JPH0225426A - Low temperature preservation of sperm - Google Patents

Abstract

PURPOSE: To preserve spermatozoons at low temperature in high survival rate of the cells by mixing a freeze-preservative agent consisting of buffer salts, antishock components and antifreezing mixtures with spermatozoons and then cooling the mixture in a high thermal diffusion powder substance previously cooled at the temperature of the cryogenic liquid. CONSTITUTION: This method is carried out by mixing a freeze-preservative agent containing glucose, buffer salts (e.g. sodium citrate), antishock components (e.g. yolk) and antifreezing mixtures (e.g. glycerol), in addition preferably biological efficient substances (e.g. choline chloride), with spermatozoons and then cooling the mixture in a high-temperature diffusive powder substances (e.g. alumina) previously cooled at the temperature of the cryogenic liquid to obtain the higher effect on the morpholigical functional preservation of spermatozoons by avoiding the crystallization of the mixture and promoting the vitrification. The antifreezing mixture and choline chloride are preferably used in concentrations of 1.5-2.5wt.% and 2wt.%, respectively.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Application The present invention relates to cryobiology and cryomedicine, and in particular to a method for cryopreservation of spermatozoa.

The present invention may find most utility when applied to the deep freezing of human sperm for clinical use in the treatment of infertility.

This helps strengthen sperm cryobanks and thus allows their selection regarding the quality of frozen cells. Moreover, the use of frozen sperm is currently extremely effective in treating various types of infertility through in vitro fertilization of eggs.

The invention can also be applied in some other areas of cryobiology, such as, for example, animal husbandry, where new breeds of domestic animals are bred within the shortest possible period of time.

Cryopreservation of sperm of commercially valuable and rare fish breeds is also a means for their rapid reproduction.

The application of cryogenic technology for sperm cryopreservation covers an even wider field of human economic activity and public health maintenance.

Given the wide range of applications currently found for sperm freezing, there is a need for further improvements in preservation methods, as existing methods have limited the ability to use some frozen cells. This is due to the inability to adequately cryoprotect spermatozoa due to the progression of the crystallization process that ultimately leads to loss of osmotic capacity.

Therefore, vitrification
)-based cryopreservation methods for spermatozoa are clearly needed, with the aim of increasing their viability.

PRIOR ART One prior art method for sperm freezing by dropping anhydrous glycerol into the ejaculate to a final concentration of 10% is available and described by Fertil and Steril.
til, and 5terj1. ), Volume 14.

No. 1, 1.963, I.K. German (1,
, Sherman) “Improved Preservation Methods for Human Sperm by Freezing and Lyophilization”]. After dispensing the sperm with cryoprotectant in 0.5 mρ portions into glass or polyethylene ampoules and hermetically sealing them, the sperm were incubated at a rate of 25 °C/min to -75 °C for 16 min.
It is cooled down to minus 180°C at a rate of 180°C. Sperm survival rate after thawing is as low as 60%.

A drawback inherent to the method is the osmotic damage to cells due to the direct addition of glycerol to the ejaculate. The potential damage thereby caused to the cell membrane organelles, in particular their microsomal caps, is exacerbated during cooling, resulting in irreversible damage to the biological structures in most of the cells involved.

Another prior art method known in the art is for sperm cryopreservation with cryoprotectant in the form of particles by placing the sperm in holes made on a dry ice surface. [Treatment of male sterility, 1982, Berlino\Idelberg New York, I-Barkay (1,8arkay).

H. Zuckerman,
See “Cryopreservation and Storage of Sperm”, pp. 263-281].

The freezing process of 0.1-0.15 mg of sample proceeds in this case at a very high speed within 60 seconds.

Freezing sperm using particles is carried out in special equipment equipped with a temperature-controlled cabinet, which controls the cryogenic freezing process. The survival rate of sperm treated in this way has reached 70-75%.

The disadvantages of this method are related to the fact that freezing the sperm involves a crystallization process, which results in the sperm being frozen in a spherical shape. Therefore, the freezing of sperm in particles cannot be carried out aseptically, since temperature gradients occur in the frozen sperm sample due to the different cooling rates of the cells located in the center of the hole and on its walls.

