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Diabetes leads to testicular damage and infertility. Mesenchymal stem cells and their secretory trophic factors have shown potential as regenerative therapies for diabetes and its associated complications. This study examined the effects of conditioned medium derived from Wharton's jelly mesenchymal stem cells (WJMSCs‐CM) on sperm parameters, reproductive hormones, biochemical parameters, and histological changes in the testes of diabetic rats. Fifty‐six male Sprague–Dawley rats (250–300 g) were assigned to eight groups: control, diabetes, and six diabetic groups receiving early or late treatments with WJMSCs‐CM (D‐CME, D‐CML), insulin (D‐INSE, D‐INSL), or DMEM (D‐DME, D‐DML). In the early treatment groups, insulin (3 U/day, subcutaneously) and WJMSCs‐CM (10 mg/week, intraperitoneally) were administered immediately after diabetes induction; in the late treatment groups, these interventions began 30 days postinduction. Blood glucose and insulin levels, along with sperm parameters, were assessed. Sex hormones, testicular antioxidant enzyme activity, malondialdehyde (MDA), and glutathione (GSH) concentrations were measured using colorimetric methods. Real‐time PCR detected Bax, Bcl‐2, and tumor necrosis factor‐alpha (TNF‐α) gene expression. Our results showed that diabetes increased blood glucose levels, decreased insulin and sex hormone levels, induced testicular oxidative stress and apoptosis, and reduced sperm parameters compared to the control. WJMSCs‐CM significantly ameliorated hyperglycemia, increased insulin and sex hormone levels, and improved sperm quality. In WJMSCs‐CM‐treated diabetic rats, MDA levels were reduced, while GSH and antioxidant enzyme activity increased. Furthermore, WJMSCs‐CM decreased the testicular Bax/Bcl‐2 ratio and TNF‐α expression, as well as enhanced spermatogenic, Sertoli, and Leydig cells. In conclusion, WJMSC‐CM administration effectively mitigated diabetes‐induced testicular damage by reducing oxidative stress, inflammation, and apoptosis. Early treatment with WJMSCs‐CM was more effective than late treatment for diabetes‐induced reproductive dysfunction. [ABSTRACT FROM AUTHOR]

Background: Sperm cryopreservation-thawing process has damaging effects on the structure and function of sperm, namely cryoinjury. Calcium overload has been reported as a postulated mechanism for sperm damage during the first steps after thawing. This study was designed to assess the intracellular calcium (Ca2+i) after cryopreservation and to clarify the role of a calcium chelator ethylene glycol-bis (2-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA) on human sperm quality. Methods: Forty semen samples were obtained from fertile men (March 2017 to 2018). The samples were randomly divided into fresh (F) and cryopreserved-thawed (CT) groups. The F and CT samples were divided into control and 1 mM EGTAtreated groups. Sperm kinematics and membrane integrity were assessed. The reactive oxygen species (ROS) and adenosine triphosphate (ATP) were measured by luminescent methods. Ca2+i, apoptotic rate, and mitochondrial membrane potential (MMP) were evaluated using flow cytometric methods. Data were compared using SPSS software, version 16.0 by ANOVA and Kruskal-Wallis test. P<0.05 was considered as significant. Results: Cryopreservation decreased sperm motility, viability, membrane integrity, Ca2+i, MMP, and induced cell apoptosis and ROS production. EGTA could not protect the cryopreserved sperm from cryoinjury. It was found to have destructive effects on fresh sperm motility and viability (P=0.009) relative to cryopreserved sperm. ATP was reduced (P=0.02) and ROS production (P=0.0001) was increased in the EGTA-treated F and CT sperms. Conclusion: Despite Ca2+i reduction by EGTA, it had no protective effects on fresh or cryopreserved sperm. We concluded that sperm cryoinjury was not dependent on calcium overload, and it was suggested that cryoinjury was mainly related to cell membranes damage. [ABSTRACT FROM AUTHOR]

