CN112956473A - Artificial transparent belt and preparation method thereof - Google Patents

Abstract

The invention provides an artificial transparent belt which is prepared from sodium alginate and polyornithine. The artificial zona pellucida provided by the invention can store a small amount of sperms (including single sperms) in the inner nuclear cavity, so that the loss of the sperms is effectively avoided, the biocompatibility of the used materials is good, the activity of the sperms is not influenced, and the problems of ethics, DNA or foreign heterologous protein pollution and the like do not exist.

Description

A kind of artificial transparent belt and preparation method

technical field

The invention belongs to the technical field of assisted reproduction, and in particular relates to an artificial zona pellucida and a preparation method.

Background technique

Infertility is a major disease that seriously endangers human reproductive health. The World Health Organization (WHO) predicts that infertility will be the third largest disease in the world after cancer and cardiovascular disease in the 21st century. WHO survey data shows that the incidence of infertility in the world accounts for about 10-15% of couples of childbearing age, of which the male factor accounts for about 40-50%. 70% of male infertility patients are caused by abnormal semen, mainly manifested as oligospermia, asthenozoospermia, oligoasthenospermia, teratospermia or azoospermia. According to statistics, the incidence of oligospermia, azoospermia and asthenospermia in men is increasing year by year. Azoospermia accounts for 10%-15% of infertile men, of which 80% are non-obstructive azoospermia, and there is currently no effective treatment. In vitro fertilization with testicular sperm retrieval combined with intrafollicular sperm injection (ICSI) technology is the only hope for genetic offspring in patients with non-obstructive azoospermia.

Clinically, the pregnancy rate of ICSI treatment cycle is only 50%. In order to obtain pregnancy, some patients need to repeat ICSI treatment several times. Since testicular sperm retrieval is an invasive operation, each sperm retrieval may cause testicular atrophy or further decline in testicular function, thereby completely losing spermatogenic function. In order to minimize the pain of repeated sperm retrieval and avoid the complete loss of testicular spermatogenic function, it is necessary to cryopreserve the micromotile sperm obtained from diagnostic puncture of azoospermia patients or remaining in the ICSI treatment cycle, so that the sperm can be collected from the woman. It will be resuscitated for ICSI treatment on egg day, which will greatly improve the flexibility and reliability of assisted fertility treatment for patients with oligospermia and azoospermia. At the same time, the sperm motility of patients with severe oligoasthenozoospermia is unstable, and preventive freezing of the sperm of patients is also very necessary, which will prevent the woman from having no sperm available on the egg retrieval day and cancel the cycle. Therefore, micro-sperm cryopreservation is a key technology to improve the success rate of pregnancy.

The cryopreservation of a small amount of sperm is a difficult problem in the field of assisted reproduction, and there is currently no ideal cryopreservation method. The zona pellucida method is a better method for cryopreserving a small amount of sperm. The zona pellucida is a layer of glycoprotein that surrounds the oocyte and plays an important role in oogenesis, egg protection, and preventing polyspermia. The contents of the zona pellucida were removed by micromanipulation, and then a small amount of sperm was injected into the empty zona pellucida with an ICSI microinjection needle for cryopreservation. In 1997, Walmsley et al. reported the birth of the first successful fertilization of infants with spermatozoa from an empty zona pellucida frozen testis, 3 out of 5 cycles, and 5 of them were twins. Empty zona pellucida freezing can significantly improve the recovery rate of sperm cryopreservation, maintain sperm motility, chromatin and DNA integrity. Studies have shown that the freezing and thawing recovery rate of zona pellucida frozen sperm is 88.3%, and the fertilization rate of ICSI motile sperm is 60%. It shows that the freezing of empty zona pellucida has low damage to sperm, and can provide good protection for freezing a small amount or single sperm. In 2005, Huang Hefeng and Xu Chenming used the zona pellucida evacuated from the cytoplasm as a cryopreservation carrier for cryopreservation of sperm. First, the biopsied testicular sperm was put into the closed zona pellucida, and then the sperm cryoprotectant was added and eluted for cryopreservation. During the recovery process, the sperm is sealed and stored in the zona pellucida to avoid the loss of sperm, and the sperm recovery rate is as high as 70%, which is much higher than the conventional cryopreservation method. Although the recovery rate of zona pellucida freezing is above 70%, there are still some problems, such as the method uses the zona pellucida of mammalian or human eggs, the source of materials is difficult (the human egg zona pellucida is difficult to obtain), and there are ethical issues Controversy (the risk of host DNA being injected into human eggs together with sperm during ICSI treatment), and the risk of zoonotic virus infection, it is difficult for this method to be widely used in assisted reproduction.

