Comparison of liquid nitrogen-free slow freezing protocols toward enabling a practical option for centralized cryobanking of ovarian tissue.

Geographically distributed ovarian tissue cryobanks remain limited due to the high facility and staff costs, and cold transportation to centers is associated with ischemia-induced tissue damage that increases with transport distance. It is ideal to perform the cryopreservation procedure at a tissue removal site or local hospital before shipment to cost-effective centralized cryobanks. However, conventional liquid nitrogen-based freezers are not portable and require expensive infrastructure. To study the possibility of an ovarian tissue cryopreservation network not dependent on liquid nitrogen, we cryopreserved bovine ovarian tissue using three cooling techniques: a controlled rate freezer using liquid nitrogen, a liquid nitrogen-free controlled rate freezer, and liquid nitrogen-free passive cooling. Upon thawing, we evaluated a panel of viability metrics in frozen and fresh groups to examine the potency of the portable liquid nitrogen-free controlled and uncontrolled rate freezers in preserving the ovarian tissue compared to the non-portable conventional controlled rate freezer. We found similar outcomes for reactive oxygen species (ROS), total antioxidant capacity (TAC), follicular morphology, tissue viability, and fibrosis in the controlled rate freezer groups. However, passive slow cooling was associated with the lowest tissue viability, follicle morphology, and TAC, and the highest tissue fibrosis and ROS levels compared to all other groups. A stronger correlation was found between follicle morphology, ovarian tissue viability, and fibrosis with the TAC/ROS ratio compared to ROS and TAC alone. The current study undergirds the possibility of centralized cryobanks using a controlled rate liquid nitrogen-free freezer to prevent ischemia-induced damage during ovarian tissue shipment.

Comparative effects of a calcium chelator (BAPTA-AM) and melatonin on cryopreservation-induced oxidative stress and damage in ovarian tissue.

Oncology treatments cause infertility, and ovarian tissue cryopreservation and transplantation (OTCT) is the only option for fertility preservation in prepubertal girls with cancer. However, OTCT is associated with massive follicle loss. Here, we aimed to determine the effect of supplementation of slow freezing and vitrification media with BAPTA-AM and melatonin alone and in combination on ovarian tissue viability, reactive oxygen species (ROS) levels, total antioxidant capacity (TAC), and follicular morphology and viability. Our results indicated that BAPTA-AM and melatonin can significantly improve ovarian tissue viability and the TAC/ROS ratio and reduce ROS generation in frozen-thawed ovarian tissues in slow freezing and vitrification procedures. BAPTA-AM was also found to be less effective on TAC compared to melatonin in vitrified ovarian tissue. While supplementation of slow freezing and vitrification media with BAPTA-AM and/or melatonin could increase the percentage of morphologically intact follicles in cryopreserved ovarian tissues, the differences were not significant. In conclusion, supplementation of cryopreservation media with BAPTA-AM or melatonin improved the outcome of ovarian tissue cryopreservation in both vitrification and slow freezing methods. Our data provide some insight into the importance of modulating redox balance and intracellular Ca2+ levels during ovarian tissue cryopreservation to optimize the current cryopreservation methods.

Ovarian tissue cryopreservation and transplantation: a review on reactive oxygen species generation and antioxidant therapy.

Cancer is the leading cause of death worldwide. Fortunately, the survival rate of cancer continues to rise, owing to advances in cancer treatments. However, these treatments are gonadotoxic and cause infertility. Ovarian tissue cryopreservation and transplantation (OTCT) is the most flexible option to preserve fertility in women and children with cancer. However, OTCT is associated with significant follicle loss and an accompanying short lifespan of the grafts. There has been a decade of research in cryopreservation-induced oxidative stress in single cells with significant successes in mitigating this major source of loss of viability. However, despite its success elsewhere and beyond a few promising experiments, little attention has been paid to this key aspect of OTCT-induced damage. As more and more clinical practices adopt OTCT for fertility preservation, it is a critical time to review oxidative stress as a cause of damage and to outline potential ameliorative interventions. Here we give an overview of the application of OTCT for female fertility preservation and existing challenges; clarify the potential contribution of oxidative stress in ovarian follicle loss; and highlight potential ability of antioxidant treatments to mitigate the OTCT-induced injuries that might be of interest to cryobiologists and reproductive clinicians.

