution, the culture solution disclosed by the invention not only can obtain higher oocyte maturation rate and better mature oocyte quality, but also is beneficial to improving the clinical oocyte utilization rate and increasing the number of available embryos of a patient.

Description

Technical Field

[0001] The present invention belongs to the field of assisted reproduction, and specifically relates to a culture medium and method for improving the in vitro maturation and quality of remaining germinal vesicle (GV) stage oocytes after ICSI. Background Art

[0002] Among the oocytes obtained after superovulation, 5%-10% are immature oocytes in the germinal vesicle (GV) stage, which need to be cultured in vitro to mature, and then the matured oocytes are fertilized in vitro again in order to obtain more available human embryos clinically. However, most of the commercially available ready-to-use reagents for in vitro maturation of oocytes on the market are applied to patients who cannot obtain mature oocytes after conventional superovulation treatment. Using such ready-to-use reagents for in vitro maturation culture of GV stage oocytes obtained after superovulation results in a low in vitro maturation rate and poor quality of the obtained embryos, which cannot be applied clinically, causing waste of oocytes and economic burden on patients. Summary of the Invention

[0003] In view of the above defects, the present invention provides a culture medium for improving the in vitro maturation and quality of remaining GV stage oocytes after ICSI. Compared with commercially available ready-to-use culture media for in vitro maturation of oocytes, it can not only obtain a higher oocyte maturation rate, but also has better quality of mature oocytes, which is beneficial to improving the clinical utilization rate of oocytes and increasing the number of available embryos for patients. The culture medium is a BM-CoE ready-to-use culture medium composed of 50 μM coenzyme Q10, 10 ng / ml epidermal growth factor, and BM basal culture medium;

[0004] The BM basal culture medium is composed of ready-to-use blastocyst culture medium 1029, 10% serum protein substitute, 29 μM sodium pyruvate, and 1.5 IU / ml human menopausal gonadotropin.

[0005] The present invention also discloses a method for preparing the above-mentioned culture medium for improving the in vitro maturation and quality of remaining GV stage oocytes after ICSI, including the following steps:

[0006] S1. Drop Con-IVM ready-to-use culture medium for in vitro maturation of oocytes and BM-CoE ready-to-use culture medium into two 6-cm dishes respectively, cover with mineral oil, and place them in an incubator at 37°C and 5% CO 2 overnight for pre-equilibration one day before ICSI injection;

[0007] S2. Retain the remaining GV stage oocytes after ICSI treatment;

[0008] S3. Randomly divide the remaining GV stage oocytes after ICSI treatment and place them in the culture dish with Con-IVM ready-to-use culture medium for in vitro maturation of oocytes and the culture dish with BM-CoE ready-to-use culture medium at 37°C and 5% CO 2Co-culture in an incubator for 24 h;

[0009] S4. After culturing for 24 h, the maturation rate was statistically analyzed with the extrusion of the first polar body as the standard in the culture dish of the Con-IVM mature oocyte in vitro maturation culture medium and the culture dish of the BM-CoE finished culture medium;

[0010] S5. Collect the matured oocytes for detecting the reactive oxygen species level;

[0011] S6. Collect the matured oocytes for detecting the mitochondrial membrane potential.

[0012] Furthermore, the statistical method in step S4 is as follows:

[0013] Taking the extrusion of the first polar body in the perivitelline space observed under an inverted microscope as the sign of mature oocytes, the oocyte maturation rate = the number of mature oocytes / the number of cultured GV-stage oocytes × 100%.

[0014] Furthermore, the specific operation of step S5 includes the following steps:

[0015] S501. Drop several prepared DCFH-DA droplets with a ratio of 1:1000 in a culture dish, and then put it into the incubator for preheating;

[0016] S502. Transfer the mature oocytes into the DCFH-DA droplets and incubate them in the 37 °C cell incubator in the dark for 25 - 30 min;

[0017] S503. Wash them 3 - 5 times in the preheated PBS dish, and observe the ROS level of the cultured oocytes at 488 mm through a laser confocal microscope device.

