RU2583949C1 - Method of increasing functional-metabolic status of sperm cells obtained from semen of healthy individual in vitro under effect of low-intensity laser - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention can be used to improve functional-metabolic status of sperm cells in vitro. Sperm cells recovered from healthy donor are washed with physiologic saline by double centrifugation for 10 minutes at 1,500 rpm. Sperm cell suspension is exposed to semiconductor laser radiation with wave length of 632 nm in alternating pulse generation mode. Pulse duration is 2 ns, frequency is 100 Hz, and power density of radiation is 0.56 J/cm². Exposure time is 1 minute.

EFFECT: increased motor activity of sperm cells, enhancing their respiratory capacity, increased Farris index, increased content of citric acid and fructose in suspension of sperm cells.

1 cl, 3 tbl

Description

A method of increasing the functional and metabolic status of sperm obtained from the seminal fluid of a healthy person in vitro, under the influence of a low-intensity laser with variable pulse generation, relates to medicine, in particular to urology, andrology, reproductive medicine, can be used to enhance the functional sperm metabolic status before the in vitro fertilization (IVF) procedure in order to increase the effectiveness of artificial intrauterine insemination.

According to the data of medical and sociological monitoring, the frequency of those infertile couples where the male factor of infertility is currently limiting ranges from 9% to 16%, with no tendency to decrease in the territory of the Russian Federation [Pushkar D.Yu. et al. Epidemiological study of the prevalence of erectile dysfunction in the Russian Federation // Urology. - 2012. - T.6. - S.5-9]. A separate category should be identified fertility disorders associated with impaired / reduced sperm motility. If a strict correlation does not always exist between the number / morphology of spermatozoa and fertility, then immobile sperm cells are absolutely incapable of conception, since they are not able to reach the place of meeting with the egg in time (the peak of ovulation in a woman). Correction and elimination of this factor of male infertility should be considered as a way to increase reproductive potential. Currently, an increase in cases of male factors of infertility associated with a violation of the kinetic capabilities of spermatozoa [Kuchkov I.N. et al., 2005]. High-tech methods and assisted reproductive technologies that exist today help only partially (15–22%) solve the problem of overcoming the male factor of infertility in asthenozoospermia [Avramenko N.V. Assisted reproductive technologies // Zaporizhzhya Medical Journal. - 2014.-№3 (84)]. Today there is no scientifically based algorithm that reveals pathogenesis factors, including immunopathogenesis of male infertility, and, as a result, there are no clearly developed technologies that can achieve high therapeutic efficacy in the treatment of male infertility associated with impaired functional and metabolic status of spermatozoa. Therefore, promising areas of recovery and stimulation of motor activity of sperm in vitro will contribute to the development of new schemes of assisted reproductive technologies in reproductology.

i крiомедицини НАН

. Заявл. 21.06.00 №2000063592. Публ. 15.05.01. Бюл. №4; Патент №68172А, Украина, МПК A61D 19/02, A01N 1/02: «Cпociб отримання сперми собак» / Грищенко B.I.,

,

, Кучков I.M., Черкашина I.B. Iнститут Проблем

i Крiомедицини НАН

. 15.07.2004. Бюл. №7. Способ обеспечивает увеличение подвижности и дыхательной способности сперматозоидов в сперме мужчин, страдающих азооспермией, и, как следствие, влияет на повышение вероятности наступления беременности после внутриматочной инсеминации обработанной перфтораном спермы. Недостатком использования способа при астенозооспермии является отсутствие данных лабораторной диагностики о влиянии перфорана на процессы ликвидации кинетических и локомоторных дисфункций сперматозоидов, введения в сперму дорогостоящего препарата Перфторан. Способ ограничен в применении хранением спермы и не позволяет применять его при астенозооспермии, поскольку перфторан не влияет на подвижность сперматозоидов.A known method of preparing sperm for intrauterine insemination [V.I. Grishchenko, I.V. Cherkashina, I.N. Kuchkov, A.A. Gapon et al., 2001], which consists in the selection of sperm taking into account the requirements of the preanalytical stage of laboratory diagnostics with the addition of physiological saline and double 10 minute centrifugation at 1500 rpm, followed by the addition of perftoran sperm into the resulting mass in a ratio of 1: 3, 3 -5 minutes exposure followed by injection of the mixture into the uterine cavity [Patent No. 380303A, Ukraine, IPC 7 A01N 1/02: “Cnocib Hypothermic Preservation of Fragments of the Fabric of the Liver” / Grishchenko V.I., Mikulinsky Yu.Yu., Goncharuk OI, Gr bman CO, IB Institut problems Cherkashina

