AFK production in ejaculate analysis transcript. Modern problems of science and education

2
1 FSBI "Polyclinic No. 1" of the Administrative Department of the Russian Federation, Moscow; NUZ "Road Clinical Hospital named after N.А. Semashko ", Moscow; State Budgetary Educational Institution of Higher Education “First Moscow State Medical University named after THEM. Sechenov "Ministry of Health of the Russian Federation; FGAOU VO "RUDN", Moscow
2 NUZ "Road Clinical Hospital named after N.А. Semashko ", Moscow
3 NUZ "Road Clinical Hospital named after N.А. Semashko ", Moscow; State Budgetary Educational Institution of Higher Education “First Moscow State Medical University named after THEM. Sechenov "Ministry of Health of the Russian Federation

The male factor occurs in half of the cases of infertile marriages. Currently, it is generally accepted that the most common cause of male infertility - 35-40% of cases - is idiopathic oligo-, astheno- or teratozoospermia, when violations in the quantitative and qualitative parameters of sperm are observed in the absence of anamnestic risk factors, the absence of violations in the results of a medical examination and hormonal research. Antioxidants are popular treatments for male infertility. However, data on their effectiveness are inconsistent.
Purpose of the study: to show the capabilities of the domestic biologically active complex AndroDoz® for the treatment of idiopathic male infertility.
Material and methods: the study involved 30 men from infertile couples aged 25–45 years. The study of ejaculate was carried out in accordance with the recommendations of the WHO. Determined the content of reactive oxygen species in the native ejaculate and washed spermatozoa. Damage to sperm chromosomes was characterized by DNA fragmentation, assessed by chromatin dispersion in agarose gel. Ejaculate analysis was performed before and during treatment with AndroDoz® orally, 4 capsules per day (2 in the morning and in the evening).
Results: after 1.5 months treatment, 2/3 of patients showed a decrease in the percentage and degree of DNA fragmentation by an average of 5 and 10%, respectively (p<0,01); уменьшилась выраженность оксидативного стресса в 70% случаев в среднем по группе более чем в 2 раза (p<0,05). Показатели стандартной спермограммы при этом не менялись.
Conclusions: the drug AndroDoz® can be used in the treatment of idiopathic male infertility with signs of oxidative stress and violations of the integrity of sperm DNA; about 2/3 of patients respond to this therapy with positive changes in sperm quality.

Keywords: male infertility, oxidative stress, DNA fragmentation, antioxidants.

For citation: Bozhedomov V.A., Lipatova N.A., Bozhedomova G.E., Shcherbakova E.V., Komarina R.A. The use of a complex of nutrients for the treatment of male infertility // BC. 2016. No. 23. S. 1546-1552

Food additive for male infertility
Bozhedomov V.A. 1-4, Lipatova N.A. 2, Bozhedomova G.E. 2,3, Shcherbakova E.V. 2, Komarina R.A. 2

1 Outpatient Department No. 1 of the Department for Presidential Affairs, Moscow
2 N.A. Semashko Road Clinical Hospital, Moscow
3 .M. Sechenov First Moscow State Medical University
4 Peoples "Friendship University of Russia, Moscow

Half of the barren marriage cases accounts for male infertility. The most common causes (35-40%) of male infertility are idiopathic oligospermia, asthenospermia, and / or teratospermia. In these cases, abnormal sperm quantity and quality are not associated with anamnestic risk factors, abnormal medical examinations or hormonal imbalances. Antioxidants are popular agents for male infertility, however, their efficacy is controversial.
Aim. To analyze the efficacy of domestic bioactive additive AndroDoz® for idiopathic male infertility.
Patients and methods. 30 men from infertile couples aged 25−45 were enrolled. Ejaculate was examined according to WHO recommendations (including ROS measurement in native ejaculate and washed spermatozoa). Chromosomal aberrations in spermatozoa were assessed by DNA fragmentation evaluated with sperm chromatin dispersion test. Ejaculate was tested before and in the course of the treatment (oral AndroDoz® 2 capsules twice a day).
Results. After 1.5 months, the percentage and the degree of DNA fragmentation reduced by 5% and 10%, respectively, in two-third of the patients (p<0.01). The severity of oxidative stress decreased more than twice in 70% of the patients (p<0.05). Standard spermogram parameters remained unchanged.
Conclusions. AndroDoz® can be recommended for idiopathic male infertility with oxidative stress and altered DNA integrity of spermatozoa. Two-third of the patients respond to this treatment demonstrating sperm quality improvement.

Key words: male infertility, oxidative stress, DNA fragmentation, antioxidants.

For citation: Bozhedomov V.A., Lipatova N.A., Bozhedomova G.E. et al. Food additive for male infertility // RMJ. 2016. No. 23. P.1546 –1552.

The article discusses the use of a complex of nutrients for the treatment of male infertility

Introduction

The male factor occurs in half of the cases of infertile marriages. Currently, it is generally accepted that the most common cause of male infertility - 35-40% of cases - is idiopathic oligo-, astheno- or teratozoospermia, when violations in the quantitative and qualitative parameters of sperm are observed in the absence of anamnestic risk factors, the absence of violations in the results of a medical examination and hormonal research.
A large number of different drugs have been tested in such cases in order to improve the quality of the sperm. In recent years, antioxidants have been actively used, which are natural or synthetic biomolecules that prevent cell damage due to oxidative stress (OS) caused by the action of excessive amounts of reactive oxygen species (ROS). Antioxidants include vitamins E, C, A, carnitines, zinc, selenium, plant extracts and some other drugs and substances. Several randomized clinical trials have shown the potential for antioxidant supplementation to treat male subfertility. According to the results of meta-analyzes M.G. Showell et al. antioxidants improve vitality, concentration and progressive motility, binding to the ovum, reduce sperm DNA fragmentation, increase the percentage of pregnancies with natural conception and assisted reproductive technology programs. However, characterizing the quality of the studies included in the analysis, the review authors note that the level of evidence is “low” and “very low”. The authors conclude: "Antioxidants may have been effective in treating subfertile men, but the presentation of the research results was too inconsistent to be certain of these results." According to E.G. Hughes et al. , the combination of antioxidants is more effective: the likelihood of spontaneous pregnancy increases 4.2 times (95% CI 2.7-6.6), childbirth - 4.9 times (95% CI 1.9-12.2). The low cost and relatively low risk of toxicity of antioxidants are attractive to patients and physicians, which is why they are recommended by the European Association of Urology (EAU) for the treatment of male infertility, however, as highlighted in the latest EAU Guidelines, not for idiopathic forms.
The purpose of this study: to show the capabilities of the domestic biologically active complex AndroDoz® for the treatment of idiopathic male infertility. AndroDose® is an additional source of L-carnosine, carnitine, coenzyme Q10, glycyrrhizic acid, selenium, zinc, vitamins E and A.