Exogenous Melatonin Ameliorates the Negative Effect of Osmotic Stress in Human and Bovine Ovarian Stromal Cells.

Ovarian tissue cryopreservation transplantation (OTCT) is the most flexible option to preserve fertility in women and children with cancer. However, OTCT is associated with follicle loss and an accompanying short lifespan of the grafts. Cryopreservation-induced damage could be due to cryoprotective agent (CPA) toxicity and osmotic shock. Therefore, one way to avoid this damage is to maintain the cell volume within osmotic tolerance limits (OTLs). Here, we aimed to determine, for the first time, the OTLs of ovarian stromal cells (OSCs) and their relationship with reactive oxygen species (ROS) and mitochondrial respiratory chain activity (MRCA) of OSCs. We evaluated the effect of an optimal dose of melatonin on OTLs, viability, MRCA, ROS and total antioxidant capacity (TAC) of both human and bovine OSCs in plated and suspended cells. The OTLs of OSCs were between 200 and 375 mOsm/kg in bovine and between 150 and 500 mOsm/kg in human. Melatonin expands OTLs of OSCs. Furthermore, melatonin significantly reduced ROS and improved TAC, MRCA and viability. Due to the narrow osmotic window of OSCs, it is important to optimize the current protocols of OTCT to maintain enough alive stromal cells, which are necessary for follicle development and graft longevity. The addition of melatonin is a promising strategy for improved cryopreservation media.

The Impact of Sex Differences on Renal Protective Effects of Lipopolysaccharide Preconditioning in Septic Shock.

BACKGROUND: Induction of septic shock by lipopolysaccharide (LPS) may lead to acute renal failure. The present study aimed to investigate the impact of sex differences on the effectiveness of low-dose LPS preconditioning (LPS-PC) on LPS-induced acute renal failure in rats. METHODS: This study was conducted at Tehran University of Medical Sciences, in 2017. A total of 48 Wistar rats were equally divided into two groups of male and female rats. The rats in each group were then allocated to three groups (n=8 per group), namely control, septic shock, and LPS-PC group. A high dose of LPS was administered for septic shock induction. LPS-PC was induced by injecting LPS before sepsis induction. The effect of sex differences on renal functional indices, renal oxidative stress markers, plasma tumor necrosis factor-α level, and renal histological changes was evaluated. Data were analyzed using two-way ANOVA followed by Tukey's post hoc test. RESULTS: In the septic shock groups, renal functional parameters (creatinine [Cr] and blood urea nitrogen [BUN]) were increased in both sexes. However, the increase was more significant in male rats (male rats: Cr=2.14±0.13, BUN=81±4.15; female rats: Cr=1.64±0.12, BUN=50±2.7). LPS-PC reduced these indices in both sexes (male rats: Cr=1.24±0.03, BUN=57±4.1; female rats: Cr=0.86±0.02, BUN=30.31±2.25). Renal superoxide dismutase (SOD) activity (male rats: 11.54±1.34, female rats: 24.4±2.04) and catalase (CAT) activity (male rats: 15±1.74, female rats: 25.75±1.97) were significantly higher in the female septic group. LPS-PC significantly increased SOD (male rats: 25.7±2.45, female rats: 42.6±3.31) and CAT (male rats: 37.25±2.34, female rats: 59.21±3.29) activities in renal tissue samples in the LPS-PC group in both sexes compared to the septic groups. In the LPS groups, plasma tumor necrosis factor-α (male rats: 375±25.65, female rats: 285.45±25.94) were significantly higher than in the LPS-PC groups (male rats: 250±21.35, female rats: 121±24.14). CONCLUSION: Male rats were more susceptible to sepsis-induced renal damage. LPS-PC had protective effects on the LPS-induced renal injury, and these effects were most prominent in female rats.

Aqueous Origanum vulgare Extract Improves the Quality of Cryopreserved Human Spermatozoa Through Its Antioxidant Effects.

