Influence of Extracellular Vesicles from the Follicular Fluid of Young Women and Women of Advanced Maternal Age with Different miRNA Profiles on Sperm Functional Properties.

We studied the effect of co-culturing of extracellular vesicles in the follicular fluid of young women and women of advanced maternal age on sperm motility. Vesicles were obtained by differential centrifugation. The sperm fraction was isolated from the seminal fluid of 18 patients (age 28-36 years). The spermatozoa were incubated with vesicles (1:2 ratio) for 60 or 120 min at 37°C in a CO2 incubator. A fraction of spermatozoa incubated without vesicles served as the control. After the incubation, the sperm samples were sedimented by centrifugation, fixed in 2.5% glutaraldehyde, and analyzed by transmission electron microscopy. RNA was isolated from the follicular fluid vesicles by column method followed by cDNA synthesis in a reaction mixture according to miScript II RT Kit protocol (Qiagen). After 60-min incubation with extracellular vesicles from the follicular fluid of women of advanced maternal age, the sperm motility and hyperactivation slightly changed in comparison with the group where incubation was performed with follicular fluid vesicles from young women and control group. Follicular fluid miRNA profiles in women of different ages varied, which suggests different functional compositions and effects of follicular fluid vesicles of different age groups on sperm characteristics. Transmission electron microscopy revealed differences in the interaction of follicular fluid vesicles from women of different age groups with spermatozoa. Further study of the effect of extracellular vesicles from the follicular fluid and analysis of their transcriptomic, proteomic, and metabolomic composition on sperm mobility and fertilizing ability will improve the effectiveness of assisted reproductive technology programs in patients with male infertility.

Influence of Extracellular Vesicles of the Follicular Fluid on Morphofunctional Characteristics of Human Sperm.

We studied the effect of extracellular vesicles of the follicular fluid on morphofunctional characteristics of human spermatozoa using CASA (computer-assisted sperm analysis) analytical system. The vesicles were obtained by sequential centrifugation at different rotational speeds and frozen at -80°C in the Sydney IVF Gamete Buffer medium. The sperm fraction was isolated from the seminal fluid of 21 patients aged 27-36 years by differential centrifugation in a density gradient. The precipitate was suspended in Sydney IVF Gamete Buffer to a concentration of 106/ml and incubated with vesicles (1:2) at 37°C in a CO2 incubator for 30 min and 1 h. Sperm fraction incubated without vesicles served as the control. After incubation, some sperm samples were centrifuged at 700g for 5 min and fixed in 2.5% glutaraldehyde in 0.1 M buffer for transmission electron microscopy. After 30-min and 1-h incubation, the progressive and total sperm motility improved, the curvilinear and linear velocity of spermatozoa did not change significantly. Incubation with vesicles significantly changed the trajectory of sperm movement, which can attest to an increase in their hyperactivation and, probably, fertilizing capacity. Analysis of the effect of extracellular vesicles of follicular fluid on sperm motility will help to improve the effectiveness of assisted reproductive technology programs with male infertility factor by improving sperm characteristics in patients with asthenozoospermia and increasing the fertilizing ability of the sperm.

[Action features of the of low-intensity electromagnetic radiation at an early stage of the experimental metabolic syndrome development induced by a diet high in carbohydrates and fats]. / Osobennosti deistviya nizkointensivnogo elektromagnitnogo izlucheniya na rannem etape razvitiya eksperimental'nogo metabolicheskogo sindroma, indutsirovannogo dietoi s vysokim soderzhaniem uglevodov i zhirov.