Material and methods

The study took place from February to July 2016. It involved 30 men from infertile couples aged 25-45 years. The inclusion criteria for the study were:
no pregnancy in marriage for more than 12 months. having sex without contraception;
the presence of sperm in the ejaculate;
idiopathic oligo-, astheno- or teratozoospermia;
absence of infections of the reproductive tract (Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma hominis, Trichomonas vaginalis) diagnosed by polymerase chain reaction;
the absence of clinical and laboratory signs of the inflammatory process of the additional sex glands;
the absence of pronounced autoimmune reactions against spermatozoa, when antisperm antibodies (ASAT) cover no more than a tenth of the mobile gametes (MAR IgG<10%);
absence of palpable varicocele;
lack of pronounced somatic pathology;
absence of psychosexual and ejaculatory dysfunctions.
The exclusion criteria were:
established genetic causes of infertility (Klinefelter's syndrome, AZF microdeletions, CFTR);
azoospermia;
pyospermia;
overproduction of follicle-stimulating hormone;
palpable varicocele, confirmed by ultrasound;
the presence of an immune factor of infertility (MAR IgG> 10%);
severe somatic pathology;
psychosexual and ejaculatory dysfunction.
The study of the ejaculate was carried out in accordance with the requirements of the WHO: the concentration, motility and proportion of normal forms were determined, the number of progressively motile spermatozoa in the ejaculate and the integral index of sperm quality (ejaculate volume x sperm concentration x proportion of progressively motile forms x proportion of normal forms) were calculated. The percentage of motile spermatozoa coated with ACAT (SpermMar Kit, FertiPro, Belgium) was determined by the method of mixed agglutination. The OS was assessed by determining the intensity of free radical processes by luminol-dependent chemiluminescence using a LKB-Wallac 1256 luminometer (Finland) and Chemiluminometer-003 (Russia). The intensity of chemiluminescence was judged by the light sum and the maximum luminescence amplitude, which corresponded to the rate of ROS formation. ROS was determined in native ejaculate and washed spermatozoa in accordance with the protocol described in the WHO Guidelines. Damage to sperm chromosomes was characterized by DNA fragmentation, assessed by chromatin dispersion (Halosperm®; Halotech DNA, Spain) in an inert agarose gel with visual assessment under a microscope for halo formation after acid denaturation of DNA and lysis of nuclear proteins. In accordance with the recommendations of the manufacturer of the test system, the percentage of spermatozoa with signs of apoptosis and the degree of halo formation disturbance were assessed on a 5-point scale.
Ejaculate analysis was performed before and during treatment with AndroDoz® orally, 4 capsules per day (2 in the morning and in the evening). Several domestic publications have already described the effects of this drug in men. The peculiarity of our study consisted in assessing not only the indices of the standard spermogram, but also the production of ROS and the state of chromatin, which is often disrupted in OS.
The empirical data were processed using the Statistica software (StatSoft, USA). Group means were presented as M ± SD, median, 25–75% percentiles, captive range. On the Box-and-whisker plots, “outliers” are points that are far from the center of the distribution and are not typical for it (possibly, these are the results of observation errors or outliers). The significance of differences between groups was checked using the Student's test for pairwise related variant (t), signs (Z), Wilcoxon (W); differences were considered significant at p<0,05.
We present data on the effects observed early (1.5 months) of treatment.

results

The average age of the patients included in the study was 34.0 + 6.95 years. Primary infertility was in 18 people (59%), the duration of infertility averaged 28.9 ± 15.9 months in the group.
The main indicators of the spermogram are the volume of ejaculate, concentration, the percentage of progressively motile and morphologically normal spermatozoa after 1.5 months. treatment did not undergo significant changes (Table 1). Accordingly, the integral calculated values ​​did not change: the number of progressively motile spermatozoa in the ejaculate and the sperm quality index (see Table 1; p> 0.05). At the same time, a statistically significant decrease in the percentage of sperm with mixed pathology was noted: in absolute values ​​- 8% for the group average and 11% for the median (Fig. 1; p<0,01); положительная динамика данного показателя имела место у 80% пациентов (p<0,01).

After 1.5 months. treatment, a significant reduction in sperm DNA damage was observed (Table 2; Fig. 2). The percentage of spermatozoa with DNA fragmentation in absolute values ​​decreased on average in the group by 4%, the median - by 5% (relative to the initial level -23% for the average, p<0,01 и −28% для медианы, р=0,01); меньше стала степень выраженности таких нарушений, оцениваемых по степени дисперсии хроматина (−10% для средней, p<0,05 и −12% для медианы, p<0,01). Положительная динамика фрагментации ДНК на фоне лечения имела место у 67% мужчин (p>0,05).


During treatment, the severity of OS significantly decreased, as evidenced by a decrease in ROS production by washed spermatozoa in 70% of cases (Table 3; p<0,05) в среднем по группе более чем в 2 раза; изменения медианы были еще более наглядны – −82% (рис. 3; p<0,05). При этом продукция АФК в нативном эякуляте изменялась статистически несущественно и даже имела тенденцию к повышению (см. табл. 3; p>0,05).