Excessive production of reactive oxygen species (ROS) during semen cryopreservation can induce structural and functional changes in spermatozoa. It is well known that antioxidants can mitigate the effect of ROS. Moreover, the application of antioxidants in freezing media is an appropriate strategy for protecting spermatozoa against deleterious effects of ROS during the cryopreservation process. As an example, oregano is a medicinal plant with important activities, with antiseptic, antibacterial, antithrombotic, and antioxidant properties. This study aimed at evaluating the antioxidant effects of oregano extract on cryopreserved human spermatozoa. In the first phase, 13 semen samples with different concentrations of oregano extract (0.0, 50, 100, 150, 300, and 500 µg/mL) were cryopreserved to achieve an optimal dose of oregano extract. Then, motility, viability, and plasma membrane integrity were evaluated. In the second phase, 20 samples were cryopreserved in freezing media supplemented with or without the optimal concentration of oregano (100 µg/mL). After thawing, motility, the levels of ROS, lipid peroxidation, and translocation of phosphatidylserine (PS) were evaluated. The results showed that 100 µg/mL oregano extract significantly increased the total motility in frozen-thawed spermatozoa in comparison with the control group (28.2 ± 4.3 vs. 42.4 ± 1.6, p < 0.05). This concentration significantly decreased the percentage of 2',7'-dichlorofluorescein-positive cells (25.53 ± 1.2 vs. 21.48 ± 1.2) and the malondialdehyde level (4.25 ± 0.7 vs. 0.82 ± 0.4 µM) (p < 0.05). In the oregano group, the percentage of vital spermatozoa without PS externalization was significantly higher than that in the control group (25.88 ± 1.6 vs. 16.8 ± 1.9, p < 0.001), while the percentage of dead spermatozoa with PS externalization spermatozoa was significantly lower than that in the control group (51.65 ± 1.4 vs. 60.36 ± 1.9, p < 0.05). In general, the addition of oregano extract to sperm freezing extender has protective effects against oxidative stress and apoptosis.

Silver-gold-apoferritin nanozyme for suppressing oxidative stress during cryopreservation.

Reactive oxygen species (ROS) cause oxidative stress, which involves in the pathogenesis of many serious diseases. Apoferittin containing gold-silver nanoparticles (Au-Ag-AFT) was designed and evaluated as a nanozyme for scavenging the ROS. The nanozyme consisting of silver-gold nanohybrid in apoferittin cage represents superoxide dismutase, catalase and peroxidase mimetic activities. The Au-Ag-AFT nanozyme was characterized by spectroscopy, FESEM, TEM and dynamic light scattering. The inhibition process for pyrogallol autoxidation was used for assaying the superoxide dismutase mimetic activity and measuring the kinetic parameters of Au-Ag-AFT nanozyme. Additionally, Aebi method and standard protocol was used for evaluating the catalase and peroxidase mimetic activity. The kcat values for superoxide dismutase, catalase and peroxidase mimetics activity were 1.4 × 106, 0.1 and 9 × 103 s-1 respectively. These values indicated that Au-Ag-AFT nanozyme could act as a suitable ROS scavenger. Additionally, Au-Ag-AFT nanozyme was examined as a protective agent for human sperm against oxidative stress induced during the cryopreservation process. Presence of the nanozyme in the sperm media significantly increased the motility and viability of the cells and also decreased the ROS, apoptosis and necrosis (P < 0.05) compare to the control group.

Surface-grafted lanthanoid complexes of the tungstosilicate polyanion [SiW12O40]4-: a synthetic, structural and computational investigation.

As an extension of our continued interest in the preparation of inorganic-organic hybrids, we report the successful hydrothermal synthesis of sodium tris[triaqua(µ-1,10-phenanthroline-2,9-dicarboxylato)dysprosium(III)] silicododecatungstate dodecahydrate, {[DyNa(C14H6N2O4)3(H2O)9(SiW12O40)]·12H2O}n or Na[Dy(PDA)(H2O)3]3[SiW12O40]·12H2O (1), and sodium aqua tris[tetraaqua(µ-4-hydroxypyridine-2,6-dicarboxylato)praseodymium(III)] silicododecatungstate dodecahydrate, {[NaPr(C7H3NO5)3(H2O)13(SiW12O40)]·12H2O}n or Na(H2O)[Pr(pydc-OH)(H2O)4]3[SiW12O40]·12H2O (2) (in which H2PDA is 1,10-phenanthroline-2,9-dicarboxylic acid and H2pydc-OH is 4-hydroxypyridine-2,6-dicarboxylic acid or chelidamic acid). Both compounds have been characterized using elemental analysis, IR spectroscopy and X-ray diffraction methods. Structural characterization by single-crystal X-ray diffraction reveals that these compounds consist of [SiW12O40]4- Keggin-type polyoxometalates (POMs), where a single {W3O13} triad is decorated with a trinuclear Ln complex. Moreover, the decorated polyanions are involved in a series of intermolecular interactions, such as hydrogen bonds and anion-π interactions, resulting in three-dimensional supramolecular architectures. Density functional theory (DFT) studies were conducted to support these intermolecular interactions in both 1 and 2, and have been rationalized using molecular electrostatic potential (MEP) surface calculations.