To sum up, although the zona pellucida method is a better method for cryopreservation of a small amount or a single sperm, and the cryopreservation of sperm is simple and fast, this method also has ethical problems and cannot avoid DNA or foreign heterologous protein contamination. limiting its clinical application.

SUMMARY OF THE INVENTION

In view of this, the technical problem to be solved by the present invention is to provide an artificial zona pellucida and a preparation method. The artificial zona pellucida provided by the present invention has good biocompatibility, does not affect sperm activity, and has no ethics, DNA or foreign Heterologous protein contamination, etc.

The invention provides an artificial zona pellucida prepared from sodium alginate and polyornithine.

Preferably, the artificial transparent belt is an outer shell with a hollow structure, and the outer shell is a composite membrane of sodium alginate and polyornithine polyelectrolyte;

The size of the artificial transparent belt is 80-200 μm, preferably 100-150 μm; the film thickness of the artificial transparent belt is 5-50 μm, preferably 10-30 μm.

Preferably, the polyornithine has a number-average molecular weight of 10 kDa to 100 kDa, preferably 10 kDa to 50 kDa.

The present invention also provides a preparation method of the above-mentioned artificial transparent zona, comprising the following steps:

A) mixing calcium alginate hydrogel microspheres with polyornithine solution, and performing a film-forming reaction to form a polyornithine polyelectrolyte complex membrane on the outside of the microspheres to obtain film-coated hydrogel microspheres;

B) Mixing the film-coated hydrogel microspheres with a sodium citrate solution to carry out a liquefaction reaction to obtain an artificial zona pellucida with a hollow structure.

Preferably, the calcium alginate hydrogel microspheres are prepared by the electrostatic drop method or the emulsification internal gelation method.

Preferably, the raw materials for preparing calcium alginate hydrogel microspheres are sodium alginate solution and calcium chloride solution, wherein the viscosity of the sodium alginate solution is 50-200 mPa·s, preferably 80-150 mPa·s The mass concentration of the calcium chloride solution is 0.5% to 2%, preferably 0.8% to 1.5%.

Preferably, the mass concentration of the polyornithine solution is 0.01% to 3%, and the preferred mass concentration is 0.05% to 2%.

Preferably, in step A), the reaction temperature is room temperature, and the reaction time is 1-20 min, preferably 3-10 min.

Preferably, the sodium citrate concentration is 30-70 mM, preferably 40-60 mM.

Preferably, the time of the liquefaction reaction is 5-30 minutes, preferably 10-20 minutes; the temperature of the liquefaction reaction is room temperature.

Compared with the prior art, the present invention provides an artificial zona pellucida prepared from sodium alginate and polyornithine. The artificial zona pellucida provided by the present invention can store a small amount of sperm (including single sperm) in the inner core cavity, effectively avoid sperm loss, and the materials used have good biocompatibility, have no impact on sperm activity, and have no ethical and DNA or foreign heterologous protein contamination and other issues.

Description of drawings

Fig. 1 is the microscope picture of the artificial zona pellucida prepared by embodiment 1;

Figure 2 shows the artificial zona pellucida loaded with sperm.

Detailed ways

The invention provides an artificial zona pellucida prepared from sodium alginate and polyornithine.

In the present invention, the artificial transparent zone is a shell with a hollow structure, and the shell is a composite membrane of sodium alginate and polyornithine polyelectrolyte.

The size of the artificial transparent belt is 80-200 μm, preferably 100-150 μm; the film thickness of the artificial transparent belt is 5-50 μm, preferably 10-30 μm.

The molecular weight of the polyornithine is 10kDa-100kDa, preferably 10kDa-50kDa.

