Method for electrically activating oocyte based on nanosecond pulse

Abstract

The invention relates to a method for electrically activating oocytes based on nanosecond pulses. Single oocytes are sequentially and uniformly arranged between two parallel electrodes of an electrode dish, the electrode dish is characterized in that the distance between the electrodes is 1 mm, the electrodes are placed in a culture dish, and the two ends of the two electrodes are sealed; a culture solution is firstly put between the electrodes which are arranged in parallel, mineral oil is covered to prevent the culture solution from being evaporated, after preheating treatment, oocytes are sequentially and uniformly put between the two electrodes which are arranged in parallel one by one, and then nanosecond pulse electric activation treatment is applied. The electrodes which are arranged in parallel are adopted, the oocytes are sequentially and uniformly placed between the electrodes in a single mode, the situation that the electric pulse effect is reduced due to adhesion and stacking of all the cells is avoided, the oocytes receive uniform and equivalent electric pulse sequences, the action effect and stability of electric pulses on the cells are obviously improved, and the operation efficiency of the oocytes is improved. And the reliability and reproducibility of a test conclusion are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and in particular to a method for activating oocytes based on nanosecond pulse electricity. Background Art

[0002] Assisted reproductive technology (ART) is an effective treatment for infertility. Intracytoplasmic sperm injection (ICSI), in particular, can overcome the inability of sperm to penetrate the zona pellucida, a common male infertility factor, leading to fertilization failure. This has increased the in vitro fertilization rate of oocytes to 70-80%. However, clinically, fertilization failure after repeated ICSI treatment, a phenomenon known as oocyte activation defect (OAD), still occurs. Abnormal calcium oscillations in the oocyte are the primary cause of oocyte activation failure and repeated ICSI failure in patients. This has led to the development of artificial oocyte activation (AOA). Current AOA techniques primarily utilize physical stimulation (especially electrical stimulation), mechanical stimulation, and chemical stimulation to artificially elevate intracellular calcium ion concentrations, thereby activating oocytes in vitro. However, to date, AOA technology can only induce single or several calcium waves in oocytes, and it is difficult to simulate the physiological, continuous, and precise calcium oscillation pattern induced by sperm. Therefore, the existing activation methods still have problems such as low oocyte activation rate, poor embryo development potential, and unclear activation mechanism, which seriously limit the application of AOA technology in clinical treatment.

[0003] Nanosecond pulsed electric fields (nsPEFs) are a novel bioelectric technology. Unlike conventional microsecond pulses that produce cell membrane electroporation, nsPEFs have shorter (nanosecond) pulse durations, which are shorter than the plasma membrane charging time. Therefore, nsPEFs have a stronger effect on internal cellular structures. Therefore, these pulse generators, with their ultrashort pulse width (ns), high electric field (kV / cm), and non-thermal effects, have the potential to modulate internal cellular structures (such as the endoplasmic reticulum, mitochondria, and lysosomes). Napotnik et al. reviewed the various intracellular effects induced by pulses of appropriate duration (11-100 ns) in eukaryotic cells, including effects on organelles, the nucleus, and intracellular calcium fluctuations. Studies have demonstrated that nsPEFs can quantitatively modulate calcium changes in various somatic cell types and can even induce calcium oscillations in cardiomyocytes, leading to cardiomyocyte excitation. Chinese patent CN107312749B discloses a physical method for oocyte activation. After placing oocytes and oocyte culture medium in an electrode cup, a two-step electrical pulse stimulation method is applied to the oocytes, followed by culture. This method can improve the activation rate and blastocyst rate of rabbit oocytes. Although this method improves the activation rate of rabbit oocytes (reaching 80%), the blastocyst rate is not high (37.5%). In practice, when this method is used on mouse oocytes, neither the activation rate nor the blastocyst rate is significantly improved, indicating that the embryonic development potential of oocytes activated using this method is not substantially improved. Furthermore, during experimental operation, it was found that the electrical pulse stimulation method of this technical solution has inconsistent effects on oocyte activation and development. Under the same experimental environment, several mature mouse oocytes were placed in an electrode cup containing culture medium and subjected to the same nanosecond electrical pulses for repeated experiments. The detected oocyte activation and development results were inconsistent, making it impossible to effectively verify the reliability and repeatability of the activation effect of this technique. However, the above patent proves that nsPEFs can safely and effectively induce calcium oscillations in oocytes. As long as the method of nanosecond pulse electrical activation can be improved and the calcium oscillation pattern in oocytes can be regulated, the embryonic development potential of oocytes can be improved, which will help optimize AOA technology. Summary of the Invention

[0004] In order to solve the above problems, the present invention proposes a method for activating oocytes based on nanosecond pulse electricity.

[0005] The present invention provides a method for activating oocytes based on nanosecond pulse electricity. The oocytes are arranged individually and evenly between two parallel electrodes of an electrode dish. The electrode dish is: the spacing between the electrodes is 1 mm, and the two electrodes are placed in a culture dish. The two ends of the two electrodes are sealed. A culture medium is first placed between the parallel electrodes and covered with mineral oil to prevent the culture medium from evaporating. After preheating, the oocytes are placed individually and evenly between the two parallel electrodes. Then, nanosecond pulse electricity activation treatment is applied, and the electrical dose is 2-4×10 6 J / g.

[0006] Furthermore, the culture dish has a diameter of 35-100 mm.

[0007] Furthermore, when the culture dish has a diameter of 100 mm and the oocytes are from mice, the electrical dose of the nanosecond pulse electrical activation treatment is 2-4×10 6 J / g, specifically: electric field strength is 8-12 kV / cm, pulse frequency is 1-2 Hz, pulse width is 10 ns, number of pulses is 10, and each pulse lasts 5-8 seconds.

[0008] Furthermore, the electrical dose is 2.5×10 6 J / g or the electric field strength is 10 kV / cm.

[0009] Furthermore, when the culture dish has a diameter of 35 mm and the oocytes are from mice, the electrical dose of the nanosecond pulse electrical activation treatment is 2-3×10 6 J / g, specifically: electric field strength is 17-20 kV / cm, pulse frequency is 1-2 Hz, pulse width is 10 ns, number of pulses is 10, and each pulse lasts 5-8 seconds.