[0018] Furthermore, the specific operation of step S6 includes the following steps:

[0019] S601. Drop several prepared JC-1 droplets with a ratio of 1:200 in a culture dish, and then put it into the incubator for preheating;

[0020] S602. Transfer the mature oocytes into the JC-1 droplets and incubate them in the 37 °C cell incubator in the dark for 25 - 30 mi n ;

[0021] S603. Wash them 3 - 5 times in the preheated PBS dish, and observe the mitochondrial membrane potential level of the cultured oocytes through a laser confocal microscope device.

[0022] The present invention has the following beneficial effects compared with the prior art:

[0023] 1. By adding a certain concentration of coenzyme Q10 and epidermal growth factor to the BM basal culture medium, compared with the commercially available finished oocyte in vitro maturation culture medium, not only can a higher oocyte maturation rate be obtained, but the quality of mature oocytes is also better, which is beneficial to improving the clinical utilization rate of oocytes and increasing the number of available embryos for patients;

[0024] 2. The preparation method of this culture medium is easy to operate, easy to repeat, has no special process and equipment requirements, and can be widely used in oocyte in vitro maturation culture reagents. Description of the Drawings

[0025] Figure 1 It is a schematic diagram comparing the results of in vitro maturation of human GV-stage oocytes by the BM-CoE finished culture medium and the Con-IVM finished oocyte in vitro maturation culture medium in the present invention.

[0026] Figure 2 It is a schematic diagram comparing the reactive oxygen species levels of human GV-stage oocytes by the BM-CoE finished culture medium and the Con-IVM finished oocyte in vitro maturation culture medium in the present invention.

[0027] Figure 3 It is a schematic diagram comparing the mitochondrial membrane potential of human GV-stage oocytes by the BM-CoE finished culture medium and the Con-IVM finished oocyte in vitro maturation culture medium in the present invention. Detailed Embodiments

[0028] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] Example 1

[0030] This example provides a culture medium for improving the in vitro maturation and quality of the remaining GV-stage eggs after ICSI, which is the BM-CoE finished culture medium composed of 50 μM coenzyme Q10 (CoQ10), 10 ng / ml epidermal growth factor (EGF), and BM basal culture medium;

[0031] The BM basal culture medium is composed of the finished blastocyst culture medium 1029, 10% serum protein substitute (SPS), 29 μM sodium pyruvate, and 1.5 IU / ml human menopausal gonadotropin (HMG).

[0032] That is to say, by using the finished blastocyst culture medium 1029 supplemented with 10% SPS + 29 μM sodium pyruvate + 1.5 IU / ml HMG to form the basic medium for in vitro maturation of immature oocytes, and on this basis, adding 50 μM CoQ10 + 10 ng / ml EGF as the culture medium for in vitro maturation of oocytes.

[0033] Example 2

[0034] A method for preparing a culture medium for improving the in vitro maturation and quality of remaining GV-stage oocytes after ICSI, comprising the following steps:

[0035] S1. Drop the Con-IVM finished oocyte in vitro maturation culture medium and the BM-CoE finished culture medium into two 6-cm dishes respectively, cover with mineral oil, and place them in a 37°C, 5% CO 2 incubator for overnight pre-equilibration one day before ICSI injection;

[0036] S2. Retain the remaining GV-stage oocytes after ICSI treatment;

[0037] S3. Randomly divide the remaining GV-stage oocytes after ICSI treatment and place them in the culture dish of the Con-IVM finished oocyte in vitro maturation culture medium and the culture dish of the BM-CoE finished culture medium, and co-culture them in a 37°C, 5% CO 2 incubator for 24 h. It should be noted that the Con-IVM finished oocyte in vitro maturation culture medium is the traditional commercially available finished oocyte in vitro maturation culture medium;

[0038] S4. After culturing for 24 h, the maturation rate is statistically analyzed with the extrusion of the first polar body as the standard in the culture dishes of the Con-IVM finished oocyte in vitro maturation culture medium and the BM-CoE finished culture medium. The statistical method is: taking the extrusion of the first polar body in the perivitelline space observed under an inverted microscope as the sign of mature oocytes, and the oocyte maturation rate = the number of mature oocytes / the number of cultured GV-stage oocytes × 100%;

[0039] S5. Retain the matured oocytes for reactive oxygen species level detection;

[0040] S501. Drop several prepared 1:1000 DCFH-DA droplets in the culture dish, and then place it in the incubator for preheating;