Background: Human follicular fluid (FF) is rich in hormones and antioxidants. Many components of FF differ in follicles of patients with polycystic ovary syndrome (PCOS). Regarding vitamin D effects on gene expression, 25(OH)D level of FF and its association with oxidative status and sex steroids dysregulation in PCOS group was evaluated and compared to controls of Non-obese healthy women. Methods: FF of 50 non-obese healthy women and 50 women with PCOS (18-36 years old) who were candidates for IVF/ICSI was aspirated on the oocyte retrieval day. Sex steroids and 25(OH)D levels were measured by ELISA. Reactive oxygen species (ROS) levels, total antioxidant capacity (TAC), and activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were assessed by chemiluminescence and spectrophotometric methods. Data were analyzed by unpaired t-test or Mann-Whitney test, and Pearson correlation coefficient. The p<0.05 was considered statistically significant. Results: Estradiol, progesterone, 25(OH)D, TAC, and activities of SOD, GPx, and CAT in FF of women with PCOS were significantly lower, whilst their free and total testosterone and ROS levels were significantly higher than controls. There were significant positive correlations between FF levels of 25(OH)D with TAC, estradiol and progesterone concentrations, SOD, GPx, and CAT activities. Negative correlations were found between 25(OH)D with free and total testosterone, and ROS levels. Conclusion: Despite different hormonal and antioxidant levels in FF of normal and cystic follicles, the correlation between 25(OH)D levels with sex steroids and oxidative stress markers showed a possible role of 25(OH)D in regulating sex hormones secretion and enhancement of antioxidant defense. [ABSTRACT FROM AUTHOR]

Background: Normal sperm function depends on appropriate intracellular calcium (Ca12+) and reactive oxygen species (ROS) levels. Calcium activates NADPH oxidase-5 (NOX5) that leads to ROS generation. The calcium channel of sperm (CatSper) is activated by progesterone and intracellular alkalization. Herein, the interactive role of CatSper, Hv1 channels, and NOX5 enzyme on Ca12+ and ROS generation in human sperm is investigated. Methods: The present laboratory in vitro study was carried out in the School of Medicine, Shiraz University of Medical Sciences (Shiraz, Iran) during 2016. Normal semen samples (n=15) were washed and diluted to 20x106 sperm/mL. The diluted samples were divided into 16 groups containing Ham's F-10 (the control group), 2 µM NNC (CatSper inhibitor), 1 mM ZnCl2 (Hv1 inhibitor), 1 µM DPI (NOX5 inhibitor), NNC+Zn, NNC+DPI, and NNC+Zn+DPI. The other 8 groups were the same as the above except that they contained 1 µM progesterone. Cell viability and Ca12+ were analyzed by flou-3 AM probe and PI staining, respectively, using flow cytometric method. ROS generation was assessed by chemiluminescence method. Statistical analysis was performed using the one-way ANOVA followed by Tukey's test. P values <0.05 were considered statistically significant. Results: Progesterone increased Ca12+ and ROS generation. The addition of NNC, Zn, or NNC+Zn significantly decreased Ca12+ in the control and progesterone containing groups. Progesterone-induced ROS generation was decreased significantly in all groups containing NNC, Zn, or DPI and reached to the control level when DPI was added to NNC or Zn. Conclusion: There is a functional relationship between CatSper and Hv1 channels in increasing Ca12+. The activity of CatSper and Hv1 channels are required for progesterone-induced ROS generation by NOX5 enzyme. [ABSTRACT FROM AUTHOR]

Cooling method was proposed to maintain the sperm quality for several days. Nevertheless, during this procedure, sperm is encountered to "cold shock", and its quality decreases time-dependently. This study was designed to improve the in vitro sperm preservation methods. Thirty normal semen samples were examined in Shiraz, Iran, 2017. Fifteen samples were incubated at 22-27 °C and 15 samples were cooled moderately to 4 °C. Each sample was divided into five subgroups; control, solvent, 200 μM Trolox, 40 μM Coenzyme Q10, and 10 mM ATP. ATP was added only 15 minutes before the analysis. Assessments of motility parameters and sperm viability were done every 24 hours. Statistical analysis was performed using SPSS 16 software. The differences between two main groups and subgroups were compared by t test and one-way ANOVA, respectively. The effect of time was analyzed by repeated measurement test. Sperm motility and viability were the same in both groups until 24 hours, except the straight line velocity was greater in the cold group. Even after 48 hours, progressive motility and sperm velocity, but not viability, were still the same. The greatest reduction in progressive motility occurred on the second day; and after 72 hours, sperm quality was better preserved in 22-27 °C. Treatment with Trolox, coenzyme-Q10, and extracellular ATP did not have effect on sperm quality. Cold temperature is recommended for in-vitro sperm preservation up to 24 hours, and 22-27 °C is preferred for longer time storage. The sperm does not need antioxidant therapy for quality maintenance, but the extender media must be supplied with nutrients and antibiotics. [ABSTRACT FROM AUTHOR]