Metabolic syndrome (MS) is a complex of interrelated metabolic disorders leading to the development of abdominal obesity, hyperglycemia, insulin resistance, and dyslipidemia. To develop new methods for the prevention and treatment of MS, it seems appropriate to simulate this disease using a high-calorie diet that induces many of its symptoms. As a therapeutic and prophylactic agent, it is promising to use low-intensity electromagnetic radiation (EMR) of ultra-high frequency (UHF), which can have a stimulating effect on metabolic and regenerative processes and strengthen the mechanisms of their regulation.Purpose of the study. To reveal the features of the effect of low-intensity microwave EMR/UHF on the development of adaptive metabolic and ultrastructural changes in the liver and blood of rats at an early stage of MS development. MATERIAL AND METHODS: The work was carried out on 40 nonlinear male rats weighing 180-220 g. In the experimental group, the rats were kept on a high-calorie diet for 2 months; in the control group, only the model was reproduced without the action of EMR/UHF; in the intact group, the animals were not exposed to any influences. The course of EMR/UHF (10 procedures) was carried out daily for 2 minutes on the lumbar region in the adrenal gland projection area using the Aquaton-2 device (power flux density less than 1 µW / cm2, frequency about 1000 MHz). The objects of the study were the liver and blood. Biochemical methods were used (RNA, DNA, total protein content, liver antioxidant activity, insulin and glucose levels in the blood), transmission electron microscopy, morphometric analysis of mitochondria (number, average and total area). RESULTS AND DISCUSSION: The use of low-intensity EMR/UHF at the background of the MS development caused an increase in a number of adaptive changes: the level of insulin in the blood increased; the content of total protein in the liver increased, and the severity of fatty degeneration decreased. The ultrastructural reorganization of hepatocytes was manifested in the activation of the protein synthesis apparatus (the phenomenon of hyperplasia of the granular endoplasmic reticulum, as well as ribosomes and polysomes). At the same time, the total area of mitochondria decreased, although their bioenergetic potential increased due to the condensation of the matrix. The revealed adaptive shifts were due to the antioxidant and membrane-stabilizing effects of EMR/UHF on general neuroendocrine and local mechanisms. CONCLUSION: Low-intensity EMR/UHF microwave can be a promising factor in the development of new methods of treatment and prevention of MS. In order to optimize the adaptive-protective and compensatory processes, as well as taking into account the decrease in the number of mitochondria, it is advisable to use EMR/UHF in more adequate modes.

Ultrastructural markers of tissue endometrial receptivity in patients with recurrent implantation failure.

The scanning electron microscopy of the endometrial surface epithelium during the 'implantation window' was performed in 119 patients with uterine factor of infertility or recurrent miscarriage due to endometrial hypoplasia. Ultramorphological picture of the surface endometrial epithelium was characterized by aplasia and hypoplasia of pinopodes (67.39%), dense cell - cell contacts (69.53%), heteromorphy of secretory cells (15.22%) in combination with atypia of microenvironment cells (50%) in patients with infertility. The asynchronous development of pinopodes (46.67%) and the absence of intercellular contacts separation during the 'implantation window' (84.44%) was observed in patients with recurrent miscarriage. The revealed disturbance determines the mechanisms of the blastocyst adhesion violation and trophoblast invasion in the different stages of implantation in patients with uterine factor of infertility and recurrent miscarriage.

[Docosahexaenoic acid in the treatment of idiopathic male infertility].

AIM: docosahexaenoic acid is one of the most common fatty acids in the cell membranes of sperm. This substance is a structural component of cell membranes, and is responsible for such properties as plasticity and fluidity which are necessary for the implementation of the process of capacitation and acrosome reaction. In addition, docosahexaenoic acid has antioxidant properties. Aim of our study was to assess the effect of dietary supplement docosahexaenoic acid (Brudi PLUS) on such markers of male fertility as the sperm DNA integrity, standard indicators of semen analysis and cryotolerance in infertile men. MATERIALS AND METHODS: a randomized, multicenter, double-blind, placebo-controlled study was conducted. A total of 109 infertile men were recruited to participate in the study. All patients underwent different analyzes, including semen analysis, sperm DNA fragmentation, MAR-test, test of cryotolerance and electron microscopy analysis of spermatozoa. In active treatment group patients were prescribed to drug containing 350 mg of docosahexaenoic acid 3 times a day for 90 days. In the control group, patients were administered to a placebo with a similar scheme. All analyzes were repeatedly performed after 3-months of therapy. RESULTS: An increase in the progressive mobility and sperm viability was determined after the test of cryotolerance in the active treatment group. According to electron microscopy analysis of spermatozoa, positive changes were observed in the number of heads of the normal form with the normal structure of chromatin and acrosome, as well as a decrease in the number of spermatozoa with insufficiently condensed chromatin. CONCLUSIONS: the therapy with drug Brudi PLUS in patients with male infertility allows to increase in cryotolerance of sperms as well as decrease in number of sperm ultrastructure defects.

High-throughput analysis of TUNEL-stained sperm using image cytometry.