[0010] Furthermore, when the diameter of the culture dish is 100 mm and the oocytes are from humans, the electrical dose of the nanosecond pulse electrical activation treatment is 3-14×10 6 The electric field strength is 11-14 kV / cm J / g, the pulse frequency is 1-2 Hz, the pulse width is 10 ns, the number of pulses is 10-30, and each pulse lasts 8-20 seconds.

[0011] Furthermore, when the culture dish has a diameter of 35 mm and the oocytes are from humans, the electrical dose of the nanosecond pulse electrical activation treatment is 3-14×10 6 The electric field strength is 20-25 kV / cm J / g, the pulse frequency is 1-2 Hz, the pulse width is 10 ns, the number of pulses is 10-30, and each pulse lasts 8-20 seconds.

[0012] Furthermore, both ends of the two parallel electrodes are sealed with neutral silicone glue.

[0013] Furthermore, the number of times the nanosecond pulse electrical activation treatment is applied is 1 or more.

[0014] The beneficial effects of the present invention are as follows:

[0015] 1. The present invention first discovered that the key to using electrical stimulation to activate oocytes is to use two parallel electrodes placed close to each other as the electric field to activate oocytes. Oocytes need to be arranged uniformly between the two parallel electrodes in sequence. Different from the processing method of the electrode cup disclosed in patent CN107312749B, the electrode spacing of the electrode cup is 4mm, and it is placed vertically. In the electrode cup, the oocytes are squeezed and stacked under the action of gravity. In fact, the electrical stimulation intensity received by each oocyte is not the same. However, the present invention uses parallel electrodes, and the oocytes use a single pattern of uniform placement between the electrodes to avoid adhesion and stacking between each cell, resulting in a decrease in the electric pulse effect. The oocytes receive a uniform and equal amount of electric pulse sequence, which reduces the influence of random errors in the experimental operation and improves the accuracy of the oocyte activation effect. At the same time, according to the field strength calculation formula "field strength = voltage / electrode spacing", under the same pulse field strength, the 1mm spacing electrode device selected by the present invention greatly reduces the voltage value required by the electric field generator, improving the safety and scalability of the operation.

[0016] 2. The present invention provides that when the diameter of the culture dish is 100mm and the culture fluid is M16 culture medium, when the oocyte is from a mouse, the electric field strength of the nanosecond pulse electric activation treatment is 8-12kV / cm, the pulse frequency is 1-2Hz, the pulse width is 10ns, the number of pulses is 10, and each pulse lasts 5-8 seconds. Using the electrical dosage of the present invention, it is possible to effectively activate mature mouse oocytes in batches and improve blastocyst development ability without inducing apoptosis caused by oxidative damage by one nanosecond pulse electric activation treatment; enhance mitochondrial activity and ATP production efficiency in oocytes, which is crucial for the normal development of early embryos; when the diameter of the culture dish is 35mm, the electric field strength of the nanosecond pulse electric activation treatment is 17-20kV / cm, which has a better activation effect on mouse oocytes and is suitable for activating application scenarios of a small number of oocytes.

[0017] 3. The present invention demonstrates that when mouse oocytes are activated and treated using the method of the present invention, when the culture dish diameter is 100 mm and the culture medium is M16 culture medium, the electric field intensity of the nanosecond pulse electric activation treatment is higher than 15 kV / cm and the oocyte activation in vitro cannot be achieved.

[0018] 4. This invention demonstrates that nanosecond pulsed electric field activation of oocytes activates mature, sperm-like oocytes by inducing a persistent calcium oscillation pattern in the oocyte cytoplasm, thereby activating mature, sperm-free oocytes and promoting their development into early embryos. This method can be used to treat ICSI patients with recurrent fertilization failure. Based on experimental results in mice, the present invention deduces that when the culture dish has a diameter of 100 mm and the oocytes are from humans, the electric field strength of the nanosecond pulsed electric field activation treatment is 11-14 kV / cm; when the culture dish has a diameter of 35 mm and the oocytes are from humans, the electric field strength of the nanosecond pulsed electric field activation treatment is 20-25 kV / cm.

[0019] 5. The present invention found that the electrode cup activation method has an unstable effect on the activation and development of oocytes. This may be related to the random distribution of oocytes in the electrode cup. Oocytes in the electrode cup are stacked at the bottom of the electrode cup under the influence of gravity or adhere to each other. It is impossible to ensure that each oocyte receives a uniform and equal sequence of electric pulses. The electric pulse effect on the oocytes gathered between the electrode plates decreases, which greatly increases the impact of random errors in the experimental operation and reduces the repeatability of the oocyte activation effect. Therefore, the inventor team developed an electrode dish with parallel electrodes with a spacing of 1mm as an oocyte activation device, and compared it with the existing oocyte electrical activation method. A comparative experiment was conducted on the recurrence rate of different electrical activation methods. It was confirmed that the electrode dish activation device using parallel electrodes to arrange oocytes in a single order significantly improved the effect and stability of the electric pulse on cells compared to the conventional electrode cup, and improved the reliability and reproducibility of the experimental conclusions. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 , an electrode dish in which the two electrodes used in Example 1 are placed in parallel;

[0021] Figure 2 , graph of oocyte cytoplasmic calcium fluctuations induced by 17 kV / cm nsPEFs treatment in Example 4;

[0022] Figure 3 , graph of oocyte cytoplasmic calcium fluctuations induced by 20 kV / cm nsPEFs treatment in Example 4;

[0023] Figure 4 , graph of oocyte cytoplasmic calcium fluctuations induced by 25 kV / cm nsPEFs treatment in Example 4;

[0024] Figure 5 , graph of oocyte cytoplasmic calcium fluctuations induced by A23187 treatment in Example 4;

[0025] Figure 6 , Under normal circumstances in the in vitro fertilization process in Example 5, oocyte Ca 2+ Oscillation mode;

[0026] Figure 7 , the oocyte Ca2+ induced by repeated 17 kV / cm nsPEF treatment in Example 5 2+ Oscillation mode;

[0027] Figure 8 , the oocyte Ca2+ induced by repeated 20 kV / cm nsPEF treatment in Example 5 2+ Oscillation mode. DETAILED DESCRIPTION

[0028] The present invention will be further described below with reference to the examples.

