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.

[QUANTITATIVE DNA EVALUATION OF THE HIGH CARCINOGENIC RISK OF HUMAN PAPILLOMA VIRUSES AND HUMAN HERPES VIRUSES IN MALES WITH FERTILITY DISORDERS].

Infertility is an actual medical and social problem. In 50% of couples it is associated with the male factor and in more than 50% of cases the etiology of the infertility remains insufficiently understood. The goal of this work was to study the prevalence and to perform quantitative analysis of the human herpes viruses (HHV) and high carcinogenic risk papilloma viruses (HR HPV) in males with infertility, as well as to assess the impact of these infections on sperm parameters. Ejaculate samples obtained from 196 males fall into 3 groups. Group 1 included men with the infertility of unknown etiology (n = 112); group 2, patients who had female partners with the history of spontaneous abortion (n = 63); group 3 (control), healthy men (n = 21). HHV and HR HPV DNA in the ejaculates were detected in a total of 42/196 (21.4%) males: in 31 and 11 patients in groups 1 and 2, respectively (p > 0.05) and in none of healthy males. HHV were detected in 24/42; HR HPV, in 18/42 males (p > 0.05) without significant difference between the groups. Among HR HPV genotypes of the clade A9 in ejaculate were more frequent (14/18, p = 0.04). Comparative analysis of the sperm parameters showed that in the ejaculates of the infected patients sperm motility as well as the number of morphologically normal cells were significantly reduced compared with the healthy men. The quantification of the viral DNA revealed that in 31% of the male ejaculates the viral load was high: > 3 Ig10/100000 cells. Conclusion. The detection of HHV and HR HPV in the ejaculate is associated with male infertility. Quantification of the viral DNA in the ejaculate is a useful indicator for monitoring viral infections in infertility and for decision to start therapy.

[Interaction of herpesviruses with mature human spermatozoa in the model system in vitro].

The DNA of human herpesviruses (HHV), including the herpes simplex virus (HSV) and cytomegalovirus (CMV), is often identified in ejaculates of patients with urogenital diseases and infertility. At least a part of viral DNA is associated with cell fraction of ejaculate. However, it remains unclear how the semen is infected by the virus. It can be located in gametes or be capable of infecting mature germ cells, including motile sperm cells. In order to resolve this issue, interactions of the CMV and HSV with human sperm cells were studied using an original optimized model of the herpesviral infection of male gametes in vitro. The analysis of the immunofluorescent staining of gametes for viral antigens has shown that CMV infected 2% gametes, while HSV infected 17.26 ± 2.58% gametes. The fraction of progressively motile sperm cells contained 13.99 ± 4.64% infected cells. Localization of HSV was studied by the confocal microscopy. Sometimes, viral gB protein was found on sperm cell membrane. In addition, optical scanning of other cells has shown the intracellular localization of the viral proteins. In the majority of spermatozoa, the viral proteins were observed in the head and neck. In some cells, they were located in the middle piece or, rarely, in the equatorial segment. In general, after in vitro infection HSV antigens were located in the same areas of the sperm cells as in ejaculates from infected patients. According to DNA-DNA hybridization in situ, gametes containing HSV DNA accounted for 16.94 ± 5.28%, which is consistent with the results obtained in the immunofluorescence assay. It can be concluded that mature male gametes are infected by HHV in the genital tract, where the virus binds to the sperm cell membrane and enters the cell. Interaction of HHV with progressively motile sperm cells implies a vertical viral transmission upon fertilization and points to the necessity of testing ejaculate for herpesviruses infections.

[Mechanisms of smooth endoplasmic reticulum aggregates creation in oocyte's cytoplasm in IVF cycles and its clinical relevance (literature review)].

A large proportion of human oocytes received from exogenous gonadotropin-stimulated cycles have different morphological attributes, or dysmorphisms. The presence of dysmorphism can affect the fertilization rate, the embryo quality and subsequently the frequency of occurrence of implantation and pregnancy. Special attention is paid to oocytes with cytoplasmic attributes such as alteration of cytoplasmic granularity, the appearance of vacuoles, lipofuscin bodies and visible (large) aggregates of smooth endoplasmic reticulum. Endoplasmic reticulum (ER) is a type of the organelle forming an interconnected network of flattened, membrane-enclosed sacs or tubes. One of the main functions of ER in the oocyte is storage and redistribution of calcium, which provides cell activation during fertilization. Furthermore, complex of ER and mitochondria is necessary for accumulation of energy, synthesis of lipids and triglycerides, as well as synthesis of cytosolic and nuclear membranes during the early stages of cleavage. The appearance of anomalously large aggregates of ER in oocytes correlates with a low fertilization rate, low embryo quality, and pregnancy rate. The aim of the manuscript is to summarize current understanding of the mechanism of formation of such pathology of oocytes, together with special aspects of their fertilization and embryo quality.

