Method for cryopreserving human sperm

FIELD: cryobiology, cryomedicine.

SUBSTANCE: method involves addition cryoprotective medium to sperm and its freezing with liquid nitrogen vapors at super high rates of freezing. Also, invention relates to composition of cryoprotective medium comprising glycerol ester, tris-buffer, EDTA, lecithin, kanamycin sulfate and water in special containers. Method provides significant enhancing viability and motility of spermatozoons and to increase frequency of pregnancy occurring after impregnation with sperm cryopreserved by proposed method.

EFFECT: improved cryopreserving method.

2 cl, 3 tbl, 3 ex

 

The present invention relates to the field of Cryobiology and cryomedicine and can be used in medical institutions in assisted reproductive technologies in the treatment of various forms of infertility.

Ensuring conditions for maintaining reproductive health is one of the most important criteria of the company's development strategy. It is known that the reproductive process can be disturbed by external factors, which include stress, deteriorating socio-economic conditions, adverse infectious diseases-epidemiology, chemical and physical effects. Due to the rapid development of new technologies the last two factors are the most dangerous. Demographics indicate the formation of a stable trend to an aging population, increase in its structure the share of elderly and senile age. These trends, combined with increasing current requirements to the quality of life in General and sexuality in particular determine the high relevance of the study of infertility in men.

Progress in the treatment of said pathology in the last decade, to a certain extent associated with the use of creamettes in the correction of the consequences of this disease. That is why cryoconserved what I reproductive human cells has now become one of the urgent problems of modern Cryobiology. Development of new methods of cryopreservation of the sperm of a man is required to create cryobanks of these cells, since only the presence of the full inventory of gametes can meet the needs of clinical medicine in this type of biological material. Available a sufficient number of cryopreserved sperm is the only way to have children, people with diseases of the reproductive system, paraplegia, progressive muscular dystrophy, myasthenia gravis, multiple sclerosis and other Organization of sperm cryobanks person solves the problem of birth full of children with parents working in hazardous industries science and technology associated with the possibility of radiation damage, chemical poisoning, and other adverse impacts. Insemination of cryopreserved sperm is also used with surgery on the genitals, forced or consciously (sterilization) leading to infertility. The sperm cryobanks provide the possibility to control the quality of ejaculates that is relevant in connection with the increase in areas of high radiation and deterioration of the epidemiological situation. Despite progress in the problem of low-temperature storage of human sperm, traditionally used methods of cryopreservation of these the notches are not effective enough.

There is a method of cryopreservation of the sperm of a man (Belous A. M., V.I. Grischenko, Parashchuk US Cryopreservation of reproductive cells. Kiev: Naukova Dumka, 1986. - 207 S.), in which the sperm add glycerol to a final concentration of 10%. Samples are poured into glass vials, sealed and cooled first at 25°C/min to minus 75°and then with a speed of 16°C/min to minus 196°C. the Samples are warmed at room temperature. The survival rate after thawing is within 60%.

The disadvantage of this method is the step of cooling the sample and as a consequence of its complexity, and low survival rate of cells after thawing.

There is a method of cryopreservation of sperm (Belous A.M., V.I. Grischenko, Parashchuk US Cryopreservation of reproductive cells. Kiev: Naukova Dumka, 1986. - 207 S.). In this way, under the protection of 10% glycerol samples were cooled at a rate of 1°C/min up to 5°With, then the sperm exhibited at this temperature for 30 min and then cooled at a rate of 20-25°C/min to minus 196°C. Survival of sperm, cryopreserved according to this method, amounted to 67.1 per cent.

The disadvantage of this method should be considered as the stage of equilibration sperm at a temperature of 5°C. At this stage is most pronounced cytotoxic effect on the cells to high concentrations of cryoprotectant used in this way.

There is a method of cryopreservation of human sperm, including the freezing of dilute glucose-citrate-yolk medium of sperm in the presence of glycerol in which to improve the survival rate of sperm freezing are 1.5-2.5%glycerol and powder of aluminum oxide (Grishchenko V., Kalugin J.V., Parashchuk US, Luchko N.A., Blackie E.N., V.F. Tarasov, Galchenko S.E. Method of cryopreservation of human sperm. A.S. 1660651 RF, MKI A 01 N 1/00 A 61 B 17/36. Publ. in B. I. 07.07.1991).