Yet another sperm cryopreservation method is known to be available [Soviet Patent (SU, A) No. 1.
1.0B, No. 837], in which case an antifreeze agent,
That is, glycerol was gradually added to the spermatozoa and treated until a final concentration of 10% was obtained, with the spermatozoa containing 0.5
Placed in a mΩ sterile tube, the latter is sealed. Freezing is carried out in two stages, first at a rate of 0.5-1.0°C/min to +4 or 5°C, and then the sperm are incubated at that temperature for 3
0 to 60 minutes, then freezing minus 180 to
Frozen spermatozoa are stored in liquid nitrogen until a temperature of 190 °C is obtained at +36 °C.
It is decompressed with C. Sperm survival rate is 66%.

A particular disadvantage of said method is that the cryopreservation of spermatozoa involves a crystallization process whose progress is hindered by freezing conditions that do not cause vitrification. Moreover, the positive influence of cryoprotectants in the above process is offset by the negative phenomena associated with the direct addition of 10% glycerol to the ejaculate. This causes damage to the cytoplasmic membrane of the sperm, which in turn reduces the freezing resistance of this membrane. Furthermore, direct addition of anhydrous glycerol to spermatozoa causes a significant osmotic shift in the ejaculate. In this case, the strong dehydration phenomenon of cells causes partial denaturation of protein molecules and dysfunction of cell organelles (djso
reentation).

Prolonged contact of cells with highly concentrated glycerol media
It is related to the development of toxic effects of the above-mentioned cryoprotectants.

PROBLEM TO BE SOLVED The present invention has as its object to provide a method for the cryopreservation of spermatozoa, in which their cooling is sufficient for the progression of vitrification of the bulk of the extracellular water. It is carried out at a high rate, and the deleterious effects that occur on the biological structure of the spermatozoa due to recrystallization during thawing will occur to a lesser extent.

The object of the present invention is to provide a method for cryopreservation of spermatozoa comprising the introduction of cryoprotectants into the spermatozoa and their cooling to a refrigerant temperature, in which, according to the invention, buffer salts and anti-shock components are introduced into the spermatozoa. and both of them together with the cryoprotectant form a cryopreservative, and the spermatozoa are cooled in the cryopreservative medium in a high heat diffusive powder substance pre-cooled to the refrigerant temperature, as described above. This is achieved based on the fact that the concentration of the cryopreservative is high enough that an amorphous phase is generated during the process of freezing the sperm and the cryopreservative.

According to the proposed method, it is possible to obtain an even higher effect in preserving the morphology and function of spermatozoa by promoting vitrification, and it is possible to achieve a survival rate as high as 85%. This may be accomplished by the use of a highly thermodiffusive powder substance to cool the sperm, or
The material prevents the refrigerant vapor from forming an insulating shell around the object being cooled without direct contact between the object and the refrigerant. The resulting significant increase in cooling rate has been found to be sufficient for vitrification to proceed, yet making it possible to reduce the antifreeze concentration to a value that contributes to the realization of the above process. There is.

The proposed method makes it possible to exclude any extreme factors that hinder and even accelerate the crystallization process in frozen spermatozoa.

At the same time, the proposed method allows to simplify the cryopreservation process by reducing the number of operations and the time required for their implementation. At the same time, the present invention contributes to higher labor productivity and lower capital costs of the process by shortening the cold storage cycle.

According to a preferred embodiment of the present invention, alumina (Al2O3
) is used as a highly thermally diffusive powder material. This is related to the fact that cooling rates high enough for amorphous freezing occur to cause vitrification of spermatozoa in the proposed cryopreservative medium, but especially for spermatozoa such as embryos. This is explained by the relatively small size of these cells compared to other cells and thus by the small amount of free water in the spermatozoa to be frozen.

It is particularly advantageous for cryoprotectants to be used at concentrations of 1.5-2.5%, since not only their toxicity but also their permeability do not develop at such concentrations. or, provided that the latter is sufficiently high for vitrification to proceed under preselected cooling conditions.

According to one aspect of the invention, the cryopreservative contains a biologically active substance.

Biologically active substances are used in the cryopreservation of spermatozoa with the aim of increasing the viability of the cells to be processed by protecting the cytoplasmic membrane from temperature shock and "lytic effects" during the phase transformation into ice. .

Advantageously, choline chloride is used as biologically active substance.