Albumin coated copper-cysteine nanozyme for reducing oxidative stress induced during sperm cryopreservation.

In the present work, SOD mimetic nanozyme (NACu-Cys) consisting of Cu-Cys complex and nano-albumin (NA) were synthesized. After characterizing the nanozyme, its superoxide dismutase (SOD) behavior was evaluated by inhibition of the pyrogallol autoxidation method. The results revealed that NACu-Cys exhibited SOD mimetic activity with a half inhibition concentration (IC50) value of 7.0 × 10-3 µM and a turnover number (kcat) of 5.4 × 107 s-1. In the next step, this nanozyme was applied as a protective agent against oxidative stress induced by sperm cryopreservation. Increasing the motility, raising the viability and reducing the apoptosis occurred as a result of NACu-Cys additions to human sperm freezing medium. Comparison between the natural SOD and SOD mimic behavior of NACu-Cys revealed that this nanoparticle has the ability to be used as oxidative stress decrescent during cryopreservation process.

Association of serum content of 25-hydroxy vitamin D with semen quality in normozoospermic and oligoasthenoteratozoospermic men.

BACKGROUND: Vitamin D has multifaceted function in human reproductive physiology. It has been revealed that vitamin D is involved in spermatogenesis, and semen quality can be linked to vitamin D status in men. OBJECTIVE: Evaluating the correlation of 25-hydroxy vitamin D (25-OHD) levels in serum with basic and advanced semen parameters and essential determinants of spermatozoa function. MATERIALS AND METHODS: Participants were categorized, based on semen parameters, into normozoospermic (NS) and oligoasthenoteratozoospermic (OAT) men. Serum level of 25-OHD was measured. Apoptotic status of spermatozoa, mitochondrial membrane potential and reactive oxygen species content of semen were assessed. RESULTS: Difference of 25-OHD concentration in serum of NS men versus OAT ones did not meet significance threshold. DNA fragmentation, reactive oxygen species content of semen and mitochondrial membrane potential state revealed significant difference between NS and OAT subjects. There were no significant differences in basic and functional semen parameters when men were stratified based on serum 25-OHD level. Taking both 25-OHD and semen categories (NS and OAT) into consideration did not indicate any significant difference in studied parameters. Total motility of spermatozoa was positively correlated with serum concentration of 25-OHD in all studied subjects. In addition, normal morphology of spermatozoa in NS men revealed a positive and significant correlation with levels of 25-OHD in serum. CONCLUSION: Vitamin D may affect motility and morphology of spermatozoa. Lower content of serum vitamin D may affect fertility of men and should be considered in examination of men with abnormal spermogram.

Effect of brain-derived neurotrophic factor (BDNF) on sperm quality of normozoospermic men.

The neurotrophin family of proteins and their receptors act as important proliferative and pro-survival factors in differentiation of nerve cells and are thought to play key roles in the development of reproductive tissues and normal function of spermatozoa. The objective of the present study was to evaluate the effect of Brain-Derived Neurotrophic Factor (BDNF) on the sperm viability and motility, lipid peroxidation (LPO), mitochondrial activity and concentration of leptin, nitric oxide (NO) and insulin in normozoospermic men. Semen samples from 20 normozoospermic men were divided into three groups: (i) control, (ii) BDNF and (iii) BDNF + K252a. BDNF and K252a were added in the dose of 0.133 and 0.1 nM, respectively. Viability was assessed by eosin-nigrosin staining technique, and motility was observed by microscopy. NO concentration and mitochondrial activity were measured with flow cytometry, and LPO was analyzed using enzyme-linked immunosorbent assay (ELISA) kits. Results showed that exogenous BDNF at 0.133 nM could significantly (p < 0.05) influence viability, motility, NO concentration, mitochondrial activity and LPO content. Secretions of insulin and leptin by human sperm were increased in cells exposed to the exogenous BDNF, whereas viability, mitochondrial activity and insulin and leptin secretions were decreased in cells exposed to the K252.