The disadvantage of this method is the use of containers, aluminum foil, non-toxic, pyrogen-free, but not tight. During storage of the samples in aluminum containers gets liquid nitrogen, which results when thawed to its rupture and loss of sample.

Known another method of cryopreservation of sperm of the same authors as the previous similar (Grishchenko V., Kalugin J.V., Parashchuk US, Luchko N.A., Blackie E.N., V.F. Tarasov, Galchenko S.E. Way of low temperature preservation of sperm. Patent 4965186, USA).

The disadvantage of this method, along with the use of containers made of aluminium foil is a small amount of frozen sample is 0.3 - 0.5 ml of This volume otogretogo suspension reproductive cells cannot be used in a number of procedures of assisted reproductive technologies.

For the prototype offers the alleged invention of the selected method of cryopreservation of human sperm, comprising adding to the sperm createsite environment, the location of the resulting biomaterial in disposable containers and freeze using liquid nitrogen vapor (Glassman AV, A.M. Karow, Benett C.E. The effects of cryopreservation and long distance transportation on human spermatozoa // Cryobiology. - 1979. - 16, No. 6. - P.615). In this way the sperm was added glycerin 100 μl every 15 seconds to 15% concentration. Cooling was carried out in containers in the form of polyvinyl vials, which, after 30-minute incubation at +4°frozen in the vapor of liquid nitrogen for 10 minutes. This has led to a 65% survival rate of sperm after thawing.

The disadvantage of this method is the long preparation period, a high concentration of cryoprotectant in the environment of freezing and low survival rate of cells after thawing.

Summarizing the description of the known methods, it should be said that the disadvantages of the prototype, and all the analogues described above, is the low survival rate of cells after thawing (60-67%). In addition, as shown by studies of the authors of the present invention, the mobility otogretogo sperms person when using the known methods is within 23±2.0%, and the frequency of pregnancy after fertilization of cryopreserved sperm donor is in the range of 11.8-21.2 percent (depending on the age of the women). You should also about the mark, the following low rates are observed against the background of such a purely technical drawbacks of the known methods, such as complexity, multiple, and excessive length.

The task of the invention is expected: improved survival and motility of sperm (after cryopreservation and thawing); increased frequency of pregnancy after fertilization of cryopreserved sperm; and reducing the complexity and duration of the method.

The task in the way cryopreservation of human sperm, which includes adding to the sperm createsite environment, the location of the resulting biomaterial in disposable containers and freeze using liquid nitrogen vapor, is achieved by the use trisamino medium of the following composition:

Onomatology ether of glycerol 2% to 20 ml

Tris - HCL buffer, pH 7,8 - 100 mm

Ethylenediaminetetraacetic acid - 1 mm

MgCl2·6N2O - 2 mm

Lecithin - 40-45 g

Kanamycin sulfate 500000 units - 0.2 g

Distilled water up to 1000 ml

Semen add trisamino environment in the ratio of 1:1, and the resulting biomaterial placed in containers with a volume of 0.5-1 ml, made of two-component polymer film thickness of 0.3-0.5 ml, consisting of polytetrafluoroethylene and polyamide, freezing spend with ultrafast MSE of the awns 3000-5000 K/min, what containers with the sample incubated for 10-15 min in a stream of vapor of liquid nitrogen, and then immersed in liquid nitrogen during storage and after storage heating is conducted in a water bath at a temperature 39-41°C.

In the basis of the method is the idea to avoid the formation on the surface of the container film boiling of the refrigerant, which is achieved by increasing the cooling rate of the sample up to 3000-5000 K/min and using cryoconserved, which promotes vitrification. When you use a different range of speeds will develop crystallization.

