A Novel Approach of Tracing in Vivo Bioluminescence Imaging Expression of Vitrified Immature Testicular Tissue Grafts Until Adulthood: A Translational Transgenic Mouse Model

Background: The optimal method for cryopreserving immature testicular tissue (ITT) remains unknown and there is no standardized protocol. Controlled slow freezing remains the mainstream method of choice in human prepubertal male fertility preservation. Currently, the outcomes for ITT vitrification are conflicting, and most data are limited to in vitro animal studies.Methods: A total of 12 pairs of donor and recipient mice were included in our experiments. The donors were immature transgenic mice, and the recipients were wild-type male mice. In the vitrification group, ITT was vitrified and thawed before transplantation. In the control group, ITT was transplanted to the recipients immediately. After thawing, we measured the expression of apoptosis-related mRNA caspase-3. More importantly, we monitored to adulthood all the transplanted grafts in vivo using noninvasive bioluminescence imaging (BLI) technology. On day 31, we removed the grafts for evaluation via hematoxylin and eosin staining and immunohistochemistry (IHC).Results: We traced the survival of the grafts by in vivo BLI on days 1, 2, 5, 7, and 31 after transplantation. In both the vitrification and the control groups, bioluminescence decreased between days 2 and 5. Subsequently, the bioluminescence showed an upward trend until day 31. Compared with day 1, the bioluminescence was significantly stronger on day 31 after transplantation (P = 0.009). The differences between the two groups were constantly insignificant after analysis. These results indicate that both fresh and frozen–thawed testicular tissues can survive for at least 31 days after transplantation. Moreover, the vitrification group showed BLI signals comparable with those of fresh tissues. Compared with the control group, expression of the caspase-3 gene was significantly increased after vitrification (P = 0.04). Histology and IHC showed that both tissue structure and protein expression were intact in both groups.Conclusions: Transplanted vitrified ITT grafts could survive till adulthood with BLI intensity comparable to that of the fresh control. Intact cells and structures for spermatogenesis in vitrified ITT grafts were as well-preserved as those in the control group. This translational model of self-repairing vitrified ITT grafts in vivo, lends weight to the role of vitrification in prepubertal male fertility preservation.

Curcumin Along With Fe3O4 Nanoparticles Improved Sperm Parameters In Rats With Testicular Ischemia

Background: Ischemic/reperfusion (I/R) in testicular tissue is one reason for the worldwide increase in male infertility. In the present study, we assessed the effects of curcumin and Fe3O4 nanoparticles (NPs) on sperm parameters in rats with I/R damage. Materials and Methods: Forty-eight adult male rats were divided into two groups (n=24 per group): control and torsion/detorsion. The control and torsion/detorsion groups were divided into four subgroups include sham, Fe3O4 NPs, curcumin, and Fe3O4 NPs+curcumin. After the rats were sacrificed, semen was collected from their epididymal tissues to assess sperm viability, motility, concentration, and morphology. Results: Curcumin significantly improved viability, motility, and normal sperm morphology in rats with I/R damage compared to the control group; however, it did not have a significant effect on sperm concentration (P<0.001). Fe3O4 NPs alone decreased all sperm parameters in the control and I/R rats (P<0.001). However, concomitant administration of Fe3O4 nanoparticles with curcumin significantly improved sperm parameters in rats with I/R damage (P<0.001). Conclusion: The increase in all semen parameters in the experimental groups with concomitant use of Fe3O4 NPs plus curcumin indicated that green synthesis of NPs could be recommended for future clinical studies.

CNV Hotspots in Testicular Seminoma Tissue and Seminal Plasma

Seminoma (SE) is the most frequent type of testicular tumour, affecting predominantly young men. Early detection and diagnosis of SE could significantly improve life quality and reproductive health after diagnosis and treatment. Copy number variation (CNV) has already been associated with various cancers as well as SE. In this study, we selected four genes (MAGEC2, NANOG, RASSF1A, and KITLG) for CNV analysis in genomic DNA (gDNA), which are located on chromosomes susceptible to gains, and whose aberrant expression was already detected in SE. Furthermore, CNV was analysed in cell-free DNA (cfDNA) from seminal plasma. Analysis was performed by droplet digital polymerase chain reaction (ddPCR) on gDNA from SE and nonmalignant testicular tissue. Seminal plasma cfDNA from SE patients before and after surgery was analysed, as well as from healthy volunteers. The CNV hotspot in gDNA from SE tissue was detected for the first time in all analysed genes, and for two genes, NANOG and KITLG it was reflected in cfDNA from seminal plasma. Although clinical value is yet to be determined, presented data emphasize a potential use of CNV as a potential SE biomarker from a liquid biopsy.