The present invention also provides a preparation method of the above-mentioned artificial transparent zona, comprising the following steps:

A) mixing calcium alginate hydrogel microspheres with polyornithine solution, and performing a film-forming reaction to form a polyornithine polyelectrolyte complex membrane on the outside of the microspheres to obtain film-coated hydrogel microspheres;

B) Mixing the film-coated hydrogel microspheres with a sodium citrate solution to carry out a liquefaction reaction to obtain an artificial zona pellucida with a hollow structure.

The present invention firstly prepares calcium alginate hydrogel microspheres. The present invention has no special limitation on the preparation method of the calcium alginate hydrogel microspheres, and the method known to those skilled in the art may be sufficient. Preferably, the present invention adopts electrostatic droplet method or emulsification internal gelation method to prepare calcium alginate hydrogel microspheres.

The raw materials for preparing calcium alginate hydrogel microspheres are sodium alginate solution and calcium chloride solution, wherein the viscosity of the sodium alginate solution is 50-200 mPa·s, preferably 80-150 mPa·s; The mass concentration of the calcium solution is 0.5% to 2%, preferably 0.8% to 1.5%. The calcification time for preparing the hydrogel microspheres is 10-30 minutes, preferably 15-25 minutes; the preparation temperature of the microspheres is room temperature.

After the calcium alginate hydrogel microspheres are obtained, the calcium alginate hydrogel microspheres are mixed with the polyornithine solution, and a film-forming reaction is carried out to form a polyornithine polyelectrolyte complex membrane outside the microspheres to obtain a coating. Membrane hydrogel microspheres. Wherein, the molecular weight of polyornithine is 10kDa～100kDa, preferably 10kDa～50kDa, the mass concentration of the solution is 0.01%～3%, and the preferred mass concentration is 0.05%～2%; the reaction time is 1～20min, and the preferred reaction time is 3～3% 10min; the reaction temperature is room temperature.

The film-coated hydrogel microspheres are mixed with a sodium citrate solution to carry out a liquefaction reaction to obtain an artificial zona pellucida with a hollow structure. in,

The concentration of the sodium citrate solution is 30-70 mM, preferably 40-60 mM; the liquefaction time is 5-30 min, optimized for 10-20 min; the reaction temperature is room temperature.

The artificial zona pellucida provided by the invention is used for cryopreserving a small amount of sperm with high sperm motility, including single sperm, such as testicular puncture sperm, epididymal puncture sperm, sperm from patients with severe oligospermia and asthenozoospermia, and the like.

The invention provides an artificial zona pellucida that can be used for cryopreservation of a small amount of sperm and a preparation method, and aims to provide a small volume, closed space, easy operation and high sperm activity suitable for cryopreservation of a small amount of sperm (including single sperm , such as testicular puncture sperm, epididymal puncture sperm, sperm from patients with severe oligospermia and asthenozoospermia, etc.) artificial zona pellucida.

The technical scheme provided by the present invention has the following technical advantages:

1. The materials used in the artificial zona pellucida provided by the present invention are non-toxic, have good biocompatibility, have no effect on the loaded sperm, and are conducive to maintaining the activity of the sperm.

2. The artificial zona pellucida provided by the present invention has a small volume, and it is easy to find and grab sperm during micromanipulation, which is beneficial to maintain the activity of the sperm.

3. The artificial zona pellucida provided by the present invention has high strength, the shape remains intact after cryopreservation, effectively avoids the loss of sperm during cryopreservation and recovery, and has a high sperm recovery rate.

4. The artificial transparent zona provided by the present invention has a simple preparation process, and is easy to enlarge and industrialize production.

In order to further understand the present invention, the artificial transparent tape provided by the present invention and the preparation method are described below with reference to the examples, and the protection scope of the present invention is not limited by the following examples.

Example 1

The invention provides an artificial zona pellucida that can be used for cryopreservation of a small amount of sperm and a preparation method thereof,

1. Preparation of polyornithine artificial zona pellucida

(1) Prepare a sodium alginate solution with a mass concentration of 1.2%, the viscosity measured by a viscometer is 90.3 mPa·s, and drop the sodium alginate solution into a calcium chloride solution with a mass concentration of 1.1% by electrostatic drop method to form seaweed Calcium acid hydrogel microspheres, calcium chloride solution is removed after calcification for 30min;

(2) The calcium alginate hydrogel microspheres were added to the polyornithine (number-average molecular weight 22kD) solution with a mass concentration of 0.1% to carry out a film-forming reaction, and the reaction was carried out under stirring conditions for 10 minutes to form a polyelectrolyte outside the microspheres The complex membrane was washed with deionized water to remove unreacted polyornithine;

(3) The cleaned coated microspheres were added to a sodium citrate solution with a mass concentration of 1.4% to liquefy for 5 minutes, the internal gel was completely liquefied to form a hollow structure, and the microspheres were washed with deionized water to remove the sodium citrate solution, that is, Hollow artificial transparent belt.