The principal motive of the use of vitrification freezing reproductive cells was the belief that vitrification is considered to be the ideal ice for preservation of biological objects at low temperatures. Only when using vitrification is possible to avoid irreversible changes in the ultrastructure of cells, caused by the formation of intracellular ice crystals, which is achieved by using verificarea solutions and special ways of lowering the temperature.

In the proposed method provides such a set of conditions, which helps to maximize the preservation of life and fertilizing properties of the male reproductive cells, scetches, in the end, and increases the survival rate of sperm (up to 85%), mobility (up to 47±3,0%) and pregnancy rate after fertilization, this sperm (to 61.9-72,4%). Essential traits for this effect are given in the claims, the characteristics that are related to the cooling rate, the procedures for cooling and annealing, the composition createsite environment and the use of special containers.

The proposed method is as follows.

For cryopreservation of sperm of the proposed method in terms of ultra-fast cooling rates used containers made of two-component polymer film consisting of Teflon and nylon, non-toxic, thickness 0.3·10-5m, calculated on the amount of 0.5-1 ml Containers are sterilized in an oven at 160°C for 1.5 hour mark and after placing them in biomaterial free side sealed.

The difference used in the proposed method, disposable, pyrogen-free containers from the existing is full of integrity, transparency (the ability of the evaluation sample, without opening the container)calculated the optimal size corresponding to the cooling rate of the sample and possible sterilization at high (160°C) temperature without loss of material containers the RA of their properties.

Used in the proposed method, the containers provide the optimum conditions of temperature sterilization, freezing and thawing, as can withstand without damage all these temperatures and contributes to optimal heat transfer and prevent the penetration of superfluid liquid nitrogen. The material of the containers, the thickness of the material, the volume of the containers is selected in the long course of special studies.

Taken for the cryopreservation of sperm first connect with createsite environment. As createsite environment using the solution of the following composition:

Onomatology ether of glycerol 2% to 20 ml

Tris - HCL buffer, pH 7,8 - 100 mm

Ethylenediaminetetraacetic acid - 1 mm

MgCl2·6N2O - 2 mm

Lecithin - 40-45 g

Kanamycin sulfate 500000 units - 0.2 g

Distilled water up to 1000 ml

The composition createsite environment is chosen in the long course of special experiments. Createdata environment is added to the sample in a 1:1 ratio.

When using more concentrated createsite environment will show a cytotoxic effect, which manifests itself in the reduction of the functional properties of the cells after thawing. By using a more dilute solution the effect of vitrification will not be attained.

Thus obtained b material placed in pre-proste-realizovannye containers, then the free end of the sealed containers.

Ultrafast cooling of frozen samples is achieved by keeping the containers in the stream of vapor of liquid nitrogen for 10-15 minutes, followed by immersion in liquid phase (i.e. liquid nitrogen) during storage (which is determined by special normative acts of the Russian Federation Ministry of health).

Upon completion of cryopreservation heating is conducted in a water bath at a temperature 39-41°C. At t 39-41°were obtained With optimal results. At a temperature of more 41°noted the increased number of deaths due to denaturation of the protein structures of cells. At a temperature of less than 39°, defrosted in the biomaterial undergo recrystallization processes, i.e. there is a formation of ice crystals, leading to rupture and disruption of the internal structures of cells.

Examples of specific performances

Example 1

For cryopreservation were collected ejaculates from donors with normospermic that met the following criteria: volume of 1 ml, containing 1 ml of ejaculate more than 80·106cells, the percentage of progressive motile forms (a+b) more than 60%, the proportion of morphologically normal forms more than 60%, cryotolerance.

Cryopreservation was performed according to the proposed method. This semen was added trisamino of the following composition:

Mon the methyl ester of glycerol and 2% 20 ml

Tris - HCL buffer, pH 7,8 - 100 mm

Ethylenediaminetetraacetic acid - 1 mm

MgCl2·6N2O - 2 mm

Lecithin - 40-45 g

Kanamycin sulfate 500000 units - 0.2 g

Distilled water up to 1000 ml

Trisamino medium were added to the sample in a 1:1 ratio.