Background: Intracellular calcium and proton concentrations are important factors for activating human sperm. Calcium ion (Ca2+) enters sperm through voltagedependent calcium channel of sperm (CatSper). Proton was extruded from sperm through voltage-gated proton channel (Hv1). In the present study, the selective inhibitors of the CatSper and Hv1 channels, NNC 55-0396 (NNC) and zinc ion, respectively, were used to investigate functions of these channels. Methods: Normal semen samples (n=24) were washed and diluted to 20×106 sperm/ ml. The diluted sample was divided into 8 groups, containing Ham’s F-10 (the control group), 2 μM NNC, 1 mM ZnCl2 and NNC+Zn. The other 4 groups were the same as above, except that they contained 1 μM progesterone. The computer assisted analysis was done by VT-Sperm 3.1 to determine the percentage of motile sperm and sperm velocity. Acrosomal status was monitored by FITC-PSA and viability assessed by Eosin–Y staining. Statistical comparisons were made using ANOVA followed by Tukey post hoc test. The p<0.05 was considered significant. Results: The percentage of viable and motile sperm, curvilinear velocity and other parameters of motility was reduced in all groups containing NNC, zinc and NNC+ zinc. Progesterone–induced acrosome reaction was abolished by each of these inhibitors. The combination effect of NNC plus zinc on motility and progesterone–induced acrosome reaction was not stronger than NNC by itself. Conclusion: CatSper and Hv1 channels play a critical role in human sperm function and viability. It seems that a functional relationship exists between CatSper and Hv1 channels. [ABSTRACT FROM AUTHOR]

Background: Low levels of reactive oxygen species (ROS) and calcium are necessary for sperm function. NADPH oxidase 5 (NOX5) is a membrane enzyme which produces ROS. This enzyme is dependent on calcium for its activity. We investigated the importance of NOX5 and an important calcium channel (CatSper) on sperm function. Methods: This laboratory in-vitro study was done in Shiraz, Iran, 2016. Normal semen samples (n=24) were washed and diluted to 20x106 sperm/mL. The diluted samples were divided into 8 groups, containing Ham's F-10 (control group), 2 µM of NNC (CatSper channel inhibitor), 1 µM DPI (NOX5 inhibitor), and NNC+DPI. The other 4 groups were the same as the 1st ones, except that they contained 1 µM of progesterone. Motility assessment was done by VT--Sperm 3.1. Acrosome status was monitored with acrosome-specific FITC-PSA using fluorescent microscopy. Sperm viability was assessed by Eosin Y. Statistical analysis was performed using SPSS 16 software. The comparison between the groups was done using the one-way ANOVA, followed by Tukey. A P<0.05 was considered significant. Results: The percentage of motile sperm, sperm velocity, and viability decreased significantly in the groups containing NNC. DPI reduced sperm progressive motility only in the progesteronestimulated condition. Progesterone induced acrosome reaction, but this effect was inhibited by NNC and DPI. Conclusion: CatSper had a prominent role in the motility, acrosome reaction, and viability of the human sperm. The function of NOX5 was important only in the stimulated sperm. We conclude that CatSper has a more prominent role than NOX5 activity. The functional relation between NOX5 and CatSper is not clear but is very probable. [ABSTRACT FROM AUTHOR]

The freezing and thawing process not only is associated with serious damage to sperm such as damage to the plasma membrane and the acrosomal membrane but also changes the membrane permeability to some ions including calcium. Also, the generation of oxygen free radicals is increased during the freezing-thawing process. The purpose of this study was to evaluate of the effects of Trolox as an antioxidant and edetic acid (EDTA) as a calcium chelator on frozen-thawed (FT) sperm and compare these effects with those on fresh sperm. This study was done on these men of 25 healthy men, who referred to Shiraz Infertility Centerbetween2012 and2013. Normal samples were transferred to the ReproductivePhysiology Laboratory, Department of Physiology,Shiraz University of Medical Sciences, Shiraz. The samples were divided into two groups randomly: fresh and FT sperm groups. Each group was divided into five subgroups: control group, the solvent group (0.1%dimethyl sulfoxide [DMSO]), Trolox group (200μM), EDTA group (1.1mM), and Trolox+EDTA group. The percentages of motility, viability, and acrosome-reacted sperm were tested. The percentages of motility and viability in the FT sperm were lower than those in the fresh sperm. The progressive motility of the FT sperm was improved nonsignificantly with Trolox+EDTA. However, the effect of Trolox+EDTA on the progressive motility of the FT sperm was much more than that on the fresh sperm. The fewest acrosome-reacted sperm were observed in the EDTA-containingFT sperm. Antioxidant supplementation or omission of extracellular calcium may partly improve motility and also reduce acrosomal damage in FT sperm. [ABSTRACT FROM AUTHOR]