i kriomeditsini NAS

. Claim 06/21/00 №2000063592. Publ. 05/15/01. Bull. No. 4; Patent No. 68172A, Ukraine, IPC A61D 19/02, A01N 1/02: “How to remove sperm dogs” / Gryshchenko BI,

,

, Kuchkov IM, Cherkashina IB Institute of Problems

i Kriomeditsini NAS

. 07/15/2004. Bull. Number 7. The method provides an increase in sperm motility and respiratory capacity in the sperm of men suffering from azoospermia, and, as a result, affects the increased likelihood of pregnancy after intrauterine insemination of sperm treated with perfluorane. The disadvantage of using the method for asthenozoospermia is the lack of laboratory diagnostic data on the effect of perforane on the processes of eliminating kinetic and locomotor dysfunctions of sperm, the introduction of the expensive drug Perftoran into sperm. The method is limited in application by storage of sperm and does not allow its use in asthenozoospermia, since perfluoran does not affect sperm motility.

Also known is a method of increasing the fertilizing ability of sperm [Application for invention No.2000119029, IPC A61D 19/02; Deryazhentsev V.I., Bortnikova A.M., Shlygin A.N., Sheiko E.I., which consists in introducing into the medium a synthetic analogue of prostaglandin F2α-sodium salt of cloprostenol at a concentration of 200 μg per 100 ml of diluted semen of bulls. The disadvantage of the claimed method is that the synthetic analogue of prostaglandin F2α-sodium salt - cloprostenol has a pronounced ability to penetrate intact skin, and therefore, laboratory assistants and researchers should be especially careful when working with the drug, since prostaglandins F2-α can cause bronchospasm in people. The method is limited in application and does not allow its use in asthenozoospermia, since the mutagenic and teratogenic effects of the synthetic analogue of prostaglandin F2α-sodium salt - cloprostenol have not been completely studied.

There is a method of improving the quality of seminal fluid in pathospermia by exposure to laser radiation in vivo [Patent RU No. 2205047, M. cl. A61N 5/067; Gavrilov Yu.A., Kuzmichev L.N., Leonov B.V., Levchuk T.N., BIPM No. 2205047, IPC A61N 5/067, BIMP No. 15, 05.27.03, p. 353], which consists in the fact that the patient with infertility is performed for two weeks: 7-10 procedures of laser stimulation in the pulsed mode of the prostate gland, laser stimulation is carried out by installing sensors simultaneously on the perineum and suprapubic region with a frequency of 0.6 kHz, power 3, 5 W, lasting 8-10 minutes and testicles with a frequency of 0.3 kHz, power 1.5 W, lasting 7-9 minutes, 5-7 procedures for stimulating massage of the prostate. Along with the laser stimulation procedure, 22 mg of zinc amino acid chelate is prescribed and 200 μg of selenium 2 times a day, 50 ME puregon intramuscularly daily for a month. The disadvantage of this method is that laser therapy, when applied in vivo, has contraindications for acute inflammatory processes, neoplasms of the prostate gland (benign and malignant), tuberculosis, auto-inflammatory pathology [Bozhedomov A.A., Sukhikh A.T. Etiopathogenesis of autoimmune reactions against sperm // Andrology and genital surgery. - 2014. - No. 4. - S. 45-53]. Puregon is contraindicated in cases of impaired liver and / or kidney function. Contraindications of zinc chelate are: acute glomerulonephritis, exacerbation of peptic ulcer, bradycardia. The above circumstances are an obstacle to the application of this method in a significant number of patients suffering from various types of pathologies.

