Impact of calcium and reactive oxygen species on human sperm function: Role of NOX5.

NOX5 is introduced as a new therapeutic target for infertility treatment. This study aimed to compare the basal and stimulated reactive oxygen species (ROS) production and sperm function in human teratozoospermic (n = 15) and normozoospermic (n = 17) semen samples following calcium overload and NOX5 activation. Washed spermatozoa incubated for 1 h under five various conditions: control group, adding a calcium ionophore A23187, phorbol myristate acetate (PMA), A23187 + PMA, and diphenylene iodonium (DPI) + A23187 + PMA. ROS generation was measured immediately after treatment for 30 min. Motility, viability, acrosome reaction, and apoptosis were evaluated after 1-h incubation. ROS production significantly increased when A23187 or PMA was added to the sperm medium. DPI had suppressive effects on ROS generation. Progressive and total motility significantly decreased following calcium elevation and NOX5 activation, which was somewhat returned by DPI. Necrotic and live cells in teratozoospermia was, respectively, higher and lower than normozoospermia samples. Incubation with A23187 significantly increased the percentage of early and late apoptosis. Teratozoosperm are more vulnerable than normal spermatozoa, and produce more basal and stimulated ROS. It seems that calcium overload induces apoptosis in spermatozoa and loss of viability through MPT pore opening and increased intracellular ROS.

The cryoprotective effect of vitamins on human spermatozoa quality: a systematic review and meta-analysis.

The Cryopreservation of spermatozoa ensures preserving fertility potential after some medical treatments such as chemotherapy and radiotherapy in cancer patients. However, many spermatozoa encounter serious damages, and their motility and viability decrease considerably after thawing. The excessive production of reactive oxygen species is one of the major causes of these damages. The supplementation of cryopreservation media with vitamins, which are well-known antioxidants, can reduce cryopreservation-induced damages. In this systematic review, we aimed to evaluate the cryoprotective effect of various vitamins on the quality of cryopreserved-thawed human spermatozoa. Two researchers searched PubMed, ISI, and Scopus databases up to March 2020. All original articles using vitamins in human spermatozoa cryopreservation media were included. We used a standardized form to extract sample size and to determine sample quality, the type and dose of vitamins, and the cryopreservation methods and their effects. We performed a meta-analysis on studies with available data (Mean + SD in cryoprotectant and cryoprotectant + cryoprotectant groups). We also performed a test of between-study heterogeneity, subgroup analysis, and meta-regression. Out of 258 studies, 16 articles were included for the analysis. Our meta-analysis revealed that using vitamins in cryopreservation media could increase motility by 4.60% (95% CI 6.16, 3.05; P = 0.0001), viability by 5.71% (95% CI 9.71, 1.72; P = 0.0001), and DNA integrity by 10.20% (95% CI 12.98, 7.42; P = 0.0001) in cryopreserved-thawed spermatozoa. We found a significant correlation between using vitamins and improved spermatozoa quality; the sperm motility and viability were improved and DNA fragmentation was reduced after thawing by vitamins. However, we could not emphasize on any type or dose of vitamins but we conclude that the anti-oxidative function of vitamins is the main reason for these benefits.

Progesterone and anandamide diminish the inhibitory effect of zinc on mature human sperm.

Zinc ion (Zn2+) homeostasis is very important for sperm capacitation and hyperactivation. Zn2+ is a specific inhibitor of the voltage-dependent proton channel (Hv1). Intracellular alkalisation of human spermatozoa is mainly dependent on opening of Hv1. Anandamide may affect spermatozoa through activation of Hv1. An increase in intracellular pH and progesterone (P4) activate cation channels of spermatozoa (CatSper). This study was designed to elucidate the interaction between ZnCl2, P4 and anandamide on human sperm function and intracellular calcium concentrations ([Ca2+]i). Human normal semen samples (n = 30) were diluted (20 × 106 spermatozoa mL-1) and divided into control and ethanol (0.01%)-, anandamide (1 nM)-, ZnCl2 (1 mM)-, P4 (10µM)-, anandamide+ZnCl2- and P4+ZnCl2-treated groups. Sperm kinematics, viability, acrosome status and [Ca2+]i were assessed. The percentage of viable and motile spermatozoa and sperm velocity was reduced in the ZnCl2-treated groups. Anandamide and P4 attenuated the inhibitory effects of ZnCl2 on sperm kinematics. Loss of the acrosome membrane was observed in all experimental groups. P4 and anandamide are present naturally in secretions of the female reproductive tract and modulate the inhibitory effects of ZnCl2 on sperm kinematics. This attenuation is probably due to a change in [Ca2+]i and prevention of Hv1 inactivation by P4 and anandamide respectively.