BACKGROUND: The number of couples that meet the definition of infertility at reproductive ages is increasing worldwide. One of the most known conditions of infertility in males is azoospermia, defined as complete absence of spermatozoa in the semen. Azoospermia manifests in two forms, namely obstructive and non-obstructive azoospermia. Although the presence of antisperm antibody (ASA) has been reported in 88% of the patients with obstructive azoospermia (OA), interestingly, there is no data regarding ASA targets in OA individuals. AIM: The present study aimed to identify sperm antibody targets in a group of OA men. SETTINGS AND DESIGN: The present study was carried out on 27 OA infertile men and 27 healthy fertile age-matched males as cases and controls, respectively. SUBJECTS AND METHODS: The sperm proteome was separated using two-dimensional gel electrophoresis technique, transferred onto the polyvinylidene fluoride membrane, and blotted with the sera of a group of OA men. Then, it was compared with the membranes blotted with the sera of a group of healthy fertile men. Matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI TOF/TOF) mass spectrometry was used to identify the different blotted spots and finally the results of the mass analysis were confirmed using reverse transcriptase polymerase chain reaction method. RESULTS: The results indicated that OA patients might produce antibody against two sperm proteins, Tektin-2 and triose phosphate isomerase. Moreover, the expressions of the two targeted proteins were confirmed at RNA level. CONCLUSIONS: The findings of the present study revealed two functionally important sperm proteins as antibody targets in azoospermic men. [ABSTRACT FROM AUTHOR]

Ischemic stroke is one of the most frequent causes of injury in the central nervous system which may lead to multiorgan dysfunction, including in the lung. The aim of this study was to investigate whether brain ischemia/reperfusion with or without mechanical ventilation leads to lung injury. Male Sprague-Dawley rats were assigned to four groups: Sham, 1-h brain ischemia (MCAO)/24-h reperfusion (I/R), mechanical ventilation with moderate tidal volume (MTV), and I/R+MTV. The pulmonary capillary permeability ( K fc ) was measured in the isolated perfused lung. Mean arterial blood pressure (MAP), heart rate (HR), blood-gas variables, histopathological parameters, lung glutathione peroxidase, and TNF-α were measured. K fc in the I/R, MTV, and I/R+MTV groups were higher than that in the Sham group. In the I/R, MTV, and I/R+MTV groups, arterial partial pressures of oxygen and the arterial partial pressure of oxygen/fraction of inspired oxygen ratios were lower, whereas arterial partial pressures of carbon dioxide were higher than those in the Sham group. The histopathological score in the I/R group was more than that in the Sham group, and in the MTV and I/R+MTV groups were higher than those in the Sham and I/R groups. Furthermore, there were stepwise rises in TNF-α in the I/R, MTV, and I/R+MTV groups, respectively. There was no significant difference in MAP between groups. However, HR in the MTV group was higher than that in the Sham group. Brain ischemia/reperfusion leads to pulmonary capillary endothelial damage and the impairment of gas exchange in the alveolar-capillary barrier, which is exacerbated by mechanical ventilation with moderate tidal volume partially linked to inflammatory reactions.

Background: Cardiovascular diseases are the major public health problem in many countries and are responsible for more than half of the deaths in above 50-year-old women. The most common curable risk factor of these disorders is hypoestrogenemia resulting from menopause. The present study aimed to investigate the effect of melatonin on plasma lipid levels in menopausal women., Materials and Methods: The present double-blind, placebo-controlled, clinical trial was conducted in 2013-2014 on 240 menopausal women between 40 and 60 years old referring to the Gynecology and obstetrics clinics of Shiraz University of Medical Sciences who were randomly divided into two groups. The intervention group received 3 mg melatonin tablets and the control group received the placebo for 3 months. The data were gathered using the demographic information questionnaire and lipid profile test before and 3 months after the intervention. Then, the data were analyzed through the SPSS statistical software (version 16). The repeated measures analysis of variance, the least significant difference, the independent-sample t , the Chi-square, and Fisher's exact tests were done for data analysis., Results: The two study groups were similar regarding the demographic and clinical variables at the beginning of the study. In the melatonin group, the amount of triglyceride increased from 140.34 ± 48.29 before the study to 151.24 ± 54.60 3 months after the intervention and no significant difference was observed between the two groups in this regard (confidence interval [CI] = 95%, P > 0.05). In addition, no significant difference was found between the two groups concerning low-density lipoprotein cholesterol level (CI = 95%, P = 0.125)., Conclusion: Melatonin was not effective in reduction of lipid levels. However, further controlled studies are needed to be conducted on the issue., Competing Interests: There are no conflicts of interest.