There is a method of preparing sperm for intrauterine insemination, including sperm sampling, preparation for fertilization by the "swim up" method [Kulakov V.I., Sukhikh G.T., Nazarenko T.A. Barren marriage. Modern approaches to diagnosis and treatment: a guide for doctors. - GEOTAR-Media, 2010], which consists in sequential washing of spermatozoa from seminal plasma (seminal plasma contains substances that suppress sperm activity) in physiological saline by double centrifugation for 10 minutes at 1500 rpm. Artificial insemination is performed with the husband’s sperm, which is introduced into the woman’s vagina without intercourse. As a result, the entire volume of sperm enters the area of the cervical pharynx, and not its insignificant part, as occurs during intercourse, which is an undoubted advantage of the proposed method. The essence of the method is that on the day of ovulation, the centrifuged and concentrated husband's sperm treated with saline in a nutrient medium is introduced into the uterine cavity through the cervix. The procedure for intrauterine insemination is painless, as the sperm is introduced using a special catheter with a very small diameter, which freely passes through the cervical canal. The duration of intrauterine insemination takes about 2 minutes. After the procedure, the woman is in a horizontal position for 20-30 minutes. The disadvantage of this method is its inefficiency (10-15%), as well as the requirements for the qualitative composition of sperm: the number of motile spermatozoa should be more than 10 million for insemination, therefore this method is completely ineffective if a man has sperm motility dysfunctions.

There is a method of increasing sperm motility and viability and a device for its implementation [Patent RU No.2012150445, IPC A61B 17/425], which consists in the fact that before intercourse a gas mixture is introduced into the woman’s vagina in the ratio of carbon dioxide 6%, nitrogen 94% under pressure above atmospheric. After the introduction of the gas mixture, the pelvis of the woman is kept in a raised state above body level, the vagina is fixed with an airtight pad. The use of the method seems problematic due to the need to carry out manipulations to hold the gas mixture in the vagina, to create, even for a short period of time, an anaerobic environment in the vagina that promotes the development of anaerobic microorganisms, which violates the natural resistance of the vagina, the presence of contraindications in women for the introduction of gas mixtures in the lower parts of the reproductive tract and most importantly - the complete inefficiency of the method in asthenozoospermia in men.

There is a method of improving sperm motility [Patent RU No. 98104562, IPC G01N 33/48], which consists in exposing the sperm suspension to a solution of 2-microglobulin, and with an increase in sperm activity more than 3 times from the original, it is assessed as high and sufficient for fertilization . The use of the method seems problematic due to the need to purchase an expensive drug-2-microglobulin and the possibility of widespread use of this methodology in andrology, perinatal and reproduction centers due to the economic component and the price of 2-microglobulin at the moment. In addition, the mutagenic and teratogenic effects of 2-microglobulin have not been fully studied.

A known method of increasing motor activity of sperm in vitro, proposed in a dissertation study Goryunova S.V. (1996) [Goryunov S.V. The effect of low-energy laser radiation on human sperm cells (experimental study) Author, diss. for the competition Art. Ph.D. 1996], the technical result of which is an increase in the physiological activity of spermatozoa, assessed by tests of utilization of fructose ejaculate and recovery of methylene blue. The results of the physiological status of spermatozoa, estimated by the total fructolysis activity, are taken into account by the rate of utilization of fructose, the oxidative activity of spermatozoa is assessed by the bleaching test of a methylene blue solution. To increase the energy-consuming activity of sperm, the author suggests using the red and infrared range of laser radiation (with wavelengths of 632.8 and 890.0 nm) at a dose of 0.6 J to 1.2 J. The author substantiates the choice of optimal parameters of laser radiation by wavelength, power and exposure for the use of laser therapy in a complex of procedures for the treatment of male infertility in order to improve the properties of spermatozoa by increasing its physiological activity. The calculation of the physiological activity of sperm, from the point of view of the author, is objective for judging the state of energy processes and indirectly motor activity of sperm.