Discussion

Antioxidants are popular drugs for treating sperm quality disorders, according to various publications. Various pharmaceutical companies offer ready-made nutrient complexes that, according to manufacturers, can improve male fertility. Our data have confirmed the positive effect of the domestic complex AndroDoz® in male infertility associated with OS. ROS production by washed spermatozoa decreased, according to our study, by an average of 2-5 times compared with the initial level. The severity of intracellular OS, assessed by the production of ROS by washed spermatozoa, is of particular importance, since the close proximity between spermatogenic free radicals and sperm DNA determines their greatest role in impaired fertility.
At the same time, there was a decrease in the percentage of spermatozoa with DNA fragmentation and the severity of such chromatin disorders. The decrease in the fragmentation index averaged 4–5% in absolute terms, or almost a quarter of the initial level. Since the relationship between the amount of ROS in the sperm, the severity of sperm OS and the fragmentation of their DNA is recognized by most specialists, such results of our study seem to be quite logical.
At the same time, it was found that positive dynamics of ROS production and DNA fragmentation during treatment took place only in 2/3 of cases. At the same time, an improvement in these indicators was not always observed at high levels of OS and DNA fragmentation, and vice versa. It remains unclear to us why the severity of intracellular OS, assessed by the production of ROS by washed spermatozoa, decreased, but the production of ROS in the native ejaculate did not change, because antioxidants had to chemically bind active radicals in both cases. Since this study included patients without signs of an infectious-inflammatory process, the effect is difficult to explain by the influence of sperm leukocytes. Elucidation of these patterns should be the subject of further research.
At the same time, our data showed that the standard indicators of spermogram - volume, concentration, motility and morphology of spermatozoa - changed insignificantly against the background of the treatment, while, according to A.A. Kamalova et al. , an increase in semen analysis occurs in 87.6%, according to M.K. Alchinbaeva et al. - in 92% of cases. E.S. Dendeberov et al. write that the use of AndroDoz after 3 months. led to an increase in ejaculate volume by 45.7%, sperm concentration by 18.5%, general motility by 33.7%, active motility by 38.4% and the number of morphologically normal forms by 50%. The data of A.A. Proskurina et al. even more optimistic: an increase in volume by 1.95 times, mobility by 7.43 times, concentration by 1.53 times and the percentage of normal forms by 6.75 times from the initial values. However, such data raise doubts: to date, there are no treatments that can increase the proportion of normal forms by 50-675%.
The absence of a significant improvement in the indices of the standard spermogram in our study (with the exception of a decrease in the percentage of spermatozoa with mixed pathology, the positive dynamics of this indicator took place in 80% of cases), possibly due to the fact that the observation period was only 1.5 months, while as the duration of the cycle of spermatogenesis, including the period of passage through the epididymis, is about 3 months. It is possible that an improvement in other indicators of the standard spermogram can occur with a longer use of the drug. It is also obvious that the initial indicators of the spermogram are important: the degree of oligo-, astheno- and teratozoospermia and their combination. Clarification of the drug's capabilities in the treatment of various forms of pathozoospermia will be the subject of discussion in subsequent publications.
The conclusion that the use of commercial vitamin and antioxidant complexes does not always lead to a pronounced improvement in the parameters of the standard spermogram is consistent with the data of a number of foreign controlled studies. Thus, the appointment of a complex of antioxidants showed an improvement in sperm motility only in 3 out of 6 such studies, the concentration increased only in 1 out of 6.
Perhaps the effectiveness of a particular antioxidant drug depends on its qualitative and quantitative composition. Effective doses of antioxidant monopreparations, according to a number of reviews, are: vitamin E> 300 mg / day, vitamin C> 1000 mg / day, carnitines (L- and acetyl-)> 3000 mg / day, selenium - 100-225 μg / day , coenzyme Q10 - 60-200 mg / day, zinc (ZnSO4) - 66-400 mg / day, glutathione - 600 mg / day, which significantly exceeds the established daily upper permissible levels of consumption for these substances and makes them unsafe for long-term use. Unbalanced antioxidant complexes can cause excessive elimination of free oxygen radicals, which are necessary for the normal course of the acrosome reaction and sperm capacity, and induce restorative stress as a rebound effect. There is evidence that with an overabundance of antioxidants, an increase in the decondensation of nuclear chromatin of spermatozoa is observed by more than 20%, which, according to F. Absalan, Y. Menezo et al., Leads to recurrent miscarriage. Changes in chromatin structure can cause changes in gene expression and affect the implantation process as a result of asynchronous condensation of chromosomes, as well as the presence of cytoplasmic fragments in the embryo. It has been established that long-term intake of such a well-known antioxidant as ascorbic acid, or its high dosages, have a very ambiguous significance for the stimulation of spermatogenesis. Vitamin C in hyperdoses destroys the disulfide bonds of proteins, contributing to their denaturation, which leads to membrane oxidation in phases I and III of spermatogenesis and improper DNA packaging.
Therefore, often commercially produced drugs represent a compromise, where low (safe for use, at the physiological level) dosages of antioxidants are compensated for by a wide range of active substances, in the hope of their synergy.
Thus, despite all the advantages of antioxidant therapy, drugs in this group should be prescribed with some caution, choosing balanced drugs with a good evidence base.
In addition, antioxidants can be effective only in the case of an excess of ROS and the development of OS. Since OS is not always the reason for the deterioration of sperm quality - in 30-80% of cases, according to M.G. Showell et al. , and about 40% according to our data - the appointment of antioxidants for the treatment of male infertility seems to be justified only in these cases.
Obviously, therefore, the EAU Guidelines also do not recommend prescribing antioxidant intake to all men in a row with idiopathic infertility. Currently, there is convincing evidence of the effectiveness of oral antioxidant intake by men only when preparing a couple for subsequent in vitro fertilization, while the role of antioxidants in the process of natural conception still needs further study.
Based on the last Cochrane review by M.G. Showell et al. including 48 studies comparing mono- and combined antioxidants with placebo, no treatment, or another antioxidant in a population of 4,179 subfertile men, antioxidants are still likely to be effective as a pregravid preparation in subfertile men. The expected clinical pregnancy rate for subfertile men who did not take any antioxidants was 6 cases out of 100, compared with 11-28 cases out of 100 men who took antioxidants. The results of the review also showed that the expected live birth rate for subfertile men in the placebo group or without treatment is 5 out of 100, compared with men taking antioxidants - from 10 to 31 out of 100.
Thus, we have shown that the use of the domestic complex AndroDoz® at a dose of 4 capsules per day after 1.5 months. treatment leads to an improvement in sperm quality - a significant decrease in the percentage of spermatozoa with mixed pathology and / or DNA fragmentation against the background of a decrease in ROS production by washed spermatozoa, which is associated with a decrease in the severity of OS in male gametes.
The integrity of male DNA is of vital importance for the interaction of the sperm and the egg, fertilization and early embryonic development, in connection with which the results obtained are of undoubted practical interest.

conclusions

1. The drug AndroDoz® can be used in the treatment of idiopathic male infertility with signs of OS and violations of the integrity of sperm DNA; about 2/3 of patients respond to this therapy with positive changes in sperm quality.
2. Against the background of this treatment, ROS production by washed spermatozoa significantly decreases, which indicates a decrease in the severity of OS in male gametes.
3. Against the background of this treatment, there is a significant improvement in the structure of sperm DNA in 67% of men.
4. There was a statistically significant decrease in the percentage of spermatozoa with mixed pathology in the spermogram in 80% of cases.
5. For 1.5 months. No significant improvement in the rest of the spermogram parameters (volume, concentration, proportion of progressively mobile and morphologically normal forms) was observed during treatment with AndroDoz®; changes in these parameters were multidirectional.
6. The study was significantly limited by the absence of a control group, the short duration of follow-up and the lack of registration of the pregnancies that occurred. Accordingly, more research is required.

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  • Special properties of the oxygen molecule and its conversion products
  • Targeted production of ROS by living cells

    • Targeted production of ROS by living cells

    All organisms are equipped with a variety of mechanisms for the targeted generation of ROS. The enzyme NADPH oxidase has been known for a long time, actively producing "toxic" superoxide, behind which the entire gamma of ROS is generated. But until very recently, it was considered a specific accessory of phagocytic cells of the immune system, explaining the need for ROS production by the critical circumstances of protection against pathogenic microorganisms and viruses. It has now become clear that this enzyme is ubiquitous. He and similar enzymes are found in the cells of all three layers of the aorta, in fibroblasts, sinocytes, chondrocytes, plant cells, yeast, in kidney cells, neurons and astrocytes of the cerebral cortex O 2 - á produce other ubiquitous enzymes: NO synthase, cytochrome P-450, gamma glutamyl transpeptidase, and the list continues to grow. It has recently been found that all antibodies are capable of producing H 2 O 2, i. E. they are also ROS generators. According to some estimates, even at rest, 10-15% of all oxygen consumed by animals undergoes one-electron reduction, and under stress conditions, when the activity of superoxide-generating enzymes sharply increases, the rate of oxygen reduction increases by another 20%. Thus, ROS must play a very important role in normal physiology.