2. Sperm cryopreservation

When freezing, first draw 1 drop of sperm culture solution (containing 30 sperm) and place it in a sperm culture dish to form a drop (about 0.5 μl), then add 10 ml of mineral oil to cover the drop, and draw 30 hollow microsphere carriers into the In the droplet of sperm culture solution, use a microinjection needle to grab 1 motile sperm and place it into 1 cryopreservation carrier, suck 30 cryopreservation tubes into the cryopreservation tube with a pipette, and add sperm cryopreservation solution to the cryopreservation tube. , after the liquid nitrogen vapor at a height of 4 cm above the liquid nitrogen surface was pre-cooled for 3 minutes (cooling rate of 20 ° C/min), the freezing tube was put into liquid nitrogen for rapid freezing and storage.

3. Sperm recovery

When resuscitated, take out the cryovial from the liquid nitrogen tank, thaw it quickly at 37°C, take out the cryopreservation solution containing the microsphere carrier from the cryotube with a pipette, and quickly add it to the petri dish (containing 5ml of sperm culture solution at 37°C), Look for sperm in the frozen carrier, grab the sperm with a microinjection needle and transfer it to another petri dish containing sperm medium for subsequent sperm immobilization and ICSI of mature eggs, see Figure 2, Figure 2 shows the loaded sperm Artificial transparent belt.

Referring to Fig. 1, Fig. 1 is a microscope picture of the artificial zona pellucida prepared in Example 1, wherein the particle size of the microspheres is 130 μm, and the film thickness is 15 μm; after cryopreservation, the integrity rate of the artificial zona pellucida is 100%, the sperm recovery rate is 100%, and the sperm survives. The complete rate of cryopreservation of human egg zona pellucida is 100%, sperm recovery rate is 100%, sperm survival rate is 86.7%, and there is no significant difference between artificial zona pellucida and artificial zona pellucida.

Example 2

The invention provides an artificial zona pellucida that can be used for cryopreservation of a small amount of sperm and a preparation method thereof,

1. Preparation of polyornithine artificial zona pellucida

(1) Prepare a sodium alginate solution with a mass concentration of 1.5%, the viscosity measured by a viscometer is 90.6 mPa·s, and the sodium alginate solution is dropped into a calcium chloride solution with a mass concentration of 1.1% by the electrostatic drop method to form seaweed Calcium acid hydrogel microspheres, calcium chloride solution is removed after calcification for 30min;

(2) The calcium alginate hydrogel microspheres were added to the polyornithine (number-average molecular weight 22kD) solution with a mass concentration of 0.1% to carry out a film-forming reaction, and the reaction was carried out under stirring conditions for 8 minutes to form a polyelectrolyte outside the microspheres The complex membrane was washed with deionized water to remove unreacted polyornithine;

(3) The cleaned coated microspheres were added to a sodium citrate solution with a mass concentration of 1.4% to liquefy for 5 minutes, the internal gel was completely liquefied to form a hollow structure, and the microspheres were washed with deionized water to remove the sodium citrate solution, that is, Hollow artificial transparent belt.

2. Sperm cryopreservation

When freezing, first draw 1 drop of sperm culture solution (containing 30 sperm) and place it in a sperm culture dish to form a drop (about 0.5 μl), then add 10 ml of mineral oil to cover the drop, and draw 30 hollow microsphere carriers into the In the droplet of sperm culture solution, use a microinjection needle to grab 1 motile sperm and place it into 1 cryopreservation carrier, use a pipette to suck up the cryopreservation tube into which 10 cryopreservation carriers are placed, and add sperm cryopreservation solution to the cryopreservation tube. , after the liquid nitrogen vapor at a height of 4 cm above the liquid nitrogen surface was pre-cooled for 3 minutes (cooling rate of 20 ° C/min), the freezing tube was put into liquid nitrogen for rapid freezing and storage.