The method has a number of undeniable advantages and allows you to take it as a prototype, as well as controversial issues that can be considered as flaws. First, the use of non-washed sperm with physiological saline may not lead to the desired increase in motor activity, since the seminal plasma contains substances that suppress sperm activity, although it is very possible that washing with saline will not affect the rate of general fructolysis and the rate of oxidative processes. Secondly, to analyze the results of the functional and metabolic potential of spermatozoa, it is necessary to take into account the combination of both clinical and laboratory (motility and varieties, viability, sperm agglutination), and biochemical parameters. What is not fully implemented S.V. Goryunov. Thirdly, S.V. Goryunov did not completely indicate the exposure parameters and the type of device generated by the laser exposure. In particular, the conclusions indicate a “corridor” of doses that stimulate the physiological potential of spermatozoa from 0.6 J to 1.2 J. (Conclusions based on the results of the dissertation research. Practical recommendations: conclusion No. 4, practical recommendation No. 3, without indicating constant or variable follow-up pulse frequency, pulse frequency, pulse duration, that is, the parameters required to take into account laser actions on biological objects.Lack of references to such a parameter as a continuous or pulsed laser action parameter, on w view is a crucial point, since with a constant follow-up of the pulse, the doses necessary to achieve energy stimulation of the cells should be lower [Moskvin SV The effectiveness of laser therapy. SV Moskvin, M., 2014], the author does not give references to the measurement of light flow during the study, which casts doubt on the calculation of the error of the measurements.

In the proposed method, the authors, for the first time, propose to use the method of increasing the functional and metabolic status of spermatozoa obtained from the seminal fluid of a healthy person, twice washed with saline to utilize the seminal plasma (the seminal plasma contains substances that inhibit sperm activity) in vitro under the action of a semiconductor laser with a wavelength of 632 nm, variable pulse generation, a duration of 2 ns, a pulse repetition rate of 100 Hz, energy density (up to th radiation) 0.56 J / cm ^2, the exposure time 1 min. The selected dose of 632 does not differ from 632.8 nm proposed by SV. Goryunov is not, if we talk about the photobiomodulating effect, but depends on the design features of the laser apparatus. In the case of our application, this is a semiconductor laser. In favor of the choice of this method of exposure, previously conducted studies revealed an increase in the functional activity of various types of cells, including sex cells under the influence of low-intensity laser radiation [Tolstykh PI, Klebanov GI Antioxidants and laser radiation in the treatment of wounds and trophic ulcers // M .: Ed. house "Eco". - 2002. - T.239. - p.21. Kozel A.I., Popov G.K. The mechanism of action of laser irradiation at the tissue and cellular levels // Vestnik RAMS. - 2000. - No. 2. - S. 41-43; Zabirova M.R., Frantseva O.V. A way to increase the functional and metabolic status of spermatozoa using low-intensity laser radiation in vitro and in vivo // Materials of the 69th inter-university (IV All-Russian) final scientific student conference M 45 with international participation dedicated to the 70th anniversary of Victory in the Great Patriotic War, Chelyabinsk, April 28, 2015 - Chelyabinsk: Publishing House of the South Ural State Medical University, 2015. - 171, [1] p. 2015. - p. 46; Baybekov I.M., Asadov X.D., Strizhkov N.A. The effect of low-intensity laser radiation on the seminiferous tubules and spermatozoa // Laser Medicine. - 2007. - T. 11. - No. 1. - S.18-21; Mazo E.B., Siluyanov K.A. The use of low-intensity laser radiation in the complex treatment of men with secretory infertility // Farmateka. 2008. - No. 9].