[0029] Experimental Statement

[0030] 1. Laboratory Animals: All experiments strictly adhered to the Chinese Laboratory Animal Welfare Act and were approved by the Laboratory Animal Ethics Committee of the College of Life Sciences, China Agricultural University. ICR (CD-1) mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. and housed in a clean-grade laboratory animal room at China Agricultural University. Each group contained at least 10 mice. The animal room was maintained at an ambient temperature of 22-24°C, with a 12-hour light-on / 12-hour dark cycle. Mice had free access to food and water.

[0031] 2. MII oocyte collection: Healthy ICR female mice aged 8-10 weeks were selected and intraperitoneally injected with 10 IU / mouse of PMSG (pregnant mare serum gonadotropin) during proestrus. 48 hours later, 10 IU / mouse of hCG (human chorionic gonadotropin) was injected intraperitoneally. Both PMSG and hCG were purchased from Ningbo Hormone Products Factory. 14-16 hours after hCG injection, the mice were sacrificed by cervical dislocation. The oviducts were removed from the peritoneal cavity and placed in warm M2 medium. Under a stereomicroscope, the ampulla of the oviduct was punctured with a 1 mL syringe needle to release the cumulus oocyte cluster. The oocytes were then transferred to 1% hyaluronidase for digestion for approximately 1 minute to completely detach the surrounding granulosa cells. MII oocytes with a distinct first polar body were collected using a glass pipette and transferred to prewarmed M16 medium pending electroporation or other treatment.

[0032] 3. Nanosecond pulse electric field stimulation (nsPEF parthenogenetic activation): The nanosecond pulse electric field uses a coaxial cable with a model of RG213 and a characteristic impedance of 50Ω as a pulse forming component. Five 2-meter-long coaxial cables are connected in parallel, and the impedance of the circuit after parallel connection is 10Ω. The circuit excitation switch uses a brass metal ball discharge method, that is, discharge is achieved through distorted electric sparks, and the output field strength and frequency are adjusted by setting the distance between the two metal balls. The circuit load part is an electrode dish, and the middle part of the electrode dish is two parallel platinum electrodes with a distance of 1mm between the electrodes. The two ends are sealed with neutral silicone glue and fixed in a culture dish (such as Figure 1 M16 culture medium was placed between the electrodes and covered with mineral oil to prevent evaporation. After preheating, oocytes were placed individually and evenly between the two electrodes under a stereomicroscope. Nanosecond pulse electric activation was then applied. The pulsed electric field intensity was monitored in real time using a digital fluorescence oscilloscope (Micsig, TO2004) and a high-voltage probe (P6015A, Tektronix). The bandwidths of the oscilloscope and high-voltage probe were 200 MHz and 75 MHz, respectively.

[0033] 4. In the following experiments, the nanosecond pulse electric field used a pulse width of 10 ns, a pulse frequency of 1-2 Hz, 10 pulses, and each pulse lasted for 5-8 seconds. Examples 1-4 all implemented one nanosecond pulse electric field stimulation treatment.

[0034] 5. A23187 Treatment of Oocytes: Calcium ionophore A23187 was purchased from MCE (1 mg / vial, CAS: 52665-69-7). A 10 mM stock concentration of A23187 was prepared with DMSO at a ratio of 5.2362 mg / ml. Oocytes were incubated in KSOM medium containing 10 μM A23187 at 37°C in the dark for 10 min. After washing three times with KSOM, the cells were cultured again at 37°C, 5% CO2, and saturated humidity.

[0035] 6. The values ​​in all the tables below are mean ± standard error. Statistical significance is indicated by different lowercase letters (P < 0.05). Different letters indicate significant differences between the groups.

[0036] Example 1: Study on the optimal electric field intensity for nanosecond pulse electrical activation of oocyte development:

[0037] The electrode dish used in this embodiment is a culture dish with a diameter of 100 mm.

[0038] Experimental Grouping: Mature mouse oocytes were stimulated with four different electric field intensities: 8 kV / cm, 10 kV / cm, 12 kV / cm, and 15 kV / cm. To reduce experimental errors caused by external factors such as the oocyte transfer procedure and electrode environment, this study used the same procedure to transfer oocytes to the electrode dish, but applied 0 kV / cm as a sham treatment. This study also used the calcium ionophore A23187, a commonly used oocyte chemical activator in clinical practice, as a positive control group.

[0039] Observation content and methods:

[0040] Mouse oocytes treated with nanosecond pulse electroactivation were placed in preheated KSOM medium and cultured in a cell culture incubator for 4-5 days. The oocytes were observed regularly after treatment. Activation was determined 6-8 hours after treatment. Oocytes were considered activated if they showed second polar body extrusion, pronuclear formation, or cleavage. Thereafter, embryonic development was monitored every 24 hours, and the embryo culture medium was changed every 48 hours. Oocytes with ruptured plasma membranes or cytoplasmic fragmentation were promptly removed and discarded.

[0041] Experimental results:

[0042] Table 1 Statistical results of oocyte activation rate and embryo development rate at each stage in different treatment groups

[0043]

[0044] Table 1 shows the results of nsPEF parthenogenetic activation of mouse oocytes at different field intensities. When a 100 mm culture dish is used as the electrode dish, nsPEF treatment with a field strength of 8-12 kV / cm, a pulse width of 10 ns, a pulse frequency of 1-2 Hz, and 10 pulses is sufficient to activate mouse oocytes and lead to blastocyst formation. When the field strength is ≤10 kV / cm, the nsPEF field strength is positively correlated with oocyte activation and development rate. However, when the field strength is greater than 10 kV / cm, the oocyte activation rate decreases with increasing field strength. Furthermore, after nsPEF stimulation at a pulsed field strength of 10 kV / cm, the oocyte activation rate was not significantly different from that of the A23187-treated group, but the blastocyst formation rate was significantly higher than that of the A23187-treated group. Furthermore, oocytes activated with a pulsed field strength of 15 kV / cm often arrested at the 2-cell and 4-cell embryonic stages and were unable to continue development.