Lipidomic profile of seminal plasma in non-obstructive azoospermia with sperm maturation arrest

Introduction. The difference between obstructive and non-obstructive azoospermia with sperm maturation arrest is important for the choice of treatment tactics and adequate counseling of a married couple.Purpose of the study. The study aimed to assess the semen lipid profile in patients with sperm maturation arrest. Materials and methods. Samples of seminal plasma for lipid composition of 24 men with normozoospermia and 64 men with azoospermia were studied. Patients with azoospermia underwent microdissection testicular biopsy followed by the detection of testicular tissue pathology. Lipid extracts were analyzed by liquid chromatography with mass spectrometry. Lipid data were compared with the results of pathomorphological studies.Results. Comparison of two groups revealed a statistically significant concentration differences for 22 lipids detected in positive-ion mode and 11 lipids detected in negative-ion mode. Those lipids mainly belong to the classes hexosylceramides, sphingomyelins and phosphatidylcholines — simple ethers and oxidized lipids. In multivariate analysis, the following lipids were found to be statistically significant predictors of sperm maturation arrest: PC 16: 0_22: 6 lipid (β-coefficient: -0.73; 95% confidence interval (95% CI): -1.42 to -0.27; odds ratio (OR): 0.48; OR CI: 0.24 to 0.76; Wald's test: -2.58; p = 0.01), SM d20: 1/22:2 lipid (β-coefficient 4.96; 95% CI 2.29 to 9.13; OR: 142.31; OR CI: 9.90 to 9.22^103; Wald's test: 2.93; p = 0.003); PG 20:3_22: 6 lipid (β-coefficient 2.52; 95% CI 1.13 to 4.49; OR: 12.37; OR CI: 3.10 to 89.27; Wald's test: 3.02; p = 0.002); PC O- 16: 1/16:0 lipid (β-coefficient 1.96; 95% CI -4.12 to 0.27; OR: 0.14; OR CI: 0.02 to 0.76; Wald's test: -2.05; p = 0.04). The prediction model characteristics of sperm maturation arrest, obtained during cross-validation in the positiveion mode composed: sensitivity 91%, specificity 85%; in negative-ion mode: sensitivity 75%; specificity 81%.Conclusions. Even though early stages of spermatogenesis are equally preserved in both fertile men and men with homogeneous sperm maturation arrest, the semen in the studied group of patients differed in its lipid profile. Patients with non-obstructive azoospermia, associated with meiosis arrest, may have unique lipidomic characteristics of seminal plasma, which in the future will make it possible to differentiate various variants of severe male infertility using non-invasive methods.

The effect of N-acetyl cysteine consumption on men with abnormal sperm parameters due to positive history of COVID-19 in the last three months

Male infertility is an important factor accounting for 40-50% of infertility cases that may be due to disturbance in one of the parameters as concentration, motility and morphology observed in one or two semen analysis with an interval of 1 and 4 weeks. COVID-19 may affect male fertility through virus division, cytotoxic effects on testicular tissue and immunopathological effect. N-acetyl cysteine (NAC) improved sperm concentration and acrosome reaction while reducing reactive oxygen species (ROS) and oxidation of sperm DNA. This interventional study was conducted on 200 men who were referred to private infertility clinics for female factor (their previous semen analysis was normal) and got COVID-19 infection in the last 3 months showing an impairment of the latest semen analysis due to COVID. Men were placed in two groups of control (n = 100) and intervention (NAC consumption). Subjects who got COVID-19 infection had a significant impairment of sperm quality (sperm concentration, sperm motility, and normal sperm morphology) compared to their semen analysis evaluated before the COVID-19 infection. NAC consumption significantly improved sperm total motility, sperm morphology and sperm concentration. COVID-19 infection has a negative effect on sperm parameters. NAC supplementation may have positive effect on sperm parameters.