Choline chloride, which functions as a methylating agent in biological processes based on the high energy potential of the nitrogen group in which three methyl groups and one hydroxyl group were present,
It is characterized by high basicity, which causes active hydration of water molecules by related substances and further enhances the antifreeze effect of the cryopreservative. Methyl groups stabilize the natural structure of biological membranes,
As a result, it contributes to their freeze resistance.

It is particularly advantageous for choline chloride to be used in a concentration of 2%.

When the concentration of choline chloride is low, the cryoprotectant cannot have high cryoprotective power, while its biologically active compounds at higher concentrations are involved in hydrophobic interactions with biopolymers.
Instead of stabilizing the membrane, it changes the membrane structure.

Other objects and advantages of the invention will become apparent from the detailed description of specific embodiments thereof below.

EMBODIMENTS To carry out the method, a metal container is immersed in a refrigerant, namely liquid nitrogen, which container is filled with a highly thermally diffusive powder substance, in which the diluted spermatozoa are placed. is thrust into it.

The present invention is carried out as follows. Once diluted, the sperm are frozen in a virtually aqueous solution containing glucose, sodium citrate, i.e., a buffer salt, egg yolk as an anti-shock component, a biologically active substance, i.e., choline chloride solution, and a cryoprotectant, i.e., glycerol. Mixed with preservatives. The diluted spermatozoa are equilibrated and then placed in a container made of food grade foil and then flared into a refrigerant, a highly thermally diffusive powder substance cooled to the temperature of liquid nitrogen. The container is then quickly transferred into liquid nitrogen for storage therein.

Sperm frozen in this way are thawed in a water bath.

Example] 2 mΩ of sperm was kept in a thermostat at 37°C for 25-30 minutes for dilution, and then added with 4.0 g of glucose, 1.0 g of sodium citrate. ．． 2g, egg yolk] 1 26.0mg, 2.0mg of 20% choline chloride solution, 3.0mM glycerol, and the remaining water.
: Add at a ratio of 1. Final glycerol concentration is 1.5%. The sperm thus diluted was held for 45 minutes,
0.5 mg each is placed in a container made of food grade foil with a back wall thickness of 0.3 mm, and the free end of the container is flared. To freeze the sperm, the container is placed in a freezing chamber, which is essentially a metal container filled with powdered alumina and immersed in liquid nitrogen, and then the container is maintained in the powdered alumina for 5 to 10 seconds; It is then transferred to liquid nitrogen for storage therein.

Thawing is performed after 3 weeks of cryopreservation by immersing the containers at 40° C. in a water bath. After thawing, the pH of sperm is 7.5.
The total sperm count was 52 million/mil, the motility rate was 66%, the active motility rate was 51.8%, and the normal morphology rate was 72%.

The viability of spermatozoa is calculated according to the following formula: Percentage of motile spermatozoa before freezing % According to the obtained results, it is 82.2%.

Example 2 2 rJl of spermatozoa were kept in a thermostat at 37°C for 25-30 minutes for dilution, then 4.0 g glucose 1 1.2 g sodium citrate, 26.0 mg egg yolk, 20%
A treatment agent containing 2.0 mg of choline chloride solution, 4.0 mg of glycerol and the remainder water is added in a 1:1 ratio. Final glycerol concentration is 2.0%. The diluted spermatozoa are held for 45 minutes and then placed in 0.5 mg portions into containers made of food grade foil with a wall thickness of 0.3 mm, and the free end of the container is flared. To freeze sperm,
The container is placed in a freezing chamber, which is essentially a metal container filled with powdered alumina and immersed in liquid nitrogen, and then the container is maintained in the powdered alumina for 5 to 10 seconds, after which it is placed in liquid nitrogen. Transfer to storage.

Thawing is performed after 3 weeks of cryopreservation by immersing the containers at 40° C. in a water bath. After thawing, sperm have a pH value of 7.5.
, total sperm count 52 million/mJ? The rate of movement was 66%, the rate of active movement was 53.1%, and the rate of normal morphology was 72%.

The sperm viability is calculated according to the following formula: % motile sperm before freezing According to the results obtained, it is 84.2%.

Example 3 Sperm 2r+dl in thermostat at 37° for dilution
C for 25-30 minutes, then glucose 4. Og
, sodium citrate 1.2g 1 egg yolk 26.0mfl,
20% choline chloride solution2. Omρ, glycerol5.
, 1=1 treatment agent containing Or+dl and remaining water
Add by ratio. Final glycerol concentration is 2.5%.