Melatonin affects membrane integrity, intracellular reactive oxygen species, caspase3 activity and AKT phosphorylation in frozen thawed human sperm.

Cryopreservation is known to induce oxidative stress in spermatozoa. Although melatonin has powerful antioxidant properties, little is known about its effects on human sperm quality during cryopreservation. The present study was undertaken to investigate the effects of melatonin treatment on human sperm parameters essential for fertilization. We first evaluated the effects of various concentrations of melatonin (0-15 mM) on human sperm parameters such as motility, viability and levels of intracellular reactive oxygen species during cryopreservation in order to identify an optimal dose with the greatest effects for further studies. Liquefied semen samples were then divided into three aliquots: cryopreserved without melatonin (control), cryopreserved with 3 mM melatonin and fresh groups. After being thawed, samples were evaluated for motility, viability, membrane integrity, intracellular reactive oxygen species levels, caspase-3 activity and AKT phosphorylation. Treatment of spermatozoa with the various concentrations of melatonin significantly increased their motility and viability and decreased their intracellular reactive oxygen species levels compared with the control group. The optimal melatonin concentration (3 mM) significantly decreased the intracellular reactive oxygen species levels, caspase-3 activity and the percentage of both dead and apoptotic-like sperm cells and increased the vitality, progressive motility and total motility and AKT phosphorylation compared with the control group. Thus, melatonin exerts protective effects against cryodamage during human spermatozoa cryopreservation and may exert its effects via the PI3K/AKT signaling pathway.

Melatonin upregulates ErbB1 and ErbB4, two primary implantation receptors, in pre-implantation mouse embryos.

OBJECTIVES: To evaluated the effects of melatonin on early embryo competence and the expression rate of the primary implantation receptors (ErbB1 and ErbB4). MATERIALS AND METHODS: Two-cell mouse embryos were cultured in 3 groups: simple media, melatonin-treated (10-9 M melatonin) and Luzindole-treated (10-9 M luzindole). Then, the rate of ErbB1 and ErbB4 gene and protein expression, the level of intracellular ROS, antioxidant capacity, and also the number of cells were evaluated and compared with the fourth group in vivo developed blastocysts (control group). RESULTS: We concluded that melatonin significantly up-regulated the ErbB1 and ErbB4 gene and protein expression, decreased intracellular ROS, increased the total antioxidant capacity, and also elevated the cell numbers in the melatonin-treated group compared with the other groups (P≤ 0.05). CONCLUSION: The use of melatonin may be a helpful factor in improving the embryo quality and enhancing the expression of ErbB1 and ErbB4, two important implantation-related genes and proteins.

Evaluation of intracellular anion superoxide level, heat shock protein A2 and protamine positive spermatozoa percentages in teratoasthenozoospermia.

BACKGROUND: Teratoasthenozoospermia (TA) is a severe form of male infertility with no clear etiology. OBJECTIVE: To compare the level of intracellular anion superoxide (O2-), heat shock protein A2 (HSPA2) and protamine deficiencies in ejaculated spermatozoa between teratoasthenozoospermic and normozoospermic men. MATERIALS AND METHODS: In this case- control study, semen samples of 20 infertile men, with TA (with normal morphology lower than 4%_ and total motility lower than 40% ) as the case group and 20 normozoospermic fertile men as the control group were evaluated for intracellular O2- and HSPA2 by flow cytometry and protamine deficiency by Chromomycin A3 (CMA3) test. RESULTS: The rate of CMA3+ spermatozoa in the case group was higher than controls (p=0.001). The percentages of HSPA2+ spermatozoa in the cases were significantly lower than controls (p=0.001). Also, intracellular O2- levels in the case group were significantly higher than controls (p=0.001) and had positive correlations with sperm apoptosis (r=0.79, p=0.01) and CMA3 positive sperm (r=0.76, p=0.01), but negative correlations with normal morphology (r=-0.81, p=0.01) and motility (r=-0.81, p=0.01). There was no significant correlation between intracellular O2- and HSPA2 in the case group (r=0.041, p=0.79). CONCLUSION: We suggest that the increase in intracellular O2-, decrease in spermatozoa HSPA2+, and high percentages of spermatozoa with immature chromatin might be considered as etiologies of infertility in TA patients.