    1

    This review article examines the current understanding of the mechanisms that underlie the generation of reactive oxygen species during permeabilization of mitochondrial membranes. The role of calcium ions and complexes of the mitochondrial respiratory chain is considered. The influence of the level of pyridine nucleotides, components of the antioxidant system, as well as the participation of matrix Ca2 + -activated dehydrogenases is discussed. There are data in the literature showing that the induction of the mitochondrial Ca2 + -dependent pore causes conformational rearrangements of the respiratory complexes I, II, and III, which enhances the generation of reactive oxygen species. The entry of calcium into the mitochondrial matrix can increase the rate of production of reactive oxygen species due to the activation of pyruvate dehydrogenase and a-ketoglutarate dehydrogenase, as well as facilitate the release of cytochrome c into the cytosol upon induction of the mitochondrial pore. The release of glutathione and reduced pyridine nucleotides through the pore reduces the antioxidant protection of the mitochondrial matrix and increases the production of superoxide anion and hydrogen peroxide. The phenomenon of a burst of reactive oxygen species caused by mitochondrial permeabilization accompanies various pathological conditions, including ischemia followed by reperfusion; therefore, an understanding of the molecular processes underlying it is necessary for the further development of methods for its pharmacological correction.

    reactive oxygen species

    mitochondrial pore

    mitochondrial respiratory chain

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    Permeabilization of the outer membrane of mitochondria is defined as a sharp increase in its permeability to ions and solutions weighing less than 1.5 kDa, leading to a loss of membrane potential, swelling of mitochondria, rupture of their outer membrane and release of apoptogenic factors. This process occurs after the opening of a megachannel known as the Ca 2+ -dependent non-specific mitochondrial pore (mPTP). The opening of mPTP appears to be a key factor causing cell death and irreversible organ damage in many pathological conditions, such as ischemia followed by reperfusion, neurodegenerative diseases, and muscular dystrophy.

    Calcium is the main activator of mPTP, and the sensitivity to the cation increases manifold under oxidative stress. Such conditions are observed during ischemia / reperfusion and are believed to be the main trigger for mPTP opening. The assumption that the main burst of reactive oxygen species (ROS) occurs during and after pore opening has been questioned for a long time, since it is known that its induction leads to uncoupling of mitochondria, and this, in turn, reduces the production of ROS. However, D. Zorov's group found that the accumulation of ROS in the mitochondrial matrix of cardiac myocytes upon photoactivation of tetramethylrhodamine derivatives triggers the induction of mPTP, which is accompanied by a multiply increased production ("burst") of ROS. The authors called this phenomenon ROS-induced ROS releas (RIRR). Subsequently, many studies have appeared demonstrating an ROS surge caused by mPTP induction. The release of ROS into the cytosol can activate redox-sensitive enzymes, as well as trigger a complex signaling response and ROS generation in neighboring mitochondria. This process is of great physiological and pathological significance, since it can induce the death of not only old and damaged mitochondria and cells, but also healthy ones. The question of the pathways of ROS formation during mPTP induction is of great scientific and practical importance, but remains open to date.

    Purpose of the study

    To review the data and hypotheses existing in the modern literature on the sites and mechanisms of ROS production during permeabilization of the outer mitochondrial membrane.

    Complex I of the mitochondrial respiratory chain

    Complex I (NADH-ubiquinone oxidoreductase) is one of the main sites of ROS production in mitochondria. It is believed that the main sites for ROS generation in it are the flavin mononucleotide NADH-binding site (site I f) and the ubisemiquinone coenzyme Q-binding site (site I q). Superoxide production at the I f site occurs during direct electron transport, when FMN is in a highly reduced state and depends on the NADH / NAD + ratio in the matrix. The inhibitor of the coenzyme Q-binding site rotenone increases superoxide production, as it causes the return of electrons to FMN. Superoxide production at complex I also occurs during reverse electron transport, when the coenzyme Q pool is fully restored.

    Under pathological conditions, an increase in the efficiency of ROS-generating sites of complex I can be associated with its conformational rearrangements. Opening of the mPTP strongly decreases the rotenone-sensitive activity of NADH-ubiquinone reductase and increases the production of H 2 O 2 in the presence of ≥50 µM NADH. NADH-ubiquinone oxidoreductase is characterized by a slow transition from an active state to an inactive state and vice versa. This suggests large conformational rearrangements of the complex, at least of that part of it that is involved in the rotenone-sensitive reduction of ubiquinone. It was shown that complex I isolated from rat hearts subjected to 30-minute anoxic perfusion became inactive and returned to active after reoxygenation. The authors suggested that these conformational rearrangements may be associated with the generation of ROS after the heart tissues subjected to coronary occlusion are reoxygenated. The transition of the complex to an inactive state is accompanied by specific unmasking of the ND3 subunit Cys39. It has been shown that nitrosating compounds reversibly modifying this cysteine ​​can be used as pharmacological protection against ROS generation during reperfusion.

    Complex II of the mitochondrial respiratory chain

    Complex II, or succinate-ubiquinone oxidoreductase, is a tetrameric flavoprotein of the inner mitochondrial membrane containing iron-sulfur clusters. It simultaneously participates in the work of the Krebs cycle and the respiratory chain, carrying out the conversion of succinate to fumarate and reducing ubiquinone to ubiquinol.