To solve the above problem, in an ejaculate obtained from 70 clinically healthy male volunteers aged 18 to 22 years old, collected taking into account the requirements of the preanalytical stage of laboratory diagnostics by masturbation, the sperm is collected in special vials and its preparation begins 20-30 minutes after liquefaction, by washing off the seminal plasma, since the seminal plasma contains substances that inhibit sperm activity. For this, physiological saline was added to the sperm, heated to 37 ° C, after which centrifugation was performed twice for 10 minutes at 1500 rpm. Inactive sperm, seminal plasma, cell debris settled, and active sperm went up. The average number of active sperm in the ejaculate was 67.34 ± 4.23 million / ml. To confirm the lack of kinetic dysfunctions of sperm in the seminal fluid in volunteers, the following laboratory parameters were determined: study of sperm motility (kinesiogram); counting the total number of sperm; assessment of sperm viability, assessment of respiratory potential. Microscopic examination of the ejaculate was carried out in 2 stages. At the 1st stage, sperm concentration, motility, aggregation and agglutination, as well as the presence of other cellular elements on unpainted native smears using light microscopy were evaluated. To assess sperm motility, native ejaculate in an amount of 20 μl was diluted in 400 μl of physiological saline heated to 37 ° C. The resulting mixture was filled in the Goryaev’s chamber and counted in 5 large squares located diagonally (only spermatozoa with heads lying inside the square were counted). The number of sperm in 1 ml of ejaculate was calculated by the formula X = (a · 4000 · 20) · 1000/80 = a · 1,000,000, where X is the number of sperm in 1 ml of ejaculate, a is the number of sperm counted in 5 large squares, 4000 - a factor that brings the result to a volume of 1 μl, based on the volume of a small square (1/4000), 20 - dilution of the ejaculate, 80 - the number of small squares, 1000 - a factor that brings the result to a volume of 1 ml. For the study of motility, 100 spermatozoa were counted, from which the percentages of actively motile, inactive (sperm making translational, rectilinear, but slow motion) and motionless sperm were calculated. Farris fertility rate was determined by the formula: 1 = V × N × M / 100, where V is the volume of ejaculate, ml; N is the number of sperm in 1 ml; M is the percentage of motile sperm. Digital material was processed using the method of variation statistics using the Statistica for Windows application package. The significance of differences in average values was judged using the nonparametric Mann-Whitney test. Differences were considered significant at p≤0.05. When studying ejaculate in healthy men, it was found: normakinesis - 60.1%, akinesis - 21.5%, hypokinesis - 18.4%, sperm viability - 81.0%. To assess the effect of low-intensity laser radiation in vitro, a suspension of spermatozoa was affected by laser radiation generated by a semiconductor laser with a wavelength of 632 nm, modulating variable pulse generation, 2 ns long, 100 Hz, radiation dose 0.56 J / cm ² . Samples containing spermatozoa during irradiation were located in thermostats specially equipped for the experiment and siliconized cuvettes with darkened walls. The laser exposure field was configured so that at any point in the action of the flux of light quanta, the deviation of its density was no more than 10% of the specified radiation parameters, measurements were made using the Anode apparatus (Russia). When studying a kinesiogram in healthy men after exposure to a low-intensity laser, it was found: normakinesis - 70.1%, akinesis - 21.5%, hypokinesis - 28.4%, sperm viability - 93.0%, an increase in Farris by 26%, which indicates a stimulating the influence of a low-intensity laser of the characteristics selected for the study with respect to the enhancement of the kinetic capabilities of sperm cells in vitro. Thus, low-intensity laser exposure with a variable pulse repetition for 1 minute leads to a statistically significant change in the functional activity of spermatozoa isolated from the seminal fluid of healthy volunteers compared with the results before laser exposure (Table 1).