[0041] S502. Transfer the mature oocytes into the DCFH-DA droplets and incubate them in the 37°C cell incubator in the dark for 25 - 30 min;

[0042] S503. Wash 3 - 5 times in a pre - warmed PBS dish, and observe the ROS level of the cultured oocytes at 488 nm through a laser confocal microscope device;

[0043] S6. Retain the matured oocytes for mitochondrial membrane potential detection;

[0044] S601. Drop several prepared JC - 1 droplets with a ratio of 1:200 in a culture dish, and then place it in an incubator for pre - warming;

[0045] S602. Transfer the mature oocytes into the JC - 1 droplets and incubate them in the dark at 37°C in a cell culture incubator for 25 - 30 min;

[0046] S603. Wash 3 - 5 times in a pre - warmed PBS dish, and observe the membrane potential level of the cultured oocytes through a laser confocal microscope device.

[0047] It should be noted that the structure described in the present invention can be implemented in many different forms and is not limited to the embodiments. Any equivalent transformation made by those of ordinary skill in the art using the specification and drawings of the present invention, or directly or indirectly applied in other related technical fields, such as the loading and unloading of other items, is included in the protection scope of the present invention.

Claims

1. A culture medium for improving the in vitro maturation and quality of remaining GV stage oocytes after ICSI, characterized in that: The culture medium is a BM-CoE finished culture medium composed of 50 μM coenzyme Q10, 10 ng / ml epidermal growth factor and BM basic culture medium; The BM basic culture medium consists of finished blastocyst culture medium 1029, 10% serum protein substitute, 29 μM sodium pyruvate and 1.5 IU / ml human menopausal gonadotropin.

2. A method for preparing a culture medium for improving in vitro maturation and quality of remaining GV stage oocytes after ICSI according to claim 1, characterized in that: The following steps are involved: S1. Add Con-IVM finished oocyte in vitro maturation culture medium and BM-CoE finished culture medium to two 6 cm dishes respectively, cover with mineral oil, and place in a 37°C, 5% CO2 incubator overnight for pre-equilibration one day before ICSI injection; S2, retain the remaining GV stage oocytes after ICSI treatment; S3. The remaining GV-stage oocytes after ICSI treatment were randomly divided into groups and placed in Con-IVM finished oocyte in vitro maturation culture medium culture dishes and BM-CoE finished oocyte culture medium culture dishes and co-cultured in a 37°C, 5% CO2 incubator for 24 h. S4. After 24 hours of culture, the maturation rates of Con-IVM finished oocyte in vitro maturation culture medium culture dishes and BM-CoE finished oocyte in vitro maturation culture medium culture dishes were calculated based on the first polar body extrusion as the standard; S5, retaining and culturing mature oocytes to detect the level of reactive oxygen species; S6. The mature oocytes are kept for culture and mitochondrial membrane potential detection is performed.

3. The method for improving in vitro maturation and quality of remaining GV stage oocytes after ICSI according to claim 2, characterized in that: The statistical method of step S4 is: The discharge of the first polar body from the perivitelline space observed under an inverted microscope was regarded as a sign of mature oocytes. Oocyte maturation rate = number of mature oocytes / number of oocytes in culture at the GV stage × 100%.

4. The method for improving in vitro maturation and quality of remaining GV stage oocytes after ICSI according to claim 2, characterized in that: The specific operation of step S5 includes the following steps: S501, adding a number of prepared 1:1000 DCFH-DA droplets to the culture dish, and then placing it in an incubator for preheating; S502, move the mature oocytes into the DCFH-DA droplets and incubate them in a cell culture incubator at 37°C in the dark for 25-30 minutes; S503. Wash 3-5 times in a preheated PBS dish and observe the ROS level of the cultured oocytes at 488 mm using a laser confocal microscope.

5. The method for improving in vitro maturation and quality of remaining GV stage oocytes after ICSI according to claim 2, characterized in that: The specific operation of step S6 includes the following steps: S601, add several drops of 1:200 JC-1 in the culture dish, and then put it in the incubator for preheating; S602, move the mature oocytes into the JC-1 droplets and incubate them in a 37°C cell culture incubator in the dark for 25-30 minutes; S603. Wash 3-5 times in a preheated PBS dish and observe the membrane potential level of the cultured oocytes using a laser confocal microscope.

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