This diluted sperm was held for 45 minutes and then the wall thickness was 0.
．． 0,5m in a container made of 3+nm food grade foil
Pour in ρ and flare the free end of the container. To freeze the sperm, place the container into a freezing chamber, which is essentially a metal container filled with powdered alumina and immersed in liquid nitrogen, and then place the container in powdered alumina for 5 to 10 minutes.
held for seconds and then transferred to liquid nitrogen for storage therein.

Thawing is done by immersing the container at 40°C in a water bath.
This is done 3 weeks after cryopreservation. After thawing, the sperm have a pH value of 7.
5. The total number of spermatozoa was 52 million/mρ, the motility rate was 66%, the active motility rate was 54.7%, and the normal morphology rate was 72%.

The viability of spermatozoa is calculated according to the following formula: according to the results obtained, it is 86.5%.

The following is a tabulation of the results of experiments carried out to establish the fact that the objects of the invention can be achieved by applying low glycerol concentrations within certain limits and high freezing rates in powdered alumina media. It is presented below, which also includes the results of the previous example.

] 6 Table 1 Viability of cryopreserved sperm versus composition of freezing medium Table 2 Sperm motility versus freezing medium 1 Natural sperm without cryopreservative and glycerol 2 Cryopreservative used but with a small amount of glycerol 31.0 41.5 52.0 62.5 73.0 85.0 87.5±1.46 49.1±2.18 67.2±3.18 82.2±2,22 84.2±1.81 86.8±1,16 78.2±1.17 71.0±3.18 1 Natural sperm without cryopreservation agent and glycerol 2 Use cryopreservation agent but with a small amount of glycerol 31.0 415 52.0 62.5 73.0 85.0 22.5±1.38 28.0±2.16 42.0±2.86 51.8±1.12 53. 1±2.16 54.7±1.61 50.2±1.56 44.0±2.74 Table 3 Sperm motility versus sperm cryopreservation method Method of the present invention 64.1±3.18 54 .68±
1.12 Conventionally known methods B5. ll±3.64 43
．． 67±2.47p<0.05 p-Student Fischer
The data in the table above demonstrate that the motility of thawed spermatozoa in both cases is reduced compared to their pre-freezing motility. There is. However, the proposed method makes it possible to obtain spermatozoa with higher activity than known methods.

The above example shows that the lowest results are obtained when natural spermatozoa are cryopreserved. Addition of cryopreservative without glycerol slightly increases the degree of cell preservation. Even better is cryoprotection with cryopreservatives containing glycerol, with the greatest degree of cryoprotection being obtained when the glycerol concentration in the freezing medium is between 1.5 and 2.5%. The use of glycerol at 2.5% concentration in this freezing method can increase the survival rate to 19.8%, which is a very good sperm preservation rate.

As mentioned above, the presence of low concentrations of glycerol and choline chloride in the cryopreservative can create favorable conditions for the development of the most pronounced cryoprotective ability without any negative phenomena occurring.

This is particularly evidenced by the fact that the cells in the applied cryopreservative have a higher vitality than in the natural material.

EFFECTS OF THE INVENTION The present invention may find application in the deep freezing of human spermatozoa for clinical use in infertility treatment. The use of such sperm stocks (or banks) has implications for experimental-clinical and practical medicine, as well as advances in the cryopreservation of sperm from domestic animals.

Claims (1)

[Claims] 1. A method for cryopreservation of sperm, comprising the introduction of cryoprotectants into the sperm and cooling them to a refrigerant temperature, wherein buffer salts and anti-shock components are introduced into the sperm, and both together with cryoprotectants constitute a cryopreservative.
Moreover, an amorphous phase is formed when the spermatozoa are cooled using a cryopreservative medium in a highly thermally diffusive powder material that has been pre-cooled to the refrigerant temperature, and the cryopreservative is frozen in the spermatozoa and the cryopreservative agent. A method characterized in that the method has a sufficiently high concentration of 2. The method according to claim 1, wherein alumina (Al_2O_3) is used as the highly thermally diffusive powder material. 3. The method according to claim 1, wherein the cryoprotectant has a concentration of 1.5-2.5%. 4. The method according to claim 1, wherein the cryopreservative contains a biologically active substance. 5. Choline chloride is used as a biologically active substance,
The method according to claim 4. 6. The method according to claim 5, wherein choline chloride is used at a concentration of 2%.