Multipotent Stem Cell and Current Application.

Stem cells are self-renewing and undifferentiated cell types that can be differentiate into functional cells. Stem cells can be classified into two main types based on their source of origin: Embryonic and Adult stem cells. Stem cells also classified based on the range of differentiation potentials into Totipotent, Pluripotent, Multipotent, and Unipotent. Multipotent stem cells have the ability to differentiate into all cell types within one particular lineage. There are plentiful advantages and usages for multipotent stem cells. Multipotent Stem cells act as a significant key in procedure of development, tissue repair, and protection. Multipotent Stem cells have been applying in treatment of different disorders such as spinal cord injury, bone fracture, autoimmune diseases, rheumatoid arthritis, hematopoietic defects, and fertility preservation.

Ovarian tissue culture in the presence of VEGF and fetuin stimulates follicle growth and steroidogenesis.

Ovarian tissue cryopreservation together with follicle culture provides a promising technique for fertility preservation in cancer patients. The study aimed to evaluate follicle parameters in a culture medium supplemented with VEGFA165 and/or fetuin. Vitrified-warmed ovarian cortical pieces were divided randomly into four culture groups consisting of basic culture medium (control), and the basic culture medium supplemented with VEGFA165, fetuin or both. After six days of culture, we evaluated the following: percentage of resting, primary and secondary growing follicles; survival rate; steroid hormones production; levels of reactive oxygen species, lipid peroxidation and total antioxidant capacity; and developmental and antioxidant gene expression. The addition of VEGFA165 alone or in combination with fetuin to the culture medium caused resting follicle activation and increased the number of growing follicles. In the VEGFA165 group, we found a significant increase in the concentrations of 17ß-estradiol at day 6 and progesterone from 4th day of the culture period. In the VEGFA165 + fetuin group, the concentration of 17ß-estradiol rose at day 4 of the culture period. The levels of BMP15, GDF9 and INHB mRNAs were increased in all treated groups. In the fetuin and fetuin + VEGFA165 groups, we observed a high level of total antioxidant capacity and expression of SOD1 and CAT genes, low reactive oxygen species and lipid peroxidation levels and increased number of viable follicles. In conclusion, the present study provides useful evidence that supplementation of culture medium with VEGFA165 + fetuin leads to primordial follicle activation and development and increased percentage of healthy secondary growing follicles.

Supplementation of freezing and thawing media with brain-derived neurotrophic factor protects human sperm from freeze-thaw-induced damage.

OBJECTIVE: To investigate the effects of brain-derived neurotrophic factor (BDNF) supplementation to freezing and thawing media on frozen-thawed human sperm parameters. DESIGN: Laboratory study. SETTING: University hospital. PATIENT(S): Semen samples from 21 healthy fertile men. INTERVENTION(S): We measured reactive oxygen species (ROS) by flow cytometry using the probes dichlorofluorescin diacetate for intracellular hydrogen peroxide (H2O2) and dihydroethidium for intracellular superoxide anion (O2-•), sperm plasma membrane integrity by flow cytometry, caspase-3 activity using ELISA, and AKT phosphorylation status using Western blot in sperm that was cryopreserved and thawed in media either supplemented with BDNF or without BDNF supplementation (control). MAIN OUTCOME MEASURE(S): Sperm motility, viability, ROS levels, caspase-3 activity and AKT phosphorylation. RESULT(S): The percentage of motile and viable sperm cells was significantly higher in BDNF-supplemented groups as compared with the nonsupplemented (control) group. There was a significant difference in AKT phosphorylation status between BDNF-supplemented groups and the control group. Moreover, the levels of intracellular H2O2 and caspase-3 activity were significantly lower in the sperm cells that were frozen and thawed in media supplemented with BDNF compared with in the control group. CONCLUSION(S): BDNF supplementation to sperm freezing or thawing media has protective effects against oxidative stress and apoptosis in frozen-thawed human spermatozoa and could improve sperm function, probably through the activation of AKT.