    The possibility of ROS formation by E. coli fumarate reductase flavin (site II f) in the presence of low concentrations of dicarboxylic acids was first shown in this work. Subsequently, ROS production was demonstrated on the mitochondrial particles of bovine heart mitochondria and skeletal muscle. The complex II inhibitor atpenin A5 and the complex III inhibitor stigmatellin, which blocks the oxidation of ubiquinol by complex III, stimulate the production of ROS by complex II in the presence of succinate. Malonate, on the other hand, inhibits the generation of ROS by complex II, which indicates that ROS are formed at the completely reduced flavin site II f, although other sites are not excluded. The dependence of the production of hydrogen peroxide on the concentration of succinate has a bell-shaped shape: the level of peroxide increases with an increase in the concentration of the substrate up to 400 μM, then significantly decreases at millimolar concentrations usually used to energize mitochondria. The reason for this phenomenon is that complex II generates ROS only when its flavin site II f is not occupied by dicarboxylic acids. Succinate and other Krebs cycle intermediates, which interact with the binding site of dicarboxylic acids, can restrict oxygen access to it and, thus, suppress ROS production by complex II. The level of succinate and fumarate in the matrix increases during ischemia / hypoxia, but this does not prevent the formation of ROS. In contrast, succinate accumulation during ischemia has been shown to strongly correlate with ROS production and reperfusion injury. The authors suggested that the main source of ROS under these conditions is the backflow of electrons through complex I. However, under conditions of prolonged ischemia, when the membranes are completely depolarized, this mechanism is hardly feasible. An alternative mechanism of ROS generation involves access of oxygen to the reduced site II f due to a decrease in the content of dicarboxylic acids in its immediate vicinity as a result of accelerated release of succinate and fumarate from the matrix upon induction of mPTP. This mechanism requires inhibition of complex II at the level of ubiquinone reduction or inhibition of ubiquinol oxidation by complex III.

    Conformational rearrangements of complex II can also contribute to the burst of ROS during membrane permeabilization. It was shown that with a decrease in the intracellular pH observed during apoptosis, dissociation of complex II occurs: the subunits of succinate dehydrogenase SDHA and SDHB, which oxidize succinate to fumarate and transfer electrons through iron-sulfur clusters, are separated from the site of reduction of coenzyme Q succinate CoQ oxidoreductase (SQR) ... This leads to inhibition of SQR activity, while the succinate dehydrogenase activity remains normal. This dissociation leads to the direct one-electron reduction of oxygen by the iron-sulfur cluster of complex II. And although it is known that low pH is an inhibitor of mPTP, nevertheless, this mechanism of an ROS surge can occur during ischemia, when a drop in pH occurs. At this time, conformational rearrangements of complex II can occur, and subsequently, during reperfusion, when the pH is restored to the initial level, mPTP opens and a burst of ROS formed on the dissociated complex is observed.

    Complex III of the mitochondrial respiratory chain

    Complex III (ubiquinol-cytochrome With oxidoreductase) is another possible site for the formation of ROS. This protein carries out the transfer of electrons from ubiquinone to cytochrome With during the functioning of the so-called Q-cycle. During this process, an unstable semiquinone is formed, which can transfer an electron to oxygen, thus forming a superoxide radical. However, under normal conditions, such a reaction is unlikely, since semiquinone is rapidly oxidized by cytochrome b. A sharp increase in the level of superoxide occurs when the complex is inhibited by antimycin A, as well as with ischemia lasting more than 30 minutes. One of the reasons for this phenomenon may be its conformational rearrangements caused by the binding of the inhibitor. It was shown on isolated heart mitochondria that complex III, inhibited by antimycin A, generates a significant amount of ROS in the presence of Mg 2+ and NAD + and in the absence of exogenous substrates upon induction of mPTP by calcium and alameticin. The authors showed that under these conditions, the production of hydrogen peroxide refers to the Mg 2+ -dependent generation of NADH by malate dehydrogenase. The production of H 2 O 2 was inhibited by stigmatellin and pyricidin, which indicates the importance of NADH-dependent reduction of ubiquinone for the generation of ROS under these conditions. These data support the hypothesis that during ischemia upon induction of mPTP, an increase in the concentration of Mg 2+, NAD + in the matrix activates malate dehydrogenase, which restores NAD + using malate, the concentration of which increases due to an increase in succinate and fumarate levels. The recovered equivalents go to the inhibited complex III, resulting in an ROS surge.

    Role pyridine nucleotides in ROS generation

    It was previously shown that oxidation of the NAD (P) H matrix in mitochondria precedes the opening of mPTP. In addition, pore induction leads to leakage of pyridine nucleotides into the cytosol of the cell. This change in the balance of NAD (P) H should affect the production of ROS during mitochondrial permeabilization. The dependence of ROS generation on the NADH concentration was investigated by A. Vinogradov's group. It has been shown that the maximum production of superoxide reaches a maximum at a NADH concentration of 10-50 μM; at millimolar concentrations, the production of the radical is inhibited. Since the physiological concentrations of NADH / NAD + matrix pairs are in the millimolar range, the contribution of complex I to ROS generation under normal conditions may be insignificant. It was found that in permeabilized mitochondria there is a high, depending on the ratio NAD (P) N / NAD (P) + and stimulated by ammonium ions, production of H2O2. At the same time, the yield of hydrogen peroxide was insensitive to dicumarol (an inhibitor of NADH-quinone oxidoreductase) and NADH-OH (an inhibitor of complex I), which indicates the matrix localization of the H 2 O 2 -generating site. The studied protein had NADH: lipoamide oxidoreductase activity and was identified as dihydrolipoamide dehydrogenase. This protein is an important component (the so-called E3 component) of two FAD-containing mitochondrial enzymes: the α-ketoglutarate dehydrogenase complex and the pyruvate dehydrogenase complex. According to the data obtained on purified complexes and isolated mitochondria, the E3 component is responsible for the production of superoxide and hydrogen peroxide. It was shown that permeabilized rat heart mitochondria, oxidizing NADH, produce about 50% of hydrogen peroxide due to the work of complex I, and the remaining 50% is accounted for by dihydrolipoamide dehydrogenase.

    The reduced forms of pyridine nucleotides not only supply electrons to the mitochondrial respiratory chain, but also regulate the redox status of the matrix through pro- and antioxidant proteins. One of these proteins is glutathione, which, together with NADPH, is a substrate for the antioxidant proteins glutathione peroxidase and glutathione reductase. When mPTP is opened, the release of NADPH and glutathione can occur, which causes the accumulation of Н 2 О 2. Moreover, under these conditions, due to a decrease in the membrane potential, nicotinamide nucleotide transhydrogenase (NADPH-transhydrogenase) cannot maintain a high level of reduced NADP +, which contributes to oxidative stress. Under physiological conditions, this enzyme regenerates NADPH in a direct reaction using NADH as a substrate. This reaction is energetically beneficial because the transhydrogenation between NADH and NADPH is associated with a proton gradient along the inner membrane. However, under pathological conditions, it can proceed in the opposite direction, regenerating NADH for the synthesis of ATP due to the utilization of NADPH. Thus, the antioxidant protection associated with the level of NADP + reduction decreases, which promotes the production of H 2 O 2.

    Role calcium in ROS generation

    It is known that an increase in the calcium concentration in the mitochondrial matrix triggers the induction of mPTP, while the pore sensitivity to the cation increases under oxidative stress, an increase in phosphate level, and a decrease in the pool of adenine nucleotides. The concentration of calcium ions in the mitochondrial matrix is ​​in the range of about 10 nM. Moreover, their calcium capacity is very high, isolated mitochondria are able to sequester more than 1M calcium from the environment, maintaining the concentration of free calcium in the micromolar range, in which the regulation of Ca 2+ -dependent enzymes occurs. These enzymes include pyruvate dehydrogenase and α-ketoglutarate dehydrogenase. Their activation leads to increased respiration and ATP synthesis and, probably, to an increase in ROS production.