(3) Sperm recovery

When resuscitated, take out the cryovial from the liquid nitrogen tank, thaw it quickly at 37°C, take out the cryopreservation solution containing the microsphere carrier from the cryotube with a pipette, and quickly add it to the petri dish (containing 5ml of sperm culture solution at 37°C), Search for sperm in the frozen carrier, grab the sperm with a microinjection needle and transfer it to another petri dish containing sperm medium for subsequent sperm immobilization and ICSI of mature eggs.

The particle size of the microspheres was 200 μm, and the membrane thickness was 15 μm; after cryopreservation, the integrity rate of the microspheres was 90%, the sperm recovery rate was 86.7%, and the sperm survival rate was 82.4%.

Example 3

The invention provides an artificial zona pellucida that can be used for cryopreservation of a small amount of sperm and a preparation method thereof,

1. Preparation of polyornithine artificial zona pellucida

(1) The sodium alginate solution with a mass concentration of 1.8% was prepared, and the viscosity measured by the viscometer was 180.9.mPa·s, and the sodium alginate solution was dropped into the calcium chloride solution with a mass concentration of 1.1% by the electrostatic drop method. Calcium alginate hydrogel microspheres are formed, and the calcium chloride solution is removed after calcification for 30 minutes;

(2) The calcium alginate hydrogel microspheres were added to the polyornithine (number-average molecular weight 22kD) solution with a mass concentration of 0.1% to carry out a film-forming reaction. Wash the microspheres with water to remove the polyornithine solution;

(3) The cleaned coated microspheres were added to a sodium citrate solution with a mass concentration of 1.4% to liquefy for 5 minutes, the internal gel was completely liquefied to form a hollow structure, and the microspheres were washed with deionized water to remove the sodium citrate solution, that is, Hollow artificial transparent belt.

2. Sperm cryopreservation

When freezing, first draw 1 drop of sperm culture solution (containing 40 sperm) and place it in a sperm culture dish to form a drop (about 0.5 μl), then add 10 ml of mineral oil to cover the drop, and draw 20 hollow microsphere carriers into the In the droplets of sperm culture solution, use a microinjection needle to grab 10 motile sperm and place them in a cryopreservation carrier, use a pipette to aspirate the cryopreservation tube into which 10 cryopreservation carriers are placed, and add sperm cryopreservation solution to the cryopreservation tube. , after the liquid nitrogen vapor at a height of 4 cm above the liquid nitrogen surface was pre-cooled for 3 minutes (cooling rate of 20 ° C/min), the freezing tube was put into liquid nitrogen for rapid freezing and storage.

(3) Sperm recovery

When resuscitated, take out the cryovial from the liquid nitrogen tank, thaw it quickly at 37°C, take out the cryopreservation solution containing the microsphere carrier from the cryotube with a pipette, and quickly add it to the petri dish (containing 5ml of sperm culture solution at 37°C), Search for sperm in the frozen carrier, grab the sperm with a microinjection needle and transfer it to another petri dish containing sperm medium for subsequent sperm immobilization and ICSI of mature eggs.

The particle size of the microspheres was 280 μm, and the film thickness was 15 μm; after cryopreservation, the integrity rate of the microspheres was 65%, the sperm recovery rate was 62.5%, and the sperm survival rate was 55%.

Example 4

The invention provides an artificial zona pellucida that can be used for cryopreservation of a small amount of sperm and a preparation method thereof,

1. Preparation of polyornithine artificial zona pellucida

(1) Prepare a sodium alginate solution with a mass concentration of 1.5%, the viscosity measured by a viscometer is 90.6 mPa·s, and the sodium alginate solution is dropped into a calcium chloride solution with a mass concentration of 1.1% by the electrostatic drop method to form seaweed Calcium acid hydrogel microspheres, calcium chloride solution is removed after calcification for 30min;

(2) The calcium alginate hydrogel microspheres were added to the polyornithine (number-average molecular weight 63kD) solution with a mass concentration of 0.1% to carry out a film-forming reaction, and the reaction was carried out under stirring conditions for 12 minutes to form a polyelectrolyte outside the microspheres The complex membrane was washed with deionized water to remove unreacted polyornithine;

(3) The cleaned coated microspheres were added to a sodium citrate solution with a mass concentration of 1.4% to liquefy for 5 minutes, the internal gel was completely liquefied to form a hollow structure, and the microspheres were washed with deionized water to remove the sodium citrate solution, that is, Hollow artificial transparent belt.