Sperm DNA integrity is an essential factor for successful fertilization and proper pregnancy progression. The terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay is commonly used for the estimation of the DNA fragmentation index. Analysis of TUNEL-stained sperm is often performed by flow cytometry, an approach that allows high-throughput analysis but in which any morphological information is lost. In this study, results of an automated image cytometry estimation of TUNEL-stained sperms were presented. The results of visual counting and automatic analysis were closely correlated, indicating that image cytometry is suitable for such analysis and may be applied in a clinical setting. © 2017 International Society for Advancement of Cytometry.

Detection of a mutation in the intron of Sperm-specific glyceraldehyde-3-phosphate dehydrogenase gene in patients with fibrous sheath dysplasia of the sperm flagellum.

The fibrous sheath is a unique cytoskeletal structure surrounding the axoneme and outer dense fibres of the sperm flagellum. Dysplasia of the fibrous sheath (DFS) is a defect of spermatozoa observed in severe asthenozoospermic patients and characterised by morphologically abnormal flagella with distorted fibrous sheaths. Sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) is a glycolytic enzyme that is tightly associated with the fibrous sheath of the sperm flagellum. The enzymatic activity of GAPDS was investigated in sperm samples of seven patients with DFS and compared to that of normal spermatozoa (n = 10). The difference in GAPDS activity in DFS and normal spermatozoa was statistically significant (0.19 ± 0.11 and 0.75 ± 0.11 µmol NADH per min per mg protein respectively). Immunochemical staining revealed irregular distribution of GAPDS in the flagellum of DFS spermatozoa. Other five samples with typical alterations in the fibrous sheath were assayed for mutations within human GAPDS gene. In all five cases, a replacement of guanine by adenine was revealed in the intron region between the sixth and the seventh exons of GAPDS. It is assumed that the deficiency in GAPDS observed in most DFS sperm samples is ascribable to a disorder in the regulation of GAPDS expression caused by the mutation in the intron region of GAPDS gene.

[Genetically Determined and Functional Human Sperm Motility Decrease].

Motility is the most important property of mammalian sperm required for fertilization. Axoneme and axoneme surrounding tail components are the morphological substrate of sperm motility. Quantitative research methods of human spermatozoa motility allowed the definition of the normative parameters for fertile men. Exogenous factors, and, rarely, genetic defects may cause a significant reduction in sperm motility. Axonemal anomalies (absence of external and/or internal dynein arms, central pair of microtubules absence) may be the cause of primary ciliary dyskinesia (PCD). PCD­a severe systemic disease of the reduction of sperm motility­is just one symptom. Dysplasia of the fibrous sheath (DFO) is also genetically determined sperm motility decrease. PCD and the DFO are multigene diseases that are inherited in an autosomal recessive manner. Modern molecular biological research methods are used to identify candidate genes. Assisted reproduction technologies (ART) allow men suffering from PCD and DFO to produce offspring. PCD and DFO symptoms appear in the homozygote. Children born after ART have the probability of being mutation carriers. We do not have complete information about etiological factor of genetically determined spermpathology. So we cannot assess the genetic risk degree. However, the possibility of mutations accumulation, which can be a risk factor for distant offspring, should be considered.

["Microbiological aging" by Mechnikov. How to interpret these ideas today?].

I.I. Mechnikov's hypothesis that the key to prolongation of life lies in the introduction of useful microflora to the gut was not proved. Any microflora needs nutrition and perceives the human body only as a nutrient substrate. Destruction of the basement membranes, that delimit the contacting with aggressive microbiological environment epithelium from the deeper parts of the body, can lead to chronic inflammatory diseases and aging of the skin as a consequence of the invasion of microorganisms. At the ultrastructural level it has been shown by the example of prostatitis and skin aging changes. Coupled with the penetration of germs flow of immune cells may cause autoimmune reactions due to abrupt changes in the molecular design of the intermembrane transport. Thus, the physiological process of macroorganism aging can be viewed as a consequence of its microbiological destruction.

Metric similarity of dynamic commutation processes in situ and in vitro.

The dynamics of string growth was studied in model homochiral solutions of biomimetics, trifluoroacetylated amino alcohols (TFAAA) in heptane, water, and inverted heptane-water emulsion. In heptane and water, a thick (~1 µ in diameter) string had a crown of thin strings on its growing terminal and these thin strings effectively adsorbed dissolved TFAAA. In emulsion, the strings grew inside the water droplets, in which this TFAAA cannot be solved, presumably due to transport of TFAAA molecules from heptane into water in the surface layer surrounding the string. Applications of these phenomena to in vivo cell commutation were discussed.