[0045] Conclusion: Using two parallel electrodes of the present invention with a 1mm spacing between them, nanosecond pulses of electric field can effectively activate mouse oocytes and significantly improve the developmental potential of parthenogenetically activated embryos. When a 100mm culture dish is used as the electrode dish, nsPEF treatment at 8-12 kV / cm can effectively activate mouse oocytes and significantly improve the developmental potential of parthenogenetically activated embryos, confirming the effectiveness of nsPEF in activating mouse oocytes. Based on the experimental results in mice, the present invention can infer that when the culture dish diameter is 100mm and the oocytes are from human origin, the electric field strength of the nanosecond pulse electroactivation treatment is 11-14 kV / cm; when the culture dish diameter is 35mm and the oocytes are from human origin, the electric field strength of the nanosecond pulse electroactivation treatment is 20-25 kV / cm.

[0046] In order to investigate the safety of nsPEFs parthenogenetic activation of mouse oocytes, we decided to continue to use 8-15 kV / cm nsPEF for safety research.

[0047] Example 2: Safety Study of Nanosecond Pulsed Electric Field Stimulation of Oocytes

[0048] The electrode dish in this embodiment is a culture dish with a diameter of 100 mm.

[0049] Experimental Grouping: Mature oocytes were stimulated with electric field intensities of 8 kV / cm, 10 kV / cm, 12 kV / cm, and 15 kV / cm, respectively. Oocytes treated with 0 kV / cm (sham treatment) served as the negative control group. Oocytes treated with the calcium ionophore A23187 served as the positive control group.

[0050] Observation content and methods: The mouse oocytes treated in each group were equilibrated in M16 medium for 1 h, and then the early apoptosis of each oocyte was detected using a cell apoptosis kit. The ROS and GSH contents and mitochondrial membrane potential level of each oocyte were calculated by live cell staining. The ATP content of each oocyte was detected using an ATP detection kit.

[0051] result:

[0052] (1) Early apoptosis detection: After 1 hour of treatment with 8-12 kV / cm nsPEFs, oocytes basically did not undergo early apoptosis. However, after treatment with 15 kV / cm and A23187, the early apoptosis rate of oocytes increased significantly. The experimental data are shown in Table 2.

[0053] Table 2 Statistical results of the incidence of early apoptosis in mouse oocytes in different treatment groups

[0054]

[0055] (2) Peroxide ROS detection: The experimental data are shown in Table 3. There was no significant statistical difference in the ROS content in the oocytes of the 8 kV / cm treatment group and the sham treatment group. Under the stimulation of 10-15 kV / cm nsPEF, the ROS content in the oocytes was significantly increased, and the level of ROS was positively correlated with the strength of the electric pulse field. Compared with the cells in the A23187 group treated with the highest field strength of 15 kV / cm, the ROS content in the oocytes was significantly increased.

[0056] Table 3 Statistical results of reactive oxygen species content in mouse oocytes in different treatment groups

[0057]

[0058] (3) Antioxidant GSH detection: As shown in Table 4, compared with the sham treatment group, except for the 8 kV / cm treatment group, the GSH content in the oocytes of the other groups decreased significantly, and the A23187 treatment group was significantly lower than the 15 kV / cm treatment group. This result corresponds to the ROS content results of the above groups.

[0059] Table 4 Statistical results of reduced glutathione content in mouse oocytes in different treatment groups

[0060]

[0061] (4) Mitochondrial membrane potential level detection: The mitochondrial membrane potential level in each group of cells was detected using the mitochondrial membrane potential probe TMRM. The mitochondrial membrane potential levels of oocytes in each treatment group are shown in Table 5. The mitochondrial membrane potential level of the 10 kV / cm treatment group was significantly higher than that of the negative control, while the mitochondrial membrane potential of oocytes treated with A23187 was significantly lower than that of the control group. This can be understood as the application of nsPEFs will charge the cell membrane and organelle membrane and generate a certain transmembrane potential. At the same time, the calcium signal generated by the intracellular calcium ion release induced by nsPEF will also increase mitochondrial activity, so the mitochondrial membrane potential will increase within a certain range. Since mitochondrial membrane potential is a double-edged sword for cell survival, moderate charging of the mitochondrial membrane to increase the membrane potential reflects the active mitochondrial energy production, while excessive electric pulses cause excessive charging effects. Excessive increase in mitochondrial membrane potential leads to abnormal electron transport chain and excessive generation of ROS that damages the normal metabolic state of the cell. Therefore, since the cells in the 15 kV / cm group showed a low activation development rate and obvious oxidative stress, the 10 kV / cm group with better activation development and cell state indicators was selected instead of the 15 kV / cm group.

[0062] Table 5 Statistical results of endometrial membrane potential levels in mouse oocytes from different treatment groups

[0063]

[0064] (5) ATP content detection: As shown in Table 6, 8-10 kV / cm nsPEFs stimulation significantly increased the ATP level in oocytes, but higher field strength stimulation (12-15 kV / cm) reduced the intracellular ATP content, just like A23187 treatment. This indicates that moderate-intensity nsPEFs stimulation can increase mitochondrial activity and ATP production, while high-intensity electrical stimulation and chemical stimulation (A23187) can damage mitochondrial function.

[0065] Table 6 Statistical results of ATP content in mouse oocytes in different treatment groups

[0066]

[0067] In summary, compared with the positive control, 10 kV / cm nsPEF stimulation significantly reduced oxidative stress and apoptosis in mouse oocytes when the electrode dish was a 100 mm diameter dish. Mitochondrial membrane potential and ATP levels were measured, demonstrating that 10 kV / cm nsPEF not only did not impair mitochondrial function but also enhanced mitochondrial activity, which is crucial for oocyte activation and development. Therefore, we demonstrate that 10 kV / cm nsPEFs, when used in a 100 mm diameter dish, are a superior parthenogenetic activation method to A23187.