NADPH oxidase 5 activation; a novel approach to human sperm cryoinjury.

Sperm cryopreservation leads to various structural and functional damages, some of which induce by oxidative stress. The reactive oxygen species (ROS) generates by mitochondria and membrane NADPH oxidases (NOXs). Among the NOXs, only NOX5 has been identified in the cell membrane of human sperm. This study was designed to clarify the possible role of NOX5 on sperm cryoinjury. Forty human semen samples were washed and randomly divided into fresh and cryopreserved groups. Each group was divided into 4 subgroups containing Ham's F10 (control), 0.1% DMSO (vehicle), 100 nM of PMA (phorbol 12-myristate 13-acetate) and 1 µM of DPI (diphenyleneiodonium), as NOX5 activator and inhibitor. The samples of cryopreserved groups were preserved in liquid nitrogen for 1 month. The sperm kinematics, membrane integrity, ROS production, apoptosis rate, mitochondrial membrane potential (MMP), intracellular ATP and calcium concentration [Ca2+]i were evaluated. The percent of sperm with intact membrane and motile sperm reduced significantly after thawing (p ≤ 0.01). The ROS production (p ≤ 0.01) and the apoptotic rate increased, MMP dissipated, and the percentage of live cells with high [Ca2+]i decreased significantly in the cryopreserved control group relative to the fresh control group. DPI, in contrast to PMA, improved sperm progressive motility (p ≤ 0.01), membrane integrity in fresh and cryopreserved groups and reduced the ROS amount in cryopreserved group (p ≤ 0.01). Apoptotic rate, [Ca2+]i, ATP, and MMP did not change with DPI and PMA in cryopreserved groups. We conclude that NOX5 activity in fresh sperm is low, and it increases during cryopreservation. NOX5 inhibition improves the cryopreserved sperm quality.

The Effects of EGTA on the Quality of Fresh and Cryopreserved-Thawed Human Spermatozoa.

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 EGTA-treated 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.

Lung injury in brain ischemia/reperfusion is exacerbated by mechanical ventilation with moderate tidal volume in rats.

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 (Kfc) 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. Kfc 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.

Effects of vitamin D on steroidogenesis, reactive oxygen species production, and enzymatic antioxidant defense in human granulosa cells of normal and polycystic ovaries.

Polycystic ovary syndrome (PCOS) is accompanied with many disturbances in hormone synthesis and antioxidant defense. Previous reports have indicated that Vitamin D (vit.D) affects gene expression and have roles in normal follicular development. Therefore, we investigated the effects of vit.D on steroidogenesis, apoptosis, reactive oxygen species (ROS) production, and antioxidant defenses of human normal granulosa cells (N-GCs) and granulosa cells from polycystic ovaries (PCO-GCs). Ovarian GCs were obtained during oocyte retrieval procedure from 120 women with PCOS and from 100 healthy women who referred to Shiraz Fertility Center. The isolated GCs were cultured in the presence or absence of vit.D (100 nM), for 48 h. Concentration of sex steroids was measured by ELISA. Glutathione peroxidase (GPx) and superoxide dismutase (SOD) expression and activities were assessed by q-PCR and photometric methods, respectively. The amount of ROS production was estimated using chemiluminescence and fluorescence methods. Cell viability and apoptosis were detected by Annexin-V/propidium iodide detection kit. Basal estrone and progesterone secretion by N-GCs was significantly higher than that of PCO-GCs. Vit.D significantly increased aromatase and 3ß-hydroxysteroid dehydrogenase activity in N-GCs and PCO-GCs. Basal expression and activity of GPx, in PCO-GCs were significantly lower than those of N-GCs. Treatment with vit.D significantly increased genes expression and enzyme activities in both groups. Basal ROS in PCO-GCs was markedly greater than that of N-GCs, which was attenuated by vit.D treatment. Cell apoptosis was directly correlated with ROS levels. We conclude that vit.D improved N-GCs and PCO-GCs functions through affecting steroidogenesis and enzymatic antioxidant defense. Under vit.D treatment, PCO-GCs could act more similar to N-GCs.

Association Between Sex Steroids and Oxidative Status with Vitamin D Levels in Follicular Fluid of Non-obese PCOS and Healthy Women.

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.

Inhibition of CatSper and Hv1 Channels and NOX5 Enzyme Affect Progesterone-Induced Increase of Intracellular Calcium Concentration and ROS Generation in Human Sperm.