[Testing system for factors damaging female and male gametes and gonads].

The paper discusses the current possibilities of testing the gameto- and gonadotoxic effects of various damaging factors, including those based on the results of a series of in vivo and in vitro experiments and clinical observations by the author et al. It gives the authors' data on the chronological evaluation of and trends in human oogenesis and spermatogenesis.

[Aneuploidy frequency in sperm of fertile men].

Analysis of sperm aneupoidy in 11 healthy men using two- or three-color FISH permitted to determine the average frequency of disomy in chromosomes 13 and 21 (0.11% and 0.2%, respectively), disomy in chromosome 18 (0.05%) and reveal gonosomal aneuploidy variants and their frequency. The frequency of XX disomy was 0.04%; XY, 0.17%; YY, 0.06%; and gonosomal nullisomy, 0.29%. We also assessed the frequency of meiotic nondisjunction of chromosomes 13, 21, 18, X, and Y and the frequency of XX, XY, and YY diploid chromosomes in sperm. The XY variant prevailed in gonosomal aneuploidy and diploidy and was associated with abnormal chromosomal segregation in meiotic anaphase I. The contribution of human sperm chromosomal imbalance to early embryonic lethality and to some chromosomal diseases of syndrome type in the offspring is discussed.

[Effect of herpes simplex virus on spermatogenesis].

To investigate the effect of Herpes Simplex virus (HSV) on spermatogenesis, HSV in ejaculate was detected by a rapid cultural method in 268 infertile males and 47 healthy ones. The number of mobile spermatozoa in HSV infected samples was less than in non-infected samples (21 mln/mlversus 40 mln/ml, p = 0.0001). The relative number of morphologically normal gametes was 13% versus 19% (p = 0.002), respectively. The quantitative karyological test discovered that males with HSV-infected ejaculate have more degenerating sex cells while in high virus contamination (more than 10 virus particles in 1 ml) the number of spermatides and spermatocytes of the 1 order at diploten stage is low. Organic testicular culture was used for more detailed study of pathogenetic mechanisms of HSV impact on spermatogenesis. Testicular explants infection was associated with reduction in the number of spermatogones, spermatocytes and spermatides on culturing week 2. The above findings reveal some pathogenetic mechanisms underling fertility disorders in males with HSV infection: a gametotoxic effect of the virus reducing populations of spermatogones, spermatocytes and spermatide; affected mobility and morphological characteristics of spermatozoa. Detection of the mechanisms of HSV action on spermatogenesis opens a perspective of antivirus drug administration in combined treatment of male infertility.

[The frequency and spectrum of mutations and the IVS8-T polymorphism of the CFTR gene in Russian infertile men].

The frequency and spectrum of mutations and the IVS8- T polymorphism of the CFTR gene have been studied in a sample of 963 in Russian infertile men. Mutations have been found in 48 out of 1926 analyzed chromosomes (2.5%) in the heterozygous state (n = 46) and in the compound heterozygote L138ins/N1303K (n = 1/n = 1). A CFTR gene mutation was combined with the 5T allele (mutCFTR/5T) in 11 patients. The following mutations have been found: F508del (n = 18), CFTRdele2,3 (21kb) (n = 9), W1282X (n = 7), 2143delT (n = 4), 3849+10kbC>T (n = 2), L138ins (n = 2), 1677delTA (n = 1), 2184insA (n = 1), 3821delT (n = 1), G542X (n = 1), N1303K (n = 1), and R334W (n = 1). The F508del mutation is the most frequent; it has been detected in 37.5% of the affected chromosomes. The total proportion of four mutations (F508del, CFTRdele2,3 (21kb), W1282X, and 2143delT) is about 79% of all mutations found. The 5T allele has been found in 10.9% infertile men and 4.8% of control men. Significant differences in the frequency of the IVS8-5T variant of the CFTR gene have been found between these groups (p = 0.005), as well as between infertile patients without mutations and control men (p = 0.019). In total, the mutations and/or 5T allele have been found in 14.6% of the patients examined. These data indicate increased frequencies of the mutations of the CFTR gene and its allele variant IVS8-5T in Russian infertile men.

[Herpes simplex virus and cytomegalovirus in male ejaculate: herpes simplex virus is more frequently encountered in idiopathic infertility and correlates with the reduction in sperm parameters].

