SUMMARYThe goal of this study was to determine seminal plasma biomarkers of testicular function in adolescents with varicocoele and to verify enriched gene ontology terms associated to these differential proteomes. An observational study was carried out in an academic research environment. A total of 77 adolescent patients were recruited from a local public school, of which 23 were without varicocoele and with normal semen analysis (control group), 37 were with varicocoele and normal semen (VNS) parameters, and 17 were with varicocoele and altered semen (VAS) parameters. Two semen collections were provided with a 1-week interval, after 2-5 days of ejaculatory abstinence. Seminal plasma proteins were identified and quantified utilizing a label-free shotgun proteomics approach, generating (i) proteins differentially expressed in each group (control, VNS, and VAS) and putative biomarkers using multivariate statistics followed by discriminant analysis. Confirmatory analysis was performed for two proteins by western blotting. Enriched biological processes and molecular functions were determined using gene ontology analysis. In total, 541 proteins were identified and quantified: 108 exclusive or overexpressed in controls, 26 in the VNS group, and 13 in the VAS group. The suggested biomarkers are Cab45/SDF4 (Q9BRK5), protein lefty-1 (O75610), DNase I (P24855), PAP2-alpha (O14494), IBP-7 (Q16270), HDC (P01860), and CRISP-3 (P54108). Western blotting results showed that Cab45 was significantly underexpressed in both varicocoele groups, and CRISP-3 was significantly overexpressed in seminal plasma of adolescents with VAS. In conclusion, specific biomarkers of spermatogenesis and homeostasis are observed in adolescents without varicocoele, and the presence of a palpable varicocoele progressively shifts these adolescents toward initially an immune response, and finally toward a chronic inflammatory profile. This shift is accompanied by decreased semen quality.

Purpose To verify if the presence of varicocele (grades II and III) with and without seminal alterations, using the 5 th centile cutoff values in table A1.1 of the World Health Organization (WHO, 2010) manual, alters the seminal plasma levels of proteins DNASE1 (deoxyribonuclease-1) and IGFBP7 (Insulin-like growth factor-binding protein 7), which are related to apoptosis regulation and cell proliferation, respectively, demonstrating that these proteins are important for correct spermatogenesis. Methods This cross sectional study was performed at the Sao Paulo Federal University Paulo between May 2014 and April 2016. A total of 61 male adolescents were included in this study, of which 20 controls without varicocele (C), 22 with varicocele and normal semen analysis (VNS) and 19 with varicocele and altered semen analysis (VAS). Seminal plasma from each patient was used for Western blotting analysis of individual protein levels. Values of each protein were normalized to a testicular housekeeping protein (PARK7-protein deglycase DJ-1). Results Levels of IGFBP7 protein are increased in varicocele. Levels of DNASE1 are progressively decreased in varicocele (lower in varicocele and normal semen analysis, lowest in varicocele and altered semen analysis) when compared to adolescents without varicocele. DNASE1 levels are positively correlated with sperm concentration and morphology (correlation values of 0.400 and 0.404, respectively; p values of 0.001 and 0.001, respectively). Conclusion In conclusion, in adolescents, seminal plasma levels of IGFBP7, responsible for proliferative activity, are increased in varicocele grades II and III, and DNASE1, responsible for apoptosis regulation, are lower in varicocele, lowest in varicocele and low semen quality. These proteins demonstrate molecular alterations brought upon by varicocele. Moreover, DNASE1 is capable of discriminating a varicocele that causes alterations to semen quality from one that does not. We propose that the initial response of varicocele is to increase proliferative activity which, if followed by regulation of apoptosis, may lead to the ejaculation of a population of sperm that are in accordance with WHO cutoff values but, in the presence of dysregulated apoptosis, leads to lower sperm concentration and morphology.

This study is the first to investigate the effects of different doses of nandrolone decanoate (ND) upon uterine tissue and fertility, and if the reproductive alterations can be restored after cessation of the treatment. Wistar female rats were treated with ND at doses of 1.87, 3.75, 7.5, and 15 mg/kg body weight, diluted in vehicle (n = 30/group), or received only mineral oil (control group, n = 45). The animals were divided into three periods of study: ND-treated receiving a daily subcutaneous injection for 15 consecutive days (1), and treatment with ND followed by 30-day recovery (2), and 60-day recovery (3). At the end of each period, five females per group were induced to death to histopathological analysis and the others were allowed to fertility evaluation (at 19th gestational day). Animals that received ND followed by 30-day recovery exhibited persistent diestrous and marked suppression of reproductive capacity. Conversely, after 60-day recovery, only lowest doses females (1.87 and 3.75 mg/kg) exhibited restoration of normal estrous cyclicity. Uterine weights were increased after ND treatment similarly to that of the controls after 60-day recovery. The ND-treated groups showed histopathological changes in the endometrium, myometrium, and perimetrium, and an increase in the thickness of both muscular and serous layers. Notably, the recovery of uterine tissue after ND treatment was dose- and period-dependent. We reported that administration of ND promoted damage in uterine tissue and fertility of rats, and the recovery periods were insufficient to restore all of the side effects caused by ND under a dose-dependent response.