2. Sperm cryopreservation

When freezing, first draw 1 drop of sperm culture solution (containing 30 sperm) and place it in a sperm culture dish to form a drop (about 0.5 μl), then add 10 ml of mineral oil to cover the drop, and draw 30 hollow microsphere carriers into the In the droplet of sperm culture solution, use a microinjection needle to grab 1 motile sperm and place it into 1 cryopreservation carrier, use a pipette to suck up the cryopreservation tube into which 10 cryopreservation carriers are placed, and add sperm cryopreservation solution to the cryopreservation tube. , after the liquid nitrogen vapor at a height of 4 cm above the liquid nitrogen surface was pre-cooled and cooled for 3 minutes (cooling rate of 20 °C/min), the freezing tube was put into liquid nitrogen for rapid freezing and storage.

(3) Sperm recovery

When resuscitated, take out the cryovial from the liquid nitrogen tank, thaw it quickly at 37°C, take out the cryopreservation solution containing the microsphere carrier from the cryotube with a pipette, and quickly add it to the petri dish (containing 5ml of sperm culture solution at 37°C), Search for sperm in the frozen carrier, grab the sperm with a microinjection needle and transfer it to another petri dish containing sperm medium for subsequent sperm immobilization and ICSI of mature eggs.

The particle size of the microspheres was 200 μm, and the film thickness was 10 μm; after cryopreservation, the integrity rate of the microspheres was 67.3%, the sperm recovery rate was 66.4%, and the sperm survival rate was 62.2%.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims (10)

1. an artificial zona pellucida, characterized in that, prepared from sodium alginate and polyornithine. 2. The artificial transparent belt according to claim 1, wherein the artificial transparent belt is an outer shell with a hollow structure, and the outer shell is a composite membrane of sodium alginate and polyornithine polyelectrolyte; The size of the artificial transparent belt is 80-200 μm, preferably 100-150 μm; the film thickness of the artificial transparent belt is 5-50 μm, preferably 10-30 μm. 3 . The artificial zona pellucida according to claim 1 , wherein the polyornithine has a number average molecular weight of 10 kDa to 100 kDa, preferably 10 kDa to 50 kDa. 4 . 4. the preparation method of the artificial transparent belt as described in any one of claim 1～3, is characterized in that, comprises the following steps: A) mixing calcium alginate hydrogel microspheres with polyornithine solution, and performing a film-forming reaction to form a polyornithine polyelectrolyte complex membrane on the outside of the microspheres to obtain film-coated hydrogel microspheres; B) Mixing the film-coated hydrogel microspheres with a sodium citrate solution to carry out a liquefaction reaction to obtain an artificial zona pellucida with a hollow structure. 5 . The preparation method according to claim 4 , wherein the calcium alginate hydrogel microspheres are prepared by an electrostatic drop method or an emulsification internal gelation method. 6 . 6. preparation method according to claim 5 is characterized in that, the raw material of described preparation calcium alginate hydrogel microspheres is sodium alginate solution and calcium chloride solution, wherein, the viscosity of described sodium alginate solution It is 50-200 mPa·s, preferably 80-150 mPa·s; the mass concentration of the calcium chloride solution is 0.5%-2%, preferably 0.8%-1.5%. 7 . The preparation method according to claim 4 , wherein the mass concentration of the polyornithine solution is 0.01% to 3%, and the preferred mass concentration is 0.05% to 2%. 8 . 8. The preparation method according to claim 4, wherein in step A), the temperature of the reaction is room temperature, and the time of the reaction is 1-20 min, preferably 3-10 min. 9 . The preparation method according to claim 4 , wherein the concentration of the sodium citrate is 30-70 mM, preferably 40-60 mM. 10 . 10 . The preparation method according to claim 4 , wherein the time of the liquefaction reaction is 5-30 min, preferably 10-20 min; the temperature of the liquefaction reaction is room temperature. 11 .

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