The present investigation was undertaken to study the detection rate of herpes simplex virus (HSV) and cytomegalovirus (CMV) in the ejaculates of males with infertility and to evaluate the impact of virus infection on the major parameters of sperm. Ejaculates from 808 patients were studied. As compared with apparently healthy individuals, the coupled males with primary infertility were found to have HSV more frequently in both the whole ejaculate (31% versus 17%; p = 0.049) and the fraction of actively motile spermatozoa (30% versus 8%; p = 0.016). Ejaculate HSV detection directly correlated with the reduced amount of actively motile spermatozoa (p = 0.0001) and the smaller proportion of morphologically normal forms of germ cells (p = 0.002). CMV was found to have no impact on the motility and morphology of spermatozoids in the ejaculate. Both HSV and CMV in the male ejaculate were significantly more frequently detectable in winter months. The findings lead to the conclusion that HSV is one of the factors for male infertility and can negatively affect the results of assisted reproductive technologies.

Hidden X chromosomal mosaicism in a 46,XX male.

We report on a 37-year-old XX male with complex hidden X chromosomal mosaicism. The patient had fully mature male genitalia with hypoplastic testes descended in the scrotum and no sign of undervirilization. Hormonal examination demonstrated hypergonadotropic hypogonadism, semen analysis showed severe oligoasthenoteratozoospermia. In situ hybridization revealed the presence of 3 SRY-positive cell lines bearing 1, 2 or 3 X chromosomes. Skewed inactivation of the paternal SRY-bearing X chromosome was detected by molecular analysis of the androgen receptor gene.

Unique mosaic X/Y translocation/insertion in infant 45,X male.

We report on a 45,X male with hydrocephaly, lobar holoprosencephaly and ichthyosis. In situ hybridization and molecular analysis have demonstrated the presence of a mosaic SRY-bearing derivative X chromosome that included Yp and heterochromatic Yq fragments.

[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.

[Asymptomatic genital herpes infection and infertility in males].

Incidence of Herpes simplex virus (HSV) was studied in ejaculates of 100 men. The examinees had neither history nor clinical symptoms of HSV genital infection. HSV was detected by a rapid cultural method in the ejaculate of 20 out of 100 examinees (20%). Of 67 males with infertility HSV was detected in 25%, in 19 males examined prophylactically and 14 patients with varicocele (a comparison group) it was found in 10.5 and 7% cases, respectively. Oligozoospermia was two times more frequent in HSV-containing ejaculates than in HSV-negative one. Mean values of the majority of sperm parameters in HSV-positive and HSV-negative groups did not differ statistically. However, it was revealed that such damage of the spermatozoa structure as microhead (consequence of the defective acrosome or reduced genome) and cytoplasm drops on the neck (a sign of immature forms) occurred more often in HSV-infected patients than in persons with HSV-negative ejaculate. Thus, asymptomatic HSV infection has a negative effect on male fertility.

[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.

[Effects of sulfate mineral water for drinking in combination with laser and magnetolaser radiation on occurrence of pathological mitoses in spermatogones of irradiated rats and their progeny].

Drinking sulfate mineral water in combination with low-intensive infra-red laser or magneto-laser radiation is shown to effectively depress development of different forms of pathological mitoses in spermatogones of irradiated rats and in their first-generation progeny.

[Types of Y chromosome deletions and their frequency in infertile men].

Deletions of Y chromosome AZF locus were analyzed during a large-scale andrological and genetic examination of 810 infertile men. The search for Yq microdeletions was carried out according to the standard EAA/EMQN guidelines. The breakpoints were mapped for the deletions in AZF locus. The Y chromosome macro- and microdeletions were detected in 61 (7.5%) infertile men. The frequencies of AZF deletions during azoospermia and severe oligozoospermia amounted to 12.2 and 8.1 %, respectively. On the whole, the frequencies of Yq microdeletions and the genophenotypic correlations characteristic of various AZF deletion types comply with the relevant published data. However, spermatozoids in the ejaculate sediment of men with completely deleted AZFa region or AZFb+c deletions (from solitary spermatozoids to several dozens) were detected for the first time. It was demonstrated that the breakpoints were localized between AZFa and AZFb regions proximally to AZFb+c microdeletions for the majority of cytogenetically detectable deletions in the Y chromosome long arm. This indicates that the mechanisms underlying Yq macro- and microdeletions are somewhat different. The issues related to the role of Y chromosome deletions in the origins of monosomy for X chromosome and X/XY mosaicism are discussed.