To verify if quality of spermatozoa from men with testicular germ cell tumours is better before or after orchiectomy. This prospective study was carried out including 24 patients with testicular germ cell tumours, who provide one semen sample before they were submitted to unilateral orchiectomy and one other semen sample 30 days after the surgery. After collection by masturbation and liquefaction, an aliquot of the semen sample was used for semen analysis and another was used to evaluate sperm mitochondrial activity, DNA fragmentation and acrosome integrity. Seminal plasma was used to evaluate lipid peroxidation levels. Pre-orchiectomy sample and post-orchiectomy sample were compared using a paired Student's t test (normal distribution) or a paired Wilcoxon test, when appropriate (p ˂ 0.05). No significant difference was observed in semen analysis. A significant decrease in DNA fragmentation and lipid peroxidation and an increase in mitochondrial activity were observed after orchiectomy. Based on our findings, the semen quality from men with testicular germ cell tumours is better after orchiectomy. K E Y W O R D SDNA fragmentation, orchiectomy, oxidative stress, spermatozoa, testicular neoplasm How to cite this article: Andrade MBR, Bertolla RP, Intasqui P, et al. Effect of orchiectomy on sperm functional aspects and semen oxidative stress in men with testicular tumours.

This study tested the hypothesis that different doses of nandrolone decanoate (ND) will cause changes in the estrous cycle and ovarian tissue of adult rats; and investigated the duration of the recovery period that is sufficient to restore the damage in the animals treated with different doses. Wistar rats were treated with ND at doses of 1.87, 3.75, 7.5 and 15 mg/kg body weight, or received mineral oil (control group) for 15 days, subcutaneously. All animals were divided into three groups according to the treatment periods: (i) ND treatment for 15 days; (ii) ND treatment followed by a 30-day recovery; and (iii) ND treatment followed by a 60-day recovery. Estrous cycle was monitored daily, and at the end of each period, the animals were euthanized for histopathological analysis. During ND treatment and after 30-day recovery, all animals exhibited persistent diestrus. After a 60-day recovery, persistent diestrus was only maintained in the group that had received the highest dose. Ovarian weight was decreased significantly after the 30-day recovery, regardless of ND doses, compared with the control group. There was a reduction (P < 0.05) in the number of corpora lutea and antral and growing follicles, in contrast to an increase (P < 0.05) in atretic follicles in a dose- and time-dependent manner. Remarkable histopathological changes occurred in the ovaries of all ND-treated groups. In conclusion, the different doses of ND caused changes in the estrous cycle and ovarian tissue of rats, and recovery periods (30 and 60 days) were insufficient to completely restore the damage in the animals treated with the highest dose.

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Matrix Metalloproteinases (MMPs) and their regulators – Tissue Inhibitors of Matrix Metalloproteinases (TIMPs) – participate in extracellular matrix remodeling, fibrosis, and semen liquefaction, as well as to inflammatory activity. Seminal plasma has been shown to contain MMPs (MMP-2 and MMP-9) and TIMPs (TIMP-1 and TIMP-2). Also, a link between MMPs gene expression and excessive reactive oxygen species (ROS) has been established. In semen, ROS are associated with altered sperm function and increased DNA fragmentation. In this study, it is hypothesized that seminal MMPs and TIMPs levels are associated with sperm DNA fragmentation due to the fact that MMPs have been associated with semen quality. We also hypothesized that these proteins could predict DNA fragmentation status in sperm. Therefore, this study set out to verify if sperm DNA fragmentation levels relate to seminal levels of members of the MMP and TIMP protein families. The High sperm DNA fragmentation group presented lower seminal plasma levels of MMP-2, MMP-7, TIMP-1, TIMP-2 and TIMP-4 when compared to Low sperm DNA fragmentation group. Also, samples in the high sperm DNA fragmentation group presented higher acrosome integrity and lower mitochondrial activity levels when compared to low sperm DNA fragmentation samples. In the logistic regression analysis, MMP-2, MMP-7, and TIMP-4 classified samples as low and high sperm DNA fragmentation, with an overall model fit of 74.5%. Results from this study may demonstrate a specific inflammatory mechanism in samples with high sperm DNA fragmentation. This, in turn, can lead to the development of new studies regarding this mechanism and, in the future, create an opportunity to treat these patients for sperm DNA fragmentation by treating inflammatory seminal activity.

Purpose: Sperm DNA fragmentation is a major cellular mechanism underlying varicocelerelated male infertility. However, the type of DNA fragmentation -whether oxidative or of another nature -remains unknown. Thus, the aim of this study was to evaluate single-and double-stranded sperm DNA fragmentation, and oxidative-induced sperm DNA damage in men with varicocele. Materials and Methods: A cross-sectional study was performed, including 94 normozoospermic adults, of which 39 men without varicocele (controls) and 55 men with varicocele grades II or III, uni-or bilaterally. All men collected semen by masturbation. After semen analysis, the remaining volume was used for evaluation of three types of sperm DNA damage: (i) total DNA fragmentation, using an alkaline comet assay, (ii) double-stranded DNA fragmentation, using a neutral comet assay, and (iii) oxidative DNA damage, using an alkaline comet assay associated with the DNA glycosylase formamidopyrimidine enzyme. In each assay, percentage of sperm with any degree of DNA fragmentation, and with high DNA fragmentation were compared between the groups using an unpaired Student's t test or a Mann-Whitney test. Results: The varicocele group presented a higher rate of sperm with fragmented DNA (both any and high DNA fragmentation), considering single-stranded DNA fragmentation, double-stranded DNA fragmentation, or a combination of both, as well as oxidativeinduced DNA fragmentation. Conclusions: Patients with varicocele have an increase in sperm DNA fragmentation levels, particularly in oxidative stress-induced sperm DNA damage.