BACKGROUND: Normal sperm function depends on appropriate intracellular calcium (Cai 2+) 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 Cai 2+ 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 20×106 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 Cai 2+ 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 Cai 2+ and ROS generation. The addition of NNC, Zn, or NNC+Zn significantly decreased Cai 2+ 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 Cai 2++. The activity of CatSper and Hv1 channels are required for progesterone-induced ROS generation by NOX5 enzyme.

Platelet-rich plasma improves impaired glucose hemostasis, disrupted insulin secretion, and pancreatic oxidative stress in streptozotocin-induced diabetic rat.

Our study aimed to investigate the effects of platelet-rich plasma (PRP) on impaired glucose homeostasis, disrupted islet insulin secretion, and pancreatic oxidative status in streptozotocin (STZ)-diabetic rats. A total of 64 Sprague-Dawley male were randomized to four groups including controls, diabetes, control-PRP, and diabetes-PRP. The rats received the PRP (0.5 ml/kg, SC injection) twice weekly for 4 weeks. Plasma glucose and insulin levels, pancreatic oxidative stress markers and islet insulin secretion and content were measured. Compared with the control group, in the diabetic group, increased plasma glucose and malondialdehyde (MDA) levels and decreased plasma insulin level, islet insulin secretion, pancreatic superoxide dismutase (SOD), and catalase activities were observed. PRP treatment significantly reduced plasma glucose and MDA levels and enhanced plasma insulin, antioxidant enzyme activity, islet insulin secretion, and content in the diabetic rats. These findings showed that PRP can improve pancreatic islet insulin secretion, pancreatic oxidative stress and regulate plasma insulin and glucose levels in diabetic rats.

Comparing Human Sperm Quality Preserved at Two Different Temperatures; Effect of Trolox, Coenzyme Q10 and Extracellular Adenosine Triphosphate.

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.

Effect of CatSper and Hv1 Channel Inhibition on Progesterone Stimulated Human Sperm.

BACKGROUND: Intracellular calcium and proton concentrations are important factors for activating human sperm. Calcium ion (Ca2+) enters sperm through voltage-dependent 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 20×106sperm/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.

Inhibition of the CatSper Channel and NOX5 Enzyme Activity Affects the Functions of the Progesterone-Stimulated Human Sperm.

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 20×106 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 progesterone-stimulated 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.

The effect of melatonin on the lipid levels in menopausal women: A double-blind, controlled, clinical trial.

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.

Kisspeptin expression features in the arcuate and anteroventral periventricular nuclei of hypothalamus of letrozole-induced polycystic ovarian syndrome in rats.

PURPOSE: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder of the reproductive system characterized by polycystic ovaries and androgen excess. Letrozole is a nonsteroidal aromatase inhibitor that is used in experimental research to induce PCOS. Kisspeptin is an essential protein in regulation of cyclicity. Kisspeptin receptor is expressed in the hypothalamus and pituitary glands, and kisspeptin containing neurons are affected from sex steroid hormones. We aimed to investigate the number of kisspeptin-positive cells in the arcuate (Arc) and anteroventral periventricular nuclei (AVPV) of hypothalamus in the letrozole-induced PCOS. METHODS: 40 female Wistar rats were divided into the proestrus control, diestrus control, proestrus vehicle, diestrus vehicle and letrozole. Animals were sacrificed after 3 weeks, and sera, ovary and brain samples were harvested for further evaluations. RESULTS: Letrozole group had high weight gain, high numbers of ovarian follicular cysts, high levels of luteinizing hormone and testosterone and increase number of kisspeptin-positive cells in the Arc nucleus, as compared with the control groups (P ≤ 0.05 vs. proestrus control and proestrus vehicle). Letrozole group showed a decrease in the number of kisspeptin-positive cells in the AVPV nucleus (P ≤ 0.05 vs. proestrus control and proestrus vehicle). CONCLUSION: Our findings show that the number of kisspeptin-positive cells may be affected from letrozole, and that the changes in the number of these cells may be in favor of the appearance of PCOS features in this group.

T helper cell subsets and related cytokines in infertile women undergoing in vitro fertilization before and after seminal plasma exposure.