Thus obtained sample was placed in a pre-sterilized in an oven at 160°C for 1.5 hours containers, made of two-component polymer film consisting of polytetrafluoroethylene and polyamide with a thickness of 0.3·10-5m, calculated on the amount of 0.5-1 ml the Free end of the containers were sealed. The container was kept in a stream of liquid nitrogen vapor for 10 minutes, followed by immersion in liquid nitrogen during storage. Heating was performed in a water bath at t41°C.

Table 1 provides data on the mobility of sperm, cryopreserved proposed method and the prototype.

Table 1

Mobility otogretogo of human sperm cryopreserved using standard methods of freezing (prototype) and with ultra-fast cooling rates (proposed method) (M±m, n=6)
The method of freezingNative suspension

Mobility, %
Createservicew the bedroom suspension

Mobility, %
The proposed method52±8,047±3,0
The standard method of freezing

(method-prototype)
52±8,023±2,0*
*the significance of differences relative to the rate of native suspension; p<0.05.

As can be seen from table data, mobility otogretogo sperms above freezing with the use of ultra-fast cooling rates

Example # 2

For cryopreservation of the proposed method was used spermin received from donors. Women on the testimony was done artificial insemination of cryopreserved sperm donor. The results are shown in table 2.

Table 2

The frequency of pregnancy after fertilization of cryopreserved sperm donor.
The METHOD of FREEZINGThe pregnancy rate, %
Up to 25 years25 -35 years
The proposed method72,461,9
The standard method of freezing (prototype)of 21.211,8

As VI is but from the data table, when using our method of freezing, the pregnancy rate was higher.

Example # 3

For cryopreservation of the proposed method was selected ejaculates from donors with normospermic that met the following criteria: volume of 1 ml, containing 1 ml of ejaculate more than 80·106cells, the percentage of progressive motile forms (a+b) more than 60%, the proportion of morphologically normal forms more than 60%, cryotolerance. Heating was performed in a water bath at 40°C. the results of freezing and thawing of cell suspension are presented in table 3.

Table 3

The survival of sperm in cryopreservation in a standard way (prototype) and when using the ultra-fast cooling rates (the proposed method).
The METHOD of FREEZINGSURVIVAL, %
The proposed method85
The standard method of freezing (prototype)60

From the table it is seen that when the cryopreservation using ultra-fast cooling rates the survival rate of the sperms was significantly higher.

Action superfine (3000-5000 K/min) cooling rates (the proposed method), which is optimal for the reproductive what's cells, associated with the best dehydrating effect, which is a prerequisite to prevent the development of intracellular crystallization. In addition, crisisit sperm, cryopreserved in this way, due to the sum of the properties of the components of cryoconserved aimed at neutralizing "effect of solution"that occur in these conditions. In connection with morphological and functional characteristics of sperm protect them when cryopreservation is achieved by an optimal combination of temperature cooling mode with low concentration range of cryoprotectant.

1. Method of cryopreservation of human sperm, which includes adding to the sperm createsite environment, the location of the resulting biomaterial in disposable containers and freeze using liquid nitrogen vapor, characterized in that use trisamino medium of the following composition:

Onomatology ether of glycerol 2%20 ml
Tris - HCL buffer pH of 7.8100 mm
Ethylenediaminetetraacetic acid1 mm
MgCL2·6N2About2 mm
Lecithin40-45 g
Kanamycin sulfate 500000 units0.2 g
In the distilled Yes To 1000 ml

semen add trisamino environment in the ratio of 1:1 and the resulting biomaterial placed in containers with a volume of 0.5-1 ml, made of two-component polymer film thickness of 0.3-0.5 ml, consisting of polytetrafluoroethylene and polyamide, freezing spend with ultrafast speeds 3000-5000 K/min, for which the container with the sample incubated for 10-15 min in a stream of vapor of liquid nitrogen, and then immersed in liquid nitrogen during storage and after storage heating is conducted in a water bath at a temperature 39-41°C.

2. The method according to claim 1, characterized in that the pre-sterilized containers in a drying Cabinet at 160°C for 1.5 h, and after placing them in biomaterial free end of the sealed container.



 

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