Since the analysis of the functional and metabolic status includes, apart from the kinesiogram, the analysis of biochemical parameters, the analysis of the biochemical parameters of the sperm suspension included the study of α-glycosidase, citric acid and fructose before and after exposure to a low-intensity laser (Table 2).

The results of a study of the biochemical potential showed that in suspension of spermatozoa there is an increase in citric acid content by 17%, fructose by 12%, an increase in the respiratory ability of sperm cells by the ability to decolorize methylene blue solution by 25% relative to indicators of biochemical activity of sperm cells without exposure to a low-intensity laser. The proposed method differs from the existing ones in that under experimental conditions, spermatozoa isolated from a healthy donor washed twice with physiological saline at 1500 rpm twice subjected to the action of a low-intensity semiconductor laser at a wavelength of 632 nm, in the mode of variable pulse generation, pulse duration 2 ns and a frequency of 100 Hz, when the radiation energy density radiation of 0.56 J / cm ² 1 min exposure time, demonstrate improved metabolic status of functionally expressing a 35% increase in motor and physiological activity of sperm, namely, an increase in motor activity (the prevalence of normakinesis over hypokinesis and akinesis) by 35%, increased sperm respiratory capacity by the ability to decolorize methylene blue solution by 25%, an increase in Farris rate by 26%, an increase in the content citric acid by 17%, fructose by 12% in relation to the functional and metabolic activity of the sperm of a healthy donor, without exposure to a low-intensity laser. Due to the cheapness of the method and the simplicity of the manipulation, this method of increasing the functional and metabolic status of spermatozoa can be successfully used in most laboratories of perinatal, andrological centers with specially equipped facilities and clinical diagnostic laboratories. The inventive method can be widely used in gynecological and andrological practice, which indicates its compliance with the criterion of "industrial applicability".

Literature

1. Giesinger O.A. Immunological aspects of physiotherapeutic immunocorrection in the complex therapy of inflammatory diseases of the urogenital tract / O.A. Giesinger, K.G. Ishpakhtina, I.I. Dolgushin // News. SUSU. - 2010. - No. 6 (182). - S.85-91. - (Ser. "Education, healthcare, physical education". Issue 22).

2. Klebanov G.I. Low-intensity laser irradiation causes priming of white blood cells / G.I. Klebanov // The use of lasers for the diagnosis and treatment of diseases: Sat. - M .: LAS, 1996. - S.11-14.

3. Kozel A.I. The mechanism of action of laser radiation at the tissue and cellular level / A.I. Kozel, G.K. Popov // Vestn. RAMS. - 2000. - No. 2. - S. 41-43.

4. Kulakov V.I. Barren marriage. Methods of assisted reproductive technologies // Guide for physicians, ed. IN AND. Kulakova.- M., Geotar Media 2005.- P.441-444.

конф. "Вiд фундаментальних дослiджень - до прогресу в медицинi". - Харкiв. - 2005. - С.183-184.5. Kuchkov I.N. The function of cryobanks of human sperm in the modern development of assisted reproductive technologies / I.N. Kuchkov, V.E. Chadayev, I.Yu. Kuzmina, I.V. Cherkashin // Materials

conf. "A View of Fundamental Reach - to Progress in Medicine." - Kharkiv. - 2005. - S.183-184.

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Claims (1)

A method of increasing the functional and metabolic status of sperm obtained from the seminal fluid of a healthy person in vitro under the action of a low-intensity laser, including increasing the motor and physiological activity of sperm obtained from the seminal fluid of a healthy person in vitro, including exposure to low-intensity laser radiation in the red wavelength range, characterized in that the sperm cells isolated from a healthy donor are washed with saline by two direct centrifugation for 10 min at 1500 rpm and effect the suspension of sperm with a semiconductor laser with a wavelength of 632 nm in the regime of variable pulse generation, a pulse duration of 2 ns and a frequency of 100 Hz, at an energy density of radiation of 0.56 J / cm ² time exposure of 1 min, expressed in increased motor and physiological activity of sperm.