[0068] Example 3: Comparison of the technical effects of nanosecond pulse stimulation applied to the electrode cup and the electrode dish:

[0069] Nanosecond pulse electric field facility: The nanosecond pulse electric field facility involved in this embodiment is the same as that in Example 1, but the circuit load parts are an electrode cup and an electrode dish respectively. The electrode cup is consistent with that disclosed in Chinese patent CN107312749B, and has an electrode spacing of 4 mm and a parallel electrode area of ​​1 cm×2 cm (Bio-Rad). M16 culture medium is added to the electrode cup.

[0070] 3.1 Electrode cup activation of oocytes: 25 degranulated mouse oocytes were transferred into the circuit-loaded electrode cup through a glass pipette. After the electrode cup was correctly connected to the electric field generator, electric pulse stimulation was applied.

[0071] 3.2 Electrode dish to activate oocytes: A homemade electrode dish used in Example 1 was connected in parallel to the positive and negative ends of the electrode cup, with an electrode spacing of 1 mm. M16 culture medium was placed between the electrodes. Under a stereomicroscope, 25 degranulated mouse oocytes were evenly arranged between the parallel electrodes and electrical pulse stimulation was applied.

[0072] The 3.3 nanosecond pulse electric field all used 10 ns pulse width, 10 kV / cm pulse intensity, 1-2 Hz pulse frequency and 10 pulses.

[0073] 3.4 The balanced oocytes were randomly divided into groups of 25 per group. In the electrode cup treatment group, the electrode cup was first correctly connected to the electric field generator, 1 mL of M16 culture medium was added to the electrode cup, and 25 oocytes were transferred into the electrode cup through a glass pipette. A pulse width of 10 ns, a pulse intensity of 10 kV / cm, a pulse frequency of 1-2 Hz, and 10 pulses were applied. Subsequently, the electrically stimulated oocytes were aspirated from the electrode cup, quickly washed with culture medium, and transferred to an embryo culture dish preheated at 37°C. The embryonic development was detected by continuous culture at 37°C, saturated humidity, and 5% CO2 for 5-6 days. In the electrode dish treatment group, a platinum electrode dish with a 1 mm spacing was properly connected to the electric field generator. 10 μL of M16 culture medium was added between the two parallel electrodes. Twenty-five oocytes were then pipetted and evenly arranged between the parallel electrodes on a 37°C incubator on a stereomicroscope, ensuring that only single cells were present. Ten pulses were applied, each with a 10 ns pulse width, a 10 kV / cm pulse intensity, and a 1-2 Hz pulse frequency. The oocytes were then quickly washed and transferred to embryo culture dishes for continuous culture and monitoring of embryonic development. This experimental procedure was repeated four times to test the reproducibility of the different electrical activation methods.

[0074] 3.5 Results: As shown in Table 7,

[0075] Table 7 Statistical results of oocyte activation and blastocyst development in mice in different treatment groups

[0076]

[0077] a The total number of oocytes processed in different treatment groups;

[0078] b The statistical data of different treatment groups are presented as mean ± SD.

[0079] In four replicates:

[0080] In Experiment 1, of 25 oocytes exposed to 10 kV / cm in an electrode cup, 16 underwent cleavage to form two-cell embryos after 24 hours of in vitro culture, while the uncleaved cells remained at the MII stage and showed no signs of degeneration. Unactivated oocytes were discarded and continued in culture. After 120 hours of in vitro culture, seven blastocysts were observed, two of which had developed to the expanded blastocyst stage, while the remaining ones were at the primary blastocyst stage. Among oocytes exposed to the same electric pulse in an electrode dish, 21 oocytes formed two-cell embryos after 24 hours, while four oocytes remained uncleaved, with no significant morphological changes. After 120 hours of continued culture, 11 activated oocytes developed into blastocysts, one of which reached the hatching blastocyst stage, six expanded blastocysts, and the remaining ones were at the primary blastocyst stage.

[0081] In experiment 2, after culturing the activated oocytes in the electrode cup for 24 hours, 5 of them developed into 2-cell embryos, and the unactivated eggs did not fragment or degenerate. After continued culture for 120 hours, no blastocyst was formed. Two oocytes developed to the 4-cell embryo stage and then arrested, and 3 arrested at the 2-cell embryo stage. After culturing the activated oocytes in the electrode dish for 24 hours, 19 of them developed into 2-cell embryos, and the unactivated eggs did not fragment or degenerate. After continued culture for 120 hours, 8 blastocysts were formed, 3 of which developed to the expanded blastocyst stage, and the rest developed to the primary blastocyst stage.

[0082] In experiment three, after culturing the activated oocytes in the electrode cup for 24 hours, 8 of them developed into 2-cell embryos, the unactivated eggs did not fragment or degenerate, and some eggs showed polar body degeneration. After continued culture for 120 hours, 2 early blastocysts were observed to form; after culturing the activated oocytes in the electrode dish for 24 hours, 19 of them developed into 2-cell embryos. After continuing to culture the activated embryos for 120 hours, 10 blastocysts were formed, of which 6 developed into the expanded blastocyst stage and 4 were in the primary blastocyst stage.

[0083] In experiment 4, after culturing the activated oocytes in the electrode cup for 24 hours, 20 of them were activated to form 2-cell embryos, and the unactivated eggs did not show any fragmentation or degeneration. After continued culture for 120 hours, 11 blastocysts were observed to form, of which 4 developed into expanded blastocysts; after culturing the activated oocytes in the electrode dish for 24 hours, 23 of them were activated to form 2-cell embryos, and after continued culture for 120 hours, 15 blastocysts were formed, of which 3 blastocysts escaped from the zona pellucida to form hatching blastocysts, 6 developed into the expanded blastocyst stage, and the remaining 6 were in the primary blastocyst stage.