    In the process of permeabilization of mitochondrial membranes, about 100 proteins are released from the intermembrane space and matrix, including such important antioxidant defense elements as glutathione and cytochrome. With.

    Cytochrome With is a positively charged protein that is associated with cardiolipin on the outside of the inner mitochondrial membrane, as well as with respiratory complexes III and IV. It has been shown that the release of cytochrome With is a two-step process involving the detachment of the protein from the intramembrane binding sites and its subsequent translocation across the outer membrane. Ca 2+ can enhance cytochrome dissociation With from the inner membrane, since it is its competitor for binding to negatively charged cardiolipin. This promotes the release of cytochrome With into the cytosol upon induction of mPTP. Moreover, ROS formed during membrane permeabilization can cause oxidation of cardiolipin, leading to a change in its physical properties, which can also increase the release of cytochrome With from mitochondria and contribute to an even greater generation of ROS. A reduced protein level slows down the transport of electrons from complex III to complex IV and, thus, increases the production of ROS in the Q-cycle. In addition, cytochrome With itself is an effective antioxidant capable of being effectively reduced by the superoxide anion. Thus, an increase in the concentration of calcium in mitochondria has a stimulating effect on ROS-producing matrix enzymes and leads to a drop in antioxidant protection, thereby increasing the total level of ROS generated by mitochondria.

    Conclusion

    Mitochondria are both a potential source and target of ROS action, leading to the loss of mitochondrial functions and, as a consequence, to irreversible cell damage in many pathological processes. In this case, mPTP plays an important role, the induction of which can lead to a powerful generation of ROS, which have a damaging effect on neighboring organelles and whole cells. Currently, the reasons for this phenomenon are poorly understood, although there are several hypotheses in the literature. It is assumed that the ROS burst may be based on conformational rearrangements of the respiratory chain complexes, activation of matrix dehydrogenases as a result of the action of Ca 2+, changes in the balance of NAD (P) N / NAD (P) + matrix, and depletion of the antioxidant system. Further study of the mechanisms and sites of ROS production during the induction of mPTP seems necessary, since their precise determination will allow the development of methods for their regulation to prevent the development of many pathological conditions of the organism.

    This work was supported by the Russian Science Foundation grant No. 17-75-10122.

    Bibliographic reference

    Kharechkina E.S., Nikiforova A.B. MECHANISMS OF GENERATION OF ACTIVE FORMS OF OXYGEN DURING PERMEABILIZATION OF MITOCHONDRIAL MEMBRANES // Modern problems of science and education. - 2018. - No. 4 .;
    URL: http: // site / ru / article / view? Id = 27719 (date accessed: 01/30/2020).

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    The seminal fluid of men, better known as semen, is called ejaculate in medicine. It is a natural fluid secreted by the testes, which has a characteristic mucous structure, viscous and opaque.

    The release of ejaculate occurs due to sexual arousal during sexual intercourse or masturbation. The ejaculate has a specific smell, similar to that of chestnut, light, almost white in color.

    The taste of the liquid depends on the food a man eats and his general health. In healthy males, it has a slightly salty taste with a bitter tinge. As with any fluid in the human body, ejaculate can be examined in a laboratory to assess the patient's health.

    Ejaculate analysis is carried out in two ways: bacteriological culture and spermogram.

    Main characteristics of sperm

    During intercourse or masturbation, a small amount of semen is released, the volume of which depends on several factors. According to medical standards, it should be in the range of two to ten milliliters.

    However, in adult men, the sperm count may be less, and its volume decreases with each intercourse, which follows with short temporary interruptions. Therefore, doctors are more often guided by the normal range from two to five milliliters.

    Very often, representatives of the stronger sex take a decrease in the amount of sperm secreted as an alarming sign, indicating a deterioration in male strength and health. At a young age, men believe that the more sperm is released during ejaculation, the greater the effect it has on a sexual partner.

    In fact, the amount of ejaculate emitted and its quality are two completely different things. A large semen volume is not always an indicator of high fertility. But the main thing in ejaculate is the number of healthy and active sperm that can reach the egg and fertilize it.


    This fertilizing capacity of sperm is calculated in laboratory conditions. According to research, there should be between 20 and 25 million healthy sperm in 1 milliliter of semen.

    The ejaculate itself consists of seminal plasma and corpuscles. The latter include not only spermatozoa, but also gonocytes. The seminal plasma is the backbone of the sperm, which is responsible for its correct structure. It stands out in the event that all internal male organs work correctly and harmoniously. It is possible to assess how healthy the semen is only by conducting laboratory tests.

    Ejaculate culture and semen analysis are prescribed to patients in the following cases.

    1. Infertility. This diagnosis is given to married couples who, during a year of active sex life, were unable to conceive a child on their own.
    2. As a survey before carrying out the in vitro fertilization procedure.
    3. If you suspect a possible loss of sperm properties due to previous diseases or injuries of the genital organs (infectious diseases, hormonal imbalance, varicocele, etc.).
    4. As a preventive examination at the request of the patient.
    5. The study of ejaculate is part of the mandatory diagnostics when planning the conception of a baby by a married couple.


    However, the main task of this study is to determine the reason that prevents a man from becoming a father, that is, infertility. The study of the ejaculate also helps to find out the reasons for the decrease in the volume of secreted fluid, a decrease in the number of active spermatozoa.

    These analyzes will determine the possible inflammations and infections that have struck the patient, which will help to start not only the treatment of infertility, but also other ailments, as well as increase the number of active sperm.

    Semen culture is one of the most frequently performed diagnostics prescribed for the study of male health. The diagnosis of "infertility" is increasingly being made to quite healthy young people who do not complain about other aspects of their health. What leads to the development of this ailment?

    First of all, it is the patient's own fault. An unhealthy lifestyle, bad habits, unhealthy diet - all this leads to hormonal imbalance in the body, as a result of which the number of healthy sperm and their activity decreases.

    Indiscriminate sexual intercourse, neglect of the rules of contraception and, as a result, diseases of the reproductive system also affect a man's ability to conceive a child.


    To these factors can be added a bad environmental situation, constant stress and high psycho-emotional stress, lack of minimal physical exertion, and harmful working conditions.

    Thanks to the analyzes, the doctor will be able to establish the exact reason why conception does not occur naturally, as well as prescribe a treatment that will eliminate these factors, increase the quality of sperm and restore the natural functions of male health. Typically, such treatment includes a number of activities: taking medications, physiotherapy procedures, lifestyle changes.

    The task of bacteriological culture is to determine the reasons why conception does not occur, to identify possible inflammations, infectious diseases and other dysfunctions of the patient's reproductive systems.