Role of prostatic epithelium basement membrane incompetence in the pathogenesis of chronic prostatitis.

Prostatic inflammation is associated with infections penetrating through the urethra. This inflammation is treated by long courses of wide-spectrum antibiotics. However, the most frequent cause of prostatitis is Escherichia coli and other enteric flora. Electron microscopy of biopsy specimens from the prostate detected gaps in the prostatic epithelium basement membrane, their size explaining the penetration of enteric flora into the prostate. These data suggest another view on the pathogenesis of prostatitis and approaches to improvement of therapy for this disease.

[Chromatin folding in human spermatozoa. I. Dynamics of chromatin remodelling in differentiating human spermatids].

Changes in chromatin structure at different stages of differentiation of human spermatids were studied. It was shown that, in nuclei of early spermatids, chromatin is loosely packed and its structural element is an 8-nm fiber. This "elementary" fiber is predominant at the initial stages of differentiation; in the course of maturation, it is replaced by globular elements approximately 60 nm in diameter. In intermediate spermatids, these globules start to condense into fibrillar aggregates and reduce their diameter to 30-40 nm. At all stages of spermatid maturation, except the final stages, these globules are convergence centers for elementary fibers. This remodelling process is vectored and directed from the apical (acrosomal) to the basal pole of the nucleus. In mature spermatids, the elementary 8-nm fibers are almost absent and the major components are 40-nm fibrillar aggregates. The nuclei of mature spermatids are structurally identical with the nuclei of spermatozoa with the so-called "immature chromatin," which are commonly found in a low proportion in sperm samples from healthy donors and may prevail over the normal cells in spermiogenetic disorders. The cause of this differentiation blockade remains unknown. Possibly, the formation of intermolecular bonds between protamines, which are required for the final stages of chromatin condensation, is blocked in a part of spermatids. The results of this study are discussed in comparison with the known models of nucleoprotamine chromatin organization in human spermatozoa.

[Culture of human amniotic fluid stem cells in 3D collagen matrix].

Most of the researchers attribute amniotic fluid stem cells (AF SCs) to mesenchymal stem cells (MSCs). However, AF SCs express both mesenchymal and epithelial markers, which distinguishes them from postnatal MSCs. Cultivation in the three-dimensional matrix provides a different look at the nature of these cells. We showed that, in 3D collagen gel, AF SCs form epithelial structures (tubules and cysts). Active contraction of the gel during the first days of cultivation, which is characteristic if mesenchymal cells, does not occur. Electron microscopic study showed that typical to epithelial cell adherent junctions are formed between AF SCs. On the other hand, AF SCs continue to express MSCs markers during cultivation in the gel. Thus, AF SCs may not be true mesenchymal cells because they can display properties of epithelial cells. Perhaps these cells undergo epithelial-mesenchymal transition, the process which actively takes place during embryogenesis.

Oxidation of glyceraldehyde-3-phosphate dehydrogenase decreases sperm motility.

The relation between the activity of the sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) and the motility of sperms was investigated. It was found that the mean value of GAPDS activity in sperm samples with low motility is 2.5-3-fold lower than that in samples with high motility. Sperm motility was shown to diminish in the presence of superoxide anion, hydroxyl radical, and hydrogen peroxide. The decrease in sperm motility in the presence of hydrogen peroxide was proportional to the concentration of the oxidant and correlated with the decrease in GAPDS activity (r = 0.96). Based on the literature data on the importance of GAPDS for the motility of sperms together with the presented observations, it was concluded that the decrease in the sperm motility in the presence of reactive oxygen species is due to the oxidation of GAPDS and inhibition of glycolysis.

[Analysis of germ cell populations in ejaculate of men infected with herpes simplex virus].

Cytological and molecular genetics methods were used to study sperm from patients with sperm infected with herpes simplex virus (HSV) as indicated by virological and immunocytochemical tests. The following methods were used: (1) sperm analysis to evaluate the morphology and functional properties of sperm; (2) fluorescence in situ hybridization (FISH) with DNA probes specific for chromosomes 1, X, and Y to evaluate nondisjunction frequencies of these chromosomes in sperm; and (3) quantitative analysis of immature germ cells in the ejaculate to identify spermatogenic abnormalities. The total sperm count and the count of sperm with normal motility proved similar to the norm. FISH analysis demonstrated no difference in the nondisjunction frequency of chromosomes 1, X, and Y between infertile patients with HSV-infected sperm and fertile donors. Comparative quantitative analysis of immature germ cells from the ejaculate has demonstrated a significant and considerable (threefold) increase in the number of spermatocytes I at the prepachytene stages of prophase I (preleptotene, leptotene, and zygotene) in HSV patients compared to normal donors. At the same time, HSV patients demonstrated a significant decrease in the number of spermatocytes I, a decrease in the proportion of spermatocytes II and spermatids, and a twofold increase in the number of unidentifiable immature germ cells. The data obtained indicate a partial spermatogenic arrest at the early stages of meiotic prophase I in HSV patients, which prompts further research into the cellular mechanisms of abnormal spermatogenesis after viral infection in humans.