[0084] Combining Table 7 with the comparison of repeated experiments 1 to 4, it can be found that under the same pulse conditions, the electrode dish can stimulate oocytes to achieve a stable high activation rate, and the activated oocytes can develop into more blastocysts under the same culture conditions, and more develop to the expanded blastocyst stage or even the hatched blastocyst stage, showing the significant advantages of the electrode dish activation method over the existing electrode cup activation method in oocyte activation efficiency and blastocyst development efficiency. At the same time, in the experiment of activating oocyte development, the stability and repeatability of the electrode dish activation method are significantly better than the electrode cup activation method. This shows that the electrode dish activation device proposed in the present invention significantly improves the effect and stability of the electric pulse on cells compared to the conventional electrode cup, and improves the reliability and reproducibility of the experimental conclusions. Therefore, the electrode dish activation method has the advantage of ensuring uniform distribution of cells between parallel electrodes, significantly improves the efficiency of oocyte activation and embryo development, reduces the influence of experimental errors, and significantly improves the problems of unstable effect and poor reproducibility of the original nsPEF oocyte activation technology, thereby improving the safety and scalability of nsPEF in the field of assisted oocyte reproduction.

[0085] Example 4: Real-time imaging technology was used to monitor changes in oocyte cytoplasmic calcium signals before and after nsPEFs treatment.

[0086] 4.1 Experimental device optimization: In order to achieve real-time monitoring of small molecules in oocytes before and after nsPEFs treatment under laser confocal microscopy, especially the important second messenger "free calcium ion Ca 2+ In order to understand the changes in the electrode dish, the electrode dish used in this example is a 35 mm dish, which differs from the electrode dish used in Examples 1-3 only in size. Specifically, a 35 mm culture dish (NEST, 801001) was used. A 25 mm long platinum electrode was adhered to the center of the bottom of the culture dish using non-corrosive, non-cytotoxic neutral silicone adhesive. The two electrodes were parallel and close to the bottom of the culture dish with a spacing of 1 mm to ensure that the suspended cells between the electrodes could be uniformly stimulated by the uniform electric field generated by the nsPEFs. After the silicone adhesive adhering to the electrodes solidified, 10-20 μL of L16 culture medium was used to test the sealing between the electrodes and the culture dish.

[0087] 4.2 Instructions: Before the experiment, add 10 μL of M16 medium containing ProLong™ Live antifade reagent between the two electrodes. Cover with mineral oil to prevent evaporation and preheat in a 37°C, 5% CO2 saturated humidity incubator for 10-20 minutes. Before nsPEFs stimulation, secure the culture dish on the stage of a confocal laser microscope. Place the oocytes loaded with the Fluo4AM calcium probe evenly between the two electrodes, ensuring that only one oocyte between the parallel electrodes is stimulated by the nsPEFs. Connect the other electrode to the nanosecond pulsed electric field generator using a 1-meter long, low-impedance, electromagnetically resistant unshielded twisted-pair cable. Before nsPEFs stimulation, scan the confocal microscope for 1-2 minutes to record the initial intracytoplasmic calcium level. After nsPEFs stimulation, scan the oocytes continuously for 20-30 minutes, using Image J software to record the real-time changes in the intracytoplasmic calcium signal.

[0088] 4.3 Calculation of electrical dose: Between parallel electrodes, the calculation formula is as follows:

[0089]

[0090] Where AD (adsorbed dose, J / g) represents the electrical dose of nsPEFs, E represents the applied nsPEFs electric field strength (kV / m), d is the pulse electrode spacing (m), W is the pulse width (s), n is the number of applied nsPEFs pulses, R is the solution impedance between the electrodes, and M is the amount of culture medium between the electrodes.

[0091] The volume of M16 culture medium applied in the 35mm electrode dish of the present embodiment and the 100mm electrode dish used in Example 1 are different from those in the solution impedance. The volume of culture fluid between the electrodes is reduced, and the number of ions in the solution is small, resulting in an increase in the impedance of the solution in the circuit. Therefore, in order to achieve the same electrical dose in the oocyte activation experiment, it is necessary to apply a larger pulsed field strength. According to the electrical dose calculation formula, the relationship between the electrical dose of nsPEFs of the electrical stimulation system in the 35mm electrode dish and the 100mm electrode dish is shown in Table 8.

[0092] Table 8 Relationship between electric field strength and electrical dose between two nsPEFs systems

[0093]

[0094] As can be seen from Table 8, the electrical dose generated by applying 17-20 kV / cm in a 35 mm electrode dish is equivalent to the dose generated by applying 10 kV / cm in a 100 mm electrode dish.

[0095] 4.4 Experimental grouping: According to the corresponding nsPEF field strength in Table 8, nsPEF treatments of 15kV / cm, 17kV / cm, 20kV / cm and 25kV / cm were applied to the oocytes in a 35mm electrode dish. Each group used a pulse width of 10 ns, a pulse frequency of 1-2 Hz and 10 pulses. Oocytes treated with the calcium ion carrier A23187 served as the positive control group. The collected MII stage mouse oocytes were pre-incubated in M16 culture medium containing 5 μM cytoplasmic calcium ion probe fluo-4 AM for 30 min. The M16 culture medium was washed three times and then placed between the two electrodes of a 35mm electrode dish for nsPEFs stimulation. The fluorescence intensity in the oocytes before and after nsPEFs stimulation was recorded and the evolution of the calcium ion signal over time was plotted. The results are shown in Tables 9 and Figure 2-5 As shown:

[0096] Table 9 Statistical results of cytoplasmic calcium fluctuation parameters in oocytes treated with nsPEFs and A23187

[0097]

[0098] When oocytes were stimulated with 15 kV / cm nsPEFs, the cytoplasmic calcium level near the positive electrode side of the oocyte increased rapidly and slightly, and then quickly returned to the initial concentration. When oocytes were stimulated with 17 kV / cm nsPEFs, a single obvious calcium peak was formed in the oocyte after 10 pulses were applied. When we increased the electric field strength to 20 kV / cm, the oocytes stimulated with 10 pulses produced self-excited continuous calcium oscillations, accompanied by 5-8 calcium fluctuations. When oocytes were stimulated with 25 kV / cm nsPEF, the cytoplasmic calcium concentration increased rapidly to form a wide calcium peak. The calcium fluctuations induced by 17-25kV / cm nsPEF are shown in the figure. Figure 2-4 After A23187 treatment, a single wide calcium fluctuation is formed in the oocyte, and its cytoplasmic calcium fluctuation pattern is shown in the figure below. Figure 5 We analyzed the cytoplasmic calcium fluctuations in each group, including the calcium peak amplitude, calcium peak period, and the rate of the ascending and descending branches. The first calcium wave parameter was analyzed for the calcium oscillation pattern in the 20 kV / cm treatment group, as shown in Table 9. This indicates that nsPEF treatments of different field strengths can induce distinct calcium fluctuation patterns within oocytes. Combined with the oocyte activation effects of 20 kV / cm nsPEF stimulation in the electrode dish shown in Table 7, this suggests that 20 kV / cm nsPEF stimulation can successfully activate oocytes and lead to embryonic development by inducing changes in intracytoplasmic calcium concentration and inducing sperm-like sustained calcium oscillations.