    Bacteriological culture of ejaculate and spermogram

    When conducting this study, it is possible to identify harmful microorganisms present in the ejaculate, as well as to find out the sensitivity of pathogenic microflora to certain types of antibiotic drugs.

    Bacteria and infections present in semen can cause changes in the structure of the ejaculate, that is, changes in its viscosity.


    This phenomenon is called viscosipathy. The reasons for its appearance: prostatitis, varicocele, orchitis, inflammatory processes in the male genitourinary organs. There are often cases when the doctor cannot establish the exact cause of these changes, then the diagnosis sounds like "idiopathic viscosipathy".

    To clarify the diagnosis, together with bacterial culture, a spermogram is carried out, which confirms or refutes the "viscous sperm syndrome". With this phenomenon, disturbances in the functioning of the internal genital organs occur in the body, as a result of which the processes responsible for the dilution of the seminal fluid proceed incorrectly.

    If the ejaculate is too viscous, dense, then the sperm cannot move freely in it, the speed of their movement decreases, they become unable to reach the fallopian tubes and are more susceptible to the effects of environmental factors, the environment of the vagina and uterus.

    It is impossible to notice such violations without clinical studies, since the sperm volume may remain the same, but the fertilizing ability is very low. Normally, the viscosity of the ejaculate should not be higher than two centimeters. Excess becomes the basis for the diagnosis of viscosipathy.


    When carrying out a spermogram, such semen data and parameters are taken into account, as in other examinations, as well as some additional characteristics: the quality of sperm, the presence or absence of red blood cells (normally they should not be), the presence or absence of mucous forms of fluid, as well as biochemical parameters ...

    When a bacteriological culture is prescribed

    Sperm culture is carried out in parallel with the study of the characteristics of the prostatic secretion. These procedures are prescribed to all patients in whom the doctor suspects the presence of an inflammatory process.

    The examination is necessary in order to identify infectious diseases and prescribe treatment that can stop these processes and prevent their transition to acute or chronic stages.

    During the study, pathogenic microorganisms are identified that can provoke ailments in the field of urology or cause sexually transmitted diseases. This is one of the highly sensitive studies that is required not only to select the tactics of drug therapy, but also to control the treatment.

    Native ejaculate and reactive oxygen species

    One of the newest methods for examining semen is the study of native (clean, untreated) spermatozoa. This technique allows you to study the ejaculate at the subcellular level, as a result of which various abnormal phenomena present in the sperm cells are determined.

    For the study are taken "live" sperm, which are emitted under a microscope, which allows them to increase 15 thousand times.

    To conduct the research correctly, it is best to donate semen directly at the clinic itself, where the research will be carried out. From the moment of collection until the beginning of laboratory diagnostics, no more than one hour should pass. Another requirement before passing this test is complete sexual rest a few days before visiting the laboratory.

    As with other studies, this analysis studies both the sperm themselves and the seminal secretion. These parameters should be consistent with healthy standards. So, the alkaline balance should be in the range from 7.2 to 7.8 pH, the volume of the liquid should be at least two ml. The number of spermatozoa in 1 ml is at least 20 million, and at least 50% of them must have a translational movement.


    The total volume of cells with a normal morphological structure should not be less than a third of the total number.

    Inactive and damaged spermatozoa should not account for more than half the volume of semen collected. If at least one of these parameters is violated, we can talk about male infertility.

    There are situations when an excess production of reactive oxygen species (ROS) is observed in the native ejaculate. ROS are the main causes of oxidative processes in semen. The reasons for this phenomenon can be diseases of the reproductive system, autoimmune disorders of the body, environmental influences.

    Also, the production of ROS increases with the age of the patient, with chronic ailments of the endocrine system, with severe physical exertion. All this affects the volume of sperm in the ejaculate, and the lack of their required number leads to infertility.


    How to prepare for the test

    To conduct the examination correctly and get the most accurate results, you should responsibly prepare for the delivery of the ejaculate. It is collected only in special sterile disposable containers, which are issued in a clinic that conducts such analyzes.

    It is forbidden to use any glass containers from under food products, condoms, plastic bags, etc. for collection.

    It is very important to mark on the container not only the date when the ejaculate was collected, but also the exact time. The accuracy of some indicators studied in the diagnostic process will depend on this. For the analysis, it is worth giving preference to the clinic recommended by the attending physician.

    Once the ejaculate is collected, it should be taken to the laboratory immediately. It is undesirable to store the collected biomaterial. But if this is not possible, the container does not need to be placed in the refrigerator.

    The optimum storage temperature is from 20 to 40 degrees. Failure to store properly can lead to false results. Also, a few days before the analysis, it is worth giving up intimate relationships.


    The test results are usually obtained within 24 hours from the moment the biomaterial is handed over to the laboratory. The received form with personal data, basic parameters, norms and studied indicators is handed over to the patient.

    The decryption of the data obtained is carried out only by the attending physician who gave a referral for the diagnosis. He also establishes the final diagnosis and prescribes therapeutic therapy. Sometimes, in addition to a reproductive physician, consultation with other specialists may be required: urologist, venereologist, surgeon, endocrinologist.

    Based on the results of laboratory tests, the doctor will determine the exact cause of infertility and prescribe a treatment designed to increase the number of active and healthy sperm in the semen. But in addition to taking medications and various physiotherapy procedures, treatment should include other parameters.

    Adherence to a healthy lifestyle will help increase sperm quality. Avoiding alcoholic beverages and cigarettes helps to improve test results in no time.

    The ejaculate will become of better quality if even minimal physical activity is included in the patient's life: morning exercises, walks, refusal of the elevator, etc.


    If it is possible to visit fitness centers, it is worth giving preference to workouts that will not lead to excessive overheating of the body. This can be swimming, yoga, stretching exercises.

    Breaks in work can also increase the number of active spermatozoa if it is associated with prolonged sitting in one place. Regular breaks every hour, during which the patient can get up and walk around the room, will not only give rest to the eyes, but also improve blood circulation in the pelvis, which directly affects the state of men's health.

    If you can't stand up, you can do a few seated exercises.

    It is important to eat properly and regularly, give up snacks, especially unhealthy foods. For the ejaculate to be of better quality, the basis of the diet should be protein and plant products, as well as fish and fermented milk drinks. You need to eat regularly, often and in small portions.

    Excessive stress and emotional stress should be avoided, which also directly affect the state of men's health.

    To increase the volume of sperm will help the refusal to visit places in which there is an increase in temperature: a bathhouse, a beach. Underwear, especially in summer, should only be made from natural fabrics.

    Synthetics increase body temperature in the groin, which decreases sperm quality.

    Such simple measures will help to supplement the treatment prescribed by the attending specialist, to improve the test results in a shorter time and to conceive the long-awaited baby faster.