Altered FoxO3 expression and apoptosis in granulosa cells of women with polycystic ovary syndrome.

PURPOSE: To determine the level of apoptosis, and alteration of FoxO3 (forkhead box O3 transcription factor) expression and phosphorylation in human granulosa cells amongst polycystic ovary syndrome (PCOS) patients and control group. METHODS: We recruited infertile women with PCOS (n = 14) and compared them with infertile women due to tubal blockage or male factor infertility (n = 14, controls). GnRH agonist and gonadotropins were used for ovarian stimulation. Follicular fluids from large follicles (>16 mm) were pooled and granulosa cells (GCs) were isolated using cell strainer methodology. Apoptosis of purified GCs was measured by flow cytometry using Annexin V and propidium iodide. Quantitative real-time PCR and western blotting were performed to assess alteration of FoxO3 expression and phosphorylation in GCs. RESULTS: There were higher percentages of early and late apoptosis in GCs of PCOS patients than in the control group. FoxO3 mRNA level and total FoxO3 protein were significantly higher in PCOS group than in the control group. The ratio of p-FoxO3/total FoxO3 decreased significantly in PCOS than in the control group. It was inferred that unphosphorylated (active form) FoxO3 was higher in GCs of PCOS patients. Apoptosis was significantly and positively correlated with the total FoxO3 and negatively correlated with the p-FoxO3 protein levels in PCOS patients. CONCLUSIONS: Activation and overexpression of FoxO3 in granulosa cells of PCOS women correlated with higher apoptosis levels in these cells suggesting that FoxO3 may be a candidate for the higher apoptosis in granulosa cells from women with PCOS.

Effect of human recombinant granulocyte colony-stimulating factor on rat busulfan-induced testis injury.

Granulocyte colony-stimulating factor (G-CSF) is known for proliferation and anti-apoptotic activities. We aimed to use this growth factor in busulfan-injured testis. 32 male Wistar rats were injected with a double dosage of 15 ml/kg busulfan with 14 days interval. Administration of human recombinant G-CSF (100 µg/kg) subcutaneously was performed in two different time periods: 3 days before and 2 days after receiving busulfan, G-CSF1; and at days 14-18 of busulfan injection, G-CSF2. Animals were sacrificed at the end of week five. Histological analysis, testis weight and sperm parameters (sperm count and viability) has been checked. Expressions of DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (DDX4), deleted in azoospermia like (DAZL), transition protein 2 (TP2), proliferating cell nuclear antigen (PCNA) and 5-Bromo-20-deoxyuridine (BrdU) were assessed. Empty seminiferous tubules were apparent in the busulfan- and G-CSF2-injected rats, but not in the G-CSF1 group. The G-CSF1-treated animals showed an increase in testis weight and sperm count and viability along with high expressions of DDX4, DAZL, TP2, PCNA and BrdU; even so, the changes were reversed in the busulfan and G-CSF2 groups (for all p < 0.05). Our results revealed that G-CSF application prior to busulfan insult is a promising approach in fertility maintenance.

Multipotent Stem Cell and Reproduction.

Stem cells are self-renewing and undifferentiated cell types that can be differentiate into functional cells. Stem cells can be classified into two main types based on their source of origin: Embryonic and Adult stem cells. Stem cells also classified based on the range of differentiation potentials into Totipotent, Pluripotent, Multipotent, and Unipotent. Multipotent stem cells have the ability to differentiate into all cell types within one particular lineage. There are plentiful advantages and usages for multipotent stem cells. Multipotent Stem cells act as a significant key in procedure of development, tissue repair, and protection. The accessibility and adaptability of these amazing cells create them a great therapeutic choice for different part of medical approaches, and it becomes interesting topic in the scientific researches to found obvious method for the most advantageous use of MSC-based therapies. Recent studies in the field of stem cell biology have provided new perspectives and opportunities for the treatment of infertility disorders.