[0099] Combining Examples 1-3 with this example, it is crucial to ensure that each oocyte receives electrical stimulation uniformly when nsPEFs are used to activate oocytes. Using an electrode dish with two electrodes placed parallel to each other with a spacing of 1 mm can ensure that oocytes can be evenly arranged in the electric field one by one and receive electrical stimulation uniformly. Depending on the application scenario, the size of the electrode dish can be large or small, but the electrical dose is 2-4×10 6 J / g can successfully activate oocytes. When the electrode dish diameter is 100 mm and the oocytes are from mice, the specific operating electric field strength is 8-12 kV / cm (preferably 10 kV / cm), the pulse frequency is 1-2 Hz, the pulse width is 10 ns, the number of pulses is 10, and each pulse lasts 5-8 seconds. When the electrode dish diameter is 35 mm and the oocytes are from mice, the specific operating electric field strength is 17-20 kV / cm (preferably 20 kV / cm), the pulse frequency is 1-2 Hz, the pulse width is 10 ns, the number of pulses is 10, and each pulse lasts 5-8 seconds.

[0100] Example 5: Exploration of the optimal treatment mode for nanosecond pulse electrical activation of oocyte development:

[0101] The above-mentioned implementation results confirmed that more than half of mouse oocytes can be activated after being subjected to a single nanosecond pulse with an electric field strength of 17-20 kV / cm in a 35mm diameter electrode dish. In this experiment, the duration of calcium oscillations during oocyte activation was prolonged by applying nanosecond electric pulses multiple times to explore the optimal electrical activation mode for mouse oocytes.

[0102] 5.1 Physiological calcium oscillation pattern in oocytes during fertilization: MII stage oocytes from mice that had undergone superovulation were collected and the granulosa cells surrounding the oocytes were stripped. The obtained oocytes were incubated with 5 μM Fluo4 AM calcium ion probe in a 37°C, 5% CO2 saturated humidity incubator for 20 minutes. The oocytes loaded with Fluo4AM calcium ion probe were incubated with pre-capacitated fresh mouse semen in an incubator for 15 minutes, and then the fertilized oocytes were immediately moved to a confocal microscope to record the real-time changes in the calcium ion signal in the cytoplasm. The calcium ion fluctuation pattern triggered by sperm-egg binding is as follows: Figure 6 As shown, it appears as a series of continuous sharp calcium peaks, with a spontaneous calcium peak formed every 200s. The characteristics of the calcium oscillation pattern changes are shown in Table 10.

[0103] Table 10 Statistical results of cytoplasmic calcium oscillation parameters in oocytes of nsPEFs and IVF groups

[0104]

[0105] Data are shown as mean ± standard error

[0106] *Indicates significant difference compared with the 17 kV / cm group (P<0.05), **P<0.01, ***P<0.001

[0107] # indicates significant difference compared with the IVF group (P<0.05), ##P<0.01, ###P<0.001

[0108] 5.2 Repeated 17 kV / cm Electric Pulse Oocyte Model Fertilization Sustained Calcium Oscillations: MII oocytes loaded with a Fluo4 AM calcium probe were transferred to a 35 mm diameter electrode dish using a glass pipette. Dynamic changes in intracytoplasmic calcium signals were continuously monitored under a confocal microscope. Approximately 1 minute after initial cytoplasmic calcium concentration in the oocyte was observed, electric pulses were applied to the oocyte between parallel electrodes at a pulse intensity of 17 kV / cm, a pulse frequency of 1-2 Hz, a pulse width of 10 ns, and a number of pulses. This generated a sharp calcium spike in the oocyte. Based on the physiological calcium oscillation characteristics shown in Table 10, a second 17 kV / cm pulse (pulse frequency of 1-2 Hz, a pulse width of 10 ns, and a number of pulses) was applied 3.5 minutes after the electric pulses were applied. Repeated 17 kV / cm electric pulses were applied to oocytes once, twice, four times, and for 2 hours, and the fluorescence intensity in oocytes before and after nsPEFs stimulation was recorded, and the evolution of calcium ion signals over time was plotted. Figure 7 The figure shows the dynamic changes in cytoplasmic calcium ion levels in oocytes during the first 40 minutes of repeated application of 17 kV / cm electric pulses for 2 hours.

[0109] 5.3 Repeated 20 kV / cm Electric Pulse Oocyte Fertilization Model Sustained Calcium Oscillations: MII-stage oocytes, with their cytoplasmic calcium levels labeled, were transferred to a custom electrode dish, and the dynamic changes in intracytoplasmic calcium signals were continuously monitored under a confocal microscope. Approximately 1 minute after monitoring the initial cytoplasmic calcium concentration in the oocyte, nanosecond pulses (20 kV / cm, 1-2 Hz, 10 ns, and 10 pulses) were applied to the oocyte between parallel electrodes. This generated a calcium oscillation pattern that lasted approximately 15 minutes. Based on the physiological calcium oscillation characteristics shown in Table 10, a second 20 kV / cm pulse (1-2 Hz, 10 ns, and 10 pulses) was applied 20 minutes after the electric pulses were applied. Repeated 20 kV / cm electric pulses were applied to oocytes once, twice, four times, and for 2 hours, and the fluorescence intensity in oocytes before and after nsPEFs stimulation was recorded, and the evolution of calcium ion signals over time was plotted. Figure 8The figure shows the dynamic changes in cytoplasmic calcium ion levels in oocytes during the first 40 minutes of repeated application of 20 kV / cm electric pulses for 2 hours.