    Doctor sexopathologist-andrologist 1st category. Head of the Kherson branch of the Ukrainian Family Planning Association.

    Andrology laboratory


    Spermogram according to WHO 2010 1607 p.

    Antisperm antibodies on sperm (Mar test indirect Ig G) 1928 p.

    Antisperm antibodies on sperm (Mar test indirect Ig A) 1928 p.

    Antisperm antibodies on spermatozoa (Mar test direct Ig G) 1499 p.

    Antisperm antibodies on spermatozoa (Mar test direct Ig A) 1499 p.

    Postcoital test (in vivo test) 2142 p.

    Kurzrock Miller test 2142 p.


    Clinical Andrology Laboratory performs special tests for men

    All boys in front of the army at the school undergo a medical examination. This helps to identify various disorders, including those related to reproductive health. Rarely, when a young man subsequently, on his own initiative, turns to a doctor for prophylaxis. Basically, they come for examination when there are already complaints, either on the initiative of the partner, or in legal marriage, when they have not been able to conceive a child for several years.

    In all these cases, both for prevention and in the presence of various problems, our task is to objectively assess the situation, choose the right list of necessary tests, conduct research and send the results to the patient's e-mail. In the future, the patient, if necessary, can discuss the data of these analyzes with the urologist-andrologist, or the gynecologist-reproductologist who work in our clinic.

    The main area of ​​work of the laboratory of clinical andrology is male infertility and combined forms of male and female infertility, as well as infectious and inflammatory processes of the male genitourinary system.

    The laboratory staff - doctors, medical technologists, laboratory assistants - have been working together for 6 years and are the leading specialists in spermatology in our country. Many have experience of work in the Scientific Center for Obstetrics and Gynecology named after V.I. V.I. Kulakov, Clinic of Urology named after V.I. R.M. Fronstein of the I.M. THEM. Sechenov, andrology clinic of the Peoples' Friendship University of Russia. Laboratory research carried out by our employees is the basis of many scientific works in andrology, dissertations and publications, incl. in leading English-language magazines.

    The qualifications, the necessary equipment and many years of experience allow us to carry out various sperm examinations at an expert level.

    The analyzes are carried out exactly according to all the requirements of the WHO-2010.

    The most modern methods for assessing sperm function have been introduced into clinical practice: oxidative stress, acrosome reaction, DNA fragmentation, chromatin packing disorders, etc.

    All analyzes are carried out here in the laboratory, they are not taken anywhere, as soon as the material is liquefied.

    There are excellent conditions for donating sperm: a separate soundproofed room with a toilet and sink, a large TV. The sperm donation room is located directly next to the laboratory.

    Ejaculate (semen) examination - basic requirements

    The study of sperm - concentration, motility and morphology of sperm, the content of leukocytes, antisperm antibodies, DNA damage, and many other functional indicators - is the main method for assessing male fertility.

    Despite the introduction of computer technology, semen examination performed by an experienced laboratory doctor is still the most accurate, reliable and reproducible diagnostic method.

    Sperm research should be carried out in specialized laboratories, and not in general laboratories (especially not in network laboratories), since the description of most of the parameters of the spermogram - motility, morphology, MAP test, etc., is subjective and largely depends on experience and qualifications laboratory doctor. Ideally, dynamic semen analysis should be performed by one specialist, since even with strict internal quality control in the laboratory, discrepancies in the results when analyzed by different doctors can occur.

    Sperm is a tricky material. Increased viscosity, defective delivery, the presence of leukocytes, immature cells of spermatogenesis, bacteria, various categories of motility can only be correctly determined by a doctor with extensive experience. Recently, the WHO standards for sperm testing have changed several times, and different reference values ​​are used in different laboratories. It is also necessary to take into account seasonal fluctuations in spermogram indicators, the patient's condition at the moment, his psychological characteristics.

    Sperm for analysis should be donated with sexual abstinence from 2 to 7 days, preferably closer to the usual rhythm of sexual activity. Eliminate alcohol, do not get sick, do not steam, do not take a hot bath, be healthy.

    If the spermogram indicators deviate from normal during the initial examination, the analysis should be repeated after 2-6 weeks to confirm the diagnosis. Since spermatogenesis - the process of formation of mature spermatogenesis - takes almost 3 months, any negative impact (fever, stress, poisoning, etc.) can affect the quality of sperm during this time.

    It is necessary to understand that even with a large number of spermatozoa, good mobility, they may not fulfill their main function - fertilization of the egg, because there is a "breakdown" at another level. According to the latest data, up to 30% of cases, male infertility occurs with "normozoospermia" - formally normal spermogram. Establishment of the male factor of infertility in this case requires the use of special functional tests.

    The basic tests for the diagnosis of "fertile" or "infertile" (may or may not have children) are currently:

      Spermogram - determination of the volume, viscosity, pH of sperm, concentration, motility and morphology of spermatozoa, their agglutination, the number of leukocytes and a number of other parameters.

      MAR test is a test for the presence of antisperm antibodies (ASAT) that lead to immune infertility. Distinguish between ACAT class IgG and class IgA, which have their own characteristics.

      Reactive oxygen species (ROS or ROS) in native ejaculate (semen) and washed sperm. An increase in the production of reactive oxygen species in the native ejaculate is a sensitive marker of an infectious-inflammatory process (more sensitive than the number of leukocytes). The production of ROS by washed spermatozoa is a sign of oxidative stress in germ cells, in which even chromosomes are affected. Sperm oxidative stress is not only the cause of male infertility, but also miscarriage and congenital anomalies in children.

    These three analyzes will help in making the correct diagnosis, outline ways of further treatment, or determine the need for additional research in case of detected violations.

    It should be noted that every day young men come to take a spermogram and do not even suspect about the presence of a purulent process, when there is a large number of leukocytes in the seminal plasma. This can lead to further blockage of the vas deferens and, as a result, the sperm do not come out or, even worse, their formation process is disrupted. As a result, the diagnosis of male fertility and the infectious process in our laboratory are inseparable. Only in stained smears can one see and distinguish leukocytes and immature cells of spermatogenesis at different stages of development. And if a purulent process is detected, then immediately the sperm can be examined for sexually transmitted infections and bacterial inoculation for aerobes and anaerobes can be done. You also need to understand that inflammation can be found in semen, and the secret of the prostate will be normal, and vice versa, in the secret of the prostate there may be inflammation, but in the semen it is normal. Sexually transmitted infections are examined from a scraping taken from the urethra, for this the patient must not urinate for more than three hours. Men of different ages turn to us for tests for an adequate diagnosis of prostatitis.

    Considering the lifestyle of a modern young man - early sexual life, a large number of sexual partners, sex without barrier contraception, a sedentary lifestyle, stress - we recommend prophylactic examination not only for infections, but also a spermogram to control the birth of healthy offspring.