OBJECTIVE: In vitro fertilization (IVF) is a well-known method for the treatment of infertility. The present study aimed to compare the differences between infertile women with successful and unsuccessful IVF outcomes regarding the expression of T helper (Th) cell transcription factors and a group of related cytokines before and after exposure to their husbands' seminal plasma. METHODS: This study was performed on 19 couples with unexplained infertility undergoing IVF treatment. Among the studied group, nine and 10 couples had successful and unsuccessful IVF outcomes, respectively. This study was carried out using real-time polymerase chain reaction. RESULTS: Before seminal plasma exposure, the expression levels of T-bet (p<0.007), interferon-γ (p=0.013), and tumor necrosis factor (TNF)-α (p=0.017) were higher in the infertile women with IVF failure than in those with successful IVF outcomes, while those of GATA3 (p<0.001), Foxp3 (p=0.001), and interleukin (IL)-35 (p<0.003) were lower. After seminal exposure, the expression of T-bet (p=0.02), Rorc (p<0.001), TNF-α (p=0.001), Foxp3 (p=0.02), and interferon-γ (p=0.001) increased in the unsuccessful IVF group, while the expression of Foxp3 (p=0.02), Rorc (p<0.001), IL-23 (p=0.04), IL-17 (p=0.02), IL-6 (p<0.001), transforming growth factor-ß (p=0.01), and IL-35 (p<0.001) increased in the successful IVF group. CONCLUSION: In summary, IVF failure was associated with imbalanced Th1/Th2/Th17/Treg responses. Moreover, our results show that seminal plasma might have a positive effect on IVF outcomes via changes in peripheral blood T cell subsets.

Combined Effect of Trolox and EDTA on Frozen-Thawed Sperm Quality.

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.

Antisperm protein targets in azoospermia men.

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.

The Effect of Melatonin on Climacteric Symptoms in Menopausal Women; A Double-Blind, Randomized Controlled, Clinical Trial.

BACKGROUND: Menopause is one of the most critical periods of woman's life. With reducing of ovarian estrogen; women are more prone to psychological and physical symptoms. The present study aimed to investigate the effect of melatonin on the climacteric symptoms. METHODS: The present double blind, placebo randomized controlled clinical trial was conducted on 240 menopausal women (40 - 60 years old) referring to the gynecology clinics of Shiraz University of Medical Sciences (January - November 2012). The participants were randomly divided into two groups through sortition. Demographic characteristics, Goldberg's general health questionnaire (GHQ), Greene Climacteric Scale and level of Follicle Stimulating Hormone (FSH) were determined for both groups before the intervention. The intervention group received one 3mg melatonin tablet each night for 3 months and the control group received the placebo in the same period. Changes of climacteric symptoms and drug complications were measured 1, 2 and 3 months after the intervention. RESULTS: We analyzed the data of 99 postmenopausal women in the intervention group and 101 postmenopausal women in the control group. In the melatonin group, the climacteric symptoms score decreased from 35.73+11.6 to 17.09+10.22 during the 3-month study period and regardless of time, a significant difference was observed between the two groups (P<0.001). In addition, a significant difference was found between the two groups regarding various dimensions of the climacteric symptoms over time (P<0.001). No significant difference was found regarding side effects between the two groups (P= 0.135). CONCLUSION: The study findings showed that using melatonin improved the climacteric symptoms.

A moderate-intensity static magnetic field enhances repair of cartilage damage in rabbits.

BACKGROUND AND AIMS: Electromagnetic fields have been proposed to enhance healing of cartilage defects by stimulation of chondrocyte proliferation, proteoglycan synthesis as well as decreasing pain and improving motion in osteoarthritic patients. However, the effects of a moderate-intensity static magnetic field on cartilage repair have not been investigated. This study tries to determine the effects of a moderate-intensity permanent magnetic field of 40 mT on cartilage repair. METHODS: Defects of 3 mm in diameter and 6 mm in depth were made on the weight bearing surface of the right medial femoral condyle of 30 rabbits. The animals were divided randomly into three equal groups (magnet, sham and control). In the magnet group, cylindrical permanent magnets were implanted subcutaneously medial to the medial femoral condyle, while in the sham group the cylindrical ceramic were not magnetized, and nothing was implanted in controls. After 12 weeks of observation, Mankin's microscopic scoring was done on all specimens, and irregularity of surface characteristics, cell colonization, hypocellularity, cartilage matrix formation, and presence of empty lacunae were investigated. RESULTS: Each of these characteristics showed significant differences in magnet group relative to control and sham groups (p <0.05). Mankin's score was 1.6 ± 0.6 in magnet group, 7.2 ± 1.6 in sham group and 7.7 ± 1 in control group (p <0.001). CONCLUSIONS: [corrected] In this animal study, microscopic Mankin's scoring depicted histological improvement in cartilage of magnet group.