Influence of quercetin on morphological changes in rats testes after 180 days during central deprivation of luteinizing hormone

A relevant and popular area of research is the protective effect of the bioflavonoid quercetin, which makes it possible to use it to correct testicular dysfunction of various origins. The aim of the study was to determine the effect of quercetin on the microscopic organization of rat testicles, nitric oxide production and the intensity of oxidative stress in rat testicles on the 180th day of the experiment, during experimental central deprivation of luteinizing hormone synthesis caused by triptorelin solution. The experiment was performed on 20 adult male white rats. Rats were divided into 2 groups of 10 animals in each group: control group (I), group with central deprivation of LH synthesis + quercetin (II). Animals from the group with central deprivation of LH synthesis were injected subcutaneously with triptorelin acetate at a dose of 0.3 mg of active substance per kg and quercetin 100 mg per kg of body weight 3 times a week, while the control group was injected with saline. Ultrathin sections made by ultramicrotome were contrasted with 1 % aqueous uranyl acetate and lead citrate according to the Reynolds method and examined by electron microscopy. According to standard methods, the material was poured into paraffin blocks, from which sections with a thickness of 4 μm were made and stained with hematoxylin and eosin. Histological specimens were examined using a Вiorex 3 light microscope with a digital microfilter with software adapted for these studies. All biochemical studies were performed in 10 % of testicular tissue homogenate using a Ulab 101 spectrophotometer. Statistical processing of the study results was performed using Microsoft Office Excel software and Real Statistics 2019 extension. The nonparametric Mann-Whitney test was used to determine the statistical significance of differences between groups. Our study of the interstitial space in the testes of white rats showed the heterogeneity of populations of endocrinocytes and macrophages and the variability of structural and functional parameters. In the tissues of the testes in conditions of prolonged central deprivation of testosterone synthesis develops oxidative stress, which on the 180th day of the experiment leads to the development of edema of the interstitial space, followed by tissue fibrosis. Changes in the polarization of macrophages in our opinion may cause oxidative stress in the testes, as evidenced by increased iNOS activity and decreased arginase activity, but use of quercetin protects rat testicular tissue from oxidative damage caused by triptorelin by increasing direct antioxidant system and effects on the lipoxygenase pathway of arachidonic acid metabolism.

Dose Dependent Degeneration of Leydig Cells Following Kisspeptin-10 Administration: An Ultrastructural Study

Background: The discovery of kisspeptin signaling as a key regulator of gonadotropin releasing hormone (GnRH) secretion from the hypothalamus enhanced our understanding of the neuroendocrine regulation of mammalian reproduction. Effects of central and peripheral administration of kisspeptin on plasma gonadotropins, testosterone and spermatogenesis are studied in detail. Objective: The present study was conducted to check the ultrastructure of Leydig cells in prepubertal male rats in response to the administration of a range of kisspeptin doses. Method: To this end, we administered a range of kisspeptin-10 doses (1µg, 1ηg and 10ρg) intraperitoneally, to prepubertal male Sprague-Dawley rats (PND 35) twice daily after every 12 hours. Control rats were injected with physiological saline in parallel. Results: At the end of the treatment, plasma concentrations of testosterone was measured by competitive binding radioimmunoassay and small pieces of rat testicular tissue were processed for electron microscopy to examine the ultrastructure of Leydig cells. Plasma testosterone concentration was reduced significantly at 1ηg (P<0.05) and 1μg (P<0.01) doses as compared to control. Distinct ultrastructural changes categorized as dilatation of cytoplasmic organelles, irregular shaped nuclei with nuclear membrane invaginations, reduced nuclear sizes, degeneration and vacuolation were observed in the kisspeptin-10 treated Leydig cells as compared to control. Quantification of the data showed reduced Leydig cell indices and hyperplasia of the interstitial cells. Conclusion: It is concluded that chronic intermittent administration of kisspeptin-10 has a dose dependent degenerative effect on the plasma testosterone levels and Leydig cells ultrastructure in prepubertal male rats.