[0110] 5.4 Effects of Repeated Electric Pulses on Oocyte Activation and Embryonic Development: Based on the above treatment method, collected MII mouse oocytes were randomly divided into groups and stimulated with electric pulses at a pulse intensity of 17 kV / cm with a 3.5-min inter-stimulation interval for one, two, four, and two hours (30-35 pulses). After stimulation, the oocytes were quickly washed and transferred to KSOM embryo culture medium preheated to 37°C for long-term culture. At a pulse intensity of 20 kV / cm with a 20-min inter-stimulation interval, the oocytes were stimulated with electric pulses for one, two, four, and two hours (6 pulses). After stimulation, the oocytes were quickly washed and transferred to KSOM embryo culture medium preheated to 37°C for long-term culture. As a control, MII oocytes from the IVF (in vitro fertilization) group were incubated with capacitated mouse semen for 2 hours, quickly washed, and similarly transferred to KSOM embryo culture medium preheated to 37°C for long-term culture to observe embryonic development. The activation and blastocyst development rates of each treatment group are shown in Table 11.

[0111] Table 11 Activation and development of oocytes in each treatment group

[0112]

[0113] Data are shown as mean ± SD;

[0114] Different superscript letters (ae) in the data indicate significant differences among the detected numbers in that group (p < 0.05).

[0115] 5.5 Results: The calcium ion fluctuation pattern triggered by sperm-egg binding is as follows Figure 1 As shown, the calcium spikes appear as a series of continuous, sharp peaks, each occurring spontaneously every 200 seconds. The results in Table 10 show that calcium spikes induced by either 17 kV / cm or 20 kV / cm pulses generally exhibit steeper rise and recovery rates. The spontaneous, sustained calcium oscillations that occur in oocytes following stimulation with 20 kV / cm nsPEFs are closer to the physiological calcium spike pattern observed in IVF, even though the peaks occur more closely.

[0116] Based on the interval duration of calcium peaks during fertilization and the characteristics of cytoplasmic calcium fluctuations caused by 17 kV / cm and 20 kV / cm electric pulses, it was determined that the cytoplasmic calcium oscillation pattern during fertilization was simulated by applying 17 kV / cm nsPEFs to oocytes every 3.5 minutes. The induced calcium ion change pattern is as follows: Figure 7The sustained calcium oscillation induced by 20 kV / cm stimulation can also form a physiological calcium oscillation pattern similar to the fertilization process through multiple pulses, as shown in Figure 8 shown.

[0117] As shown in Table 11, oocytes activated under sustained physiological calcium oscillations induced by multiple nsPEF pulses achieved higher parthenogenetic activation efficiency and blastocyst formation rates. Furthermore, oocyte development activated by 17-20 kV / cm nsPEFs stimulation lasting 2 hours showed no statistically significant difference in fertilization and blastocyst development rates compared to those achieved with IVF. This suggests that the longer-lasting calcium oscillations induced by repeated electrical pulses lead to better activation efficiency and embryo development rates, demonstrating that nsPEF electrical activation has broad potential for application and promotion in the field of AOA.

[0118] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0119] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for activating oocytes based on nanosecond pulse electrical stimulation, characterized in that: The oocytes were arranged uniformly between two parallel electrodes of an electrode dish. The electrode dish was placed in a culture dish with a spacing of 1 mm between the electrodes. The two electrodes were sealed at both ends. Culture medium was first placed between the parallel electrodes and covered with mineral oil to prevent evaporation of the culture medium. After preheating, the oocytes were placed uniformly between the two parallel electrodes. Nanosecond pulse electrical activation was then applied with an electrical dose of 2-4×10 6 J / g.

2. The method for activating oocytes based on nanosecond pulse electricity according to claim 1, characterized in that: The diameter of the culture dish is 35-100 mm.

3. The method for activating oocytes based on nanosecond pulse electrical stimulation according to claim 2, characterized in that: When the culture dish has a diameter of 100 mm and the oocytes are from mice, the electrical dose of the nanosecond pulse electrical activation treatment is 2-4×10 6 J / g, specifically: electric field strength is 8-12 kV / cm, pulse frequency is 1-2 Hz, pulse width is 10 ns, number of pulses is 10, and each pulse lasts 5-8 seconds.

4. The method for activating oocytes based on nanosecond pulse electricity according to claim 3, characterized in that: The electrical dose is 2.5×10 6 J / g or the electric field strength is 10 kV / cm.

5. The method for activating oocytes based on nanosecond pulse electricity according to claim 2, characterized in that: When the culture dish has a diameter of 35 mm and the oocytes are from mice, the electrical dose of the nanosecond pulse electrical activation treatment is 2-3×10 6 J / g, specifically: electric field strength is 17-20 kV / cm, pulse frequency is 1-2 Hz, pulse width is 10 ns, number of pulses is 10, and each pulse lasts 5-8 seconds.

6. The method for activating oocytes based on nanosecond pulse electricity according to claim 2, characterized in that: When the culture dish has a diameter of 100 mm and the oocytes are from humans, the electrical dose of the nanosecond pulse electrical activation treatment is 3-14×10 6 The electric field strength is 11-14 kV / cm J / g, the pulse frequency is 1-2 Hz, the pulse width is 10 ns, the number of pulses is 10-30, and each pulse lasts 8-20 seconds.

7. The method for activating oocytes based on nanosecond pulse electricity according to claim 2, characterized in that: When the culture dish has a diameter of 35 mm and the oocytes are from humans, the electrical dose of the nanosecond pulse electrical activation treatment is 3-14×10 6 The electric field strength is 20-25 kV / cm J / g, the pulse frequency is 1-2 Hz, the pulse width is 10 ns, the number of pulses is 10-30, and each pulse lasts 8-20 seconds.

8. The method for activating oocytes based on nanosecond pulse electricity according to claim 1, characterized in that: Both ends of the two parallel electrodes are sealed with neutral silicone glue.

9. The method for activating oocytes based on nanosecond pulse electrical stimulation according to claim 1, wherein: The number of times of applying the nanosecond pulse electrical activation treatment is 1 or more.

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