Investigation of glyceraldehyde-3-phosphate dehydrogenase from human sperms.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDs) was purified from human sperms and properties of the enzyme were investigated. After sonication of sperms, the most part of GAPDs is associated with the insoluble cell fraction. Trypsin treatment results in the cleavage of part of the N-terminal domain of the enzyme yielding a soluble fragment that was purified by hydrophobic chromatography on Phenyl-Sepharose. The isolated fragment was shown to be a tetramer with molecular weight of approximately 150 kD (according to Blue Native PAGE) and composed of subunits of 40 kD (according to SDS-PAGE). The specific activity of the isolated fragment reached 374 U/mg. It is supposed that GAPDs exists in sperms as the tetrameric molecule bound to the fibrous sheath of the flagellum through the N-terminus of one or two subunits. Comparative study of the amino acid sequences of mammalian GAPDs revealed conservative cysteine residues (C21, C94, and C150) that are specific for the sperm isoenzyme and absent in the somatic isoenzyme. Residue C21 can be involved in the formation of the disulfide bond between the N-terminal domain of GAPDs and fibrous sheath proteins.

[Herpetic infection of spermatozoa in failure of reproductive technologies and spontaneous abortion].

Herpes simplex virus (HSV) was studied in ejaculate of 36 men whose wives had the history of spontaneous abortions, or failure of artificial insemination (AI), or extracorporeal fertilization (ECF). The control group consisted of 222 patients examined prophylactically after urogenital inflammation or for primary infertility. HSV was detected with immunofluorescence (IF) reaction with monoclonal antibodies to HSV antigens, rapid culture method (RCM) and transmission electronic microscopy. HSV antigens were detected in fraction of mobile spermatozoa in 13 (65%) of 20 samples from patients of the study group. HSV detectability in the study group was significantly higher than in the controls (p = 0.003). Intragametic capsides of HSV were detected at ultrastructural investigation in 13 of 19 (68%) ejaculate samples from patients of the study group in whom IF and/ or RCM results were positive. The study of 6 ejaculate samples of this group in which HSV was not detected by IF or RCM and 24 control samples HSV capsides were not detected. The following conclusions can be made: mobile spermatozoa of normal morphology can be infected with HSV; as shown morphologically, samples of ejaculate from men whose wives had spontaneous abortions contained intragametic HSV capsides; intragametic HSV infection of spermatozoa had a negative effect on the course of pregnancy and/or embryon vitality.

Detection of type 2 herpes simplex virus in cells of spermatogenic epithelium in infected testes of guinea pigs.

We developed a model of herpetic orchitis in guinea pigs. Intratesticular inoculation of type 2 herpes simplex virus suspension results in infection of the testicular spermatocytes and spermatides. The possibility of viral infection dissemination from infected into intact testis is proven.

[The use of endobiotic ciliates from old collections in transmission electron microscopic investigations].

The opportunity of the use of formalin-fixed endobiotic ciliates from old collections in transmission electron microscopic investigations (TEM) has been studied. Ciliates from the following species were examined: Ditoxum funinucleum Gassovsky, 1919 from the hindgut of Equus hemionus kulan Groves et Mazak, 1967 preserved in a collection during 19 years, Blepharoprosthium pireum Bundle, 1895 and Cochliatoxum periachtum Gassovsky, 1919 from the hindgut of the Yakut horse Equus caballus L. stored during 1.5 years, and Triplumaria heterofasciculata Timoshenko et Imai, 1995 from faeces of the Asian elephant Elephas maximus L. stored during 2.5 years. It is shown, that the main taxonomically important characters of the cortex ultrastructure, ciliature, and internal fibril structure of the cell of Trichostomatia keep well during a long-term storage in formalin.