Assessment of developmental rate of mouse embryos yielded from in vitro fertilization of the oocyte with treatment of melatonin and vitamin C simultaneously.

BACKGROUND: In recent decades, in vitro fertilization (IVF) has been widely used as a method of assisted reproductive technology (ART) to improve fertility in individuals. To be more successful in this laboratory method, we used the presence of two common types of antioxidants (melatonin and vitamin C) simultaneously and exclusively in IVF medium. METHODS: The cumulus-oocyte complexes (COCs) were obtained from Gonadotropin-releasing hormone (GnRH) and Human Chorionic Gonadotropin (HMG) -stimulated mice. Subsequently, metaphase II (MII) oocytes were fertilized in vitro. In the experiment, the IVF medium was randomly divided into two equal groups: The control group did not receive any antioxidants. In the treatment group, 100 µM melatonin and 5 mM vitamin C were added to the IVF medium. Finally, oocytes and putative embryos transferred into developmental medium and cultured 120 h after IVF to the blastocyst stage. After and before IVF, oocytes and putative embryos were stained with dichlorodihydrofluorescein diacetate (DCFDA) and the H2O2 level was measured with an inverted fluorescence microscope using ImageJ software. At the end of the fifth day after IVF, the expression of Bax and B cell lymphoma 2 (Bcl2) was evaluated using real-time PCR. RESULTS: The levels of reactive oxygen species (ROS) in oocytes and putative embryos observed in the treatment group demonstrated a significant reduce compared to the control group (p ≤ 0.01. (.Furthermore, the number of embryos in the blastocycte stage(P < 0.05), the expression level of the Bcl2 (P < 0.05) gene, the Bax unlike gene, significantly increased compared with the control group. CONCLUSION: We conclude that the presence of melatonin and vitamin C antioxidants simultaneously and exclusively in the IVF medium leads to a reduction in ROS and ,as a result, improves the growth of the embryo up to the blastocyst stage.

Epi-miRNAs: Regulators of the Histone Modification Machinery in Human Cancer.

Cancer is a leading cause of death and disability worldwide. Epigenetic deregulation is one of the most critical mechanisms in carcinogenesis and can be classified into effects on DNA methylation and histone modification. MicroRNAs are small noncoding RNAs involved in fine-tuning their target genes after transcription. Various microRNAs control the expression of histone modifiers and are involved in a variety of cancers. Therefore, overexpression or downregulation of microRNAs can alter cell fate and cause malignancies. In this review, we discuss the role of microRNAs in regulating the histone modification machinery in various cancers, with a focus on the histone-modifying enzymes such as acetylases, deacetylases, methyltransferases, demethylases, kinases, phosphatases, desumoylases, ubiquitinases, and deubiquitinases. Understanding of microRNA-related aberrations underlying histone modifiers in pathogenesis of different cancers can help identify novel therapeutic targets or early detection approaches that allow better management of patients or monitoring of treatment response.

Orbital Lateral Cantal Distance Can Predict the Cephalometric Characteristic of Mandible in Iranian Population.

ABSTRACT: Orbitofacial anthropometrics have become an important tool used in reconstructive surgery. The authors attempt to evaluate the relation between orbital lateral canthal distance and the cephalometric characteristic of mandible in Iranian population.In a cross-sectional study, anthropometric parameters of face in 200 subjects (100 males and 100 females) with mean age of 34.39 ±â€Š18.83 were evaluated by three-dimensional computed tomography imaging.In this study, there was not a significant difference in the age of sex groups (P = 0.183). Also, there was no significant difference in the left and right mandible angle in different sex groups (P = 0.25, P  =  0.124, respectively). There were significant differences in the anterior mandible distance, inferior mandible angle distance (P = 0.0001) and lateral cantus distance of sex groups (P = 0.0001). There was a significant correlation between lateral contuse distance and left mandible angle (r = 0.226, P = 0.001), right mandible angle (r = 0.283, P = 0.00), mandible angle (r = -0.266, P = 0.00), anterior mandible angle distance (r = 0.655, P = 0.00), and inferior mandible angle distance (r = 0.582, P = 0.00).Here, we conclude that orbital lateral canthal distance can predict the cephalometric characteristic of mandible in Iranian population.

The Therapeutic Potential of Conditioned Medium from Human Breast Milk Stem Cells in Treating Spinal Cord Injury.

STUDY DESIGN: Experimental animal study. PURPOSE: This study investigated the therapeutic effects of human breast milk stem cell (BMSC)-conditioned medium (BMSC-CM) in a model of spinal cord injury (SCI) in male Sprague-Dawley rats. OVERVIEW OF LITERATURE: SCI is one of the leading causes of disability in addition to sensory and motor impairment. So far, there have been no successful treatments for SCI. Given the positive outcomes associated with using stem cells and their derivatives as a treatment for various diseases, there is a growing interest in using them as an SCI treatment. Recent research has demonstrated that CM from stem cells has therapeutic advantages. METHODS: Human BMSCs were isolated and characterized, and CM was subsequently collected. Animals received an intrathecal administration of BMSC-CM after SCI. The activity of caspase-3 was measured to assess apoptosis, and levels of tumor necrosis factor-α and interleukin-1ß were measured to assess inflammation. Also, sensory and locomotor performances were assessed after SCI and BMSC-CM administration. RESULTS: Administration of CM from BMSC reduced apoptosis and inflammation at the site of injury in a rat model of SCI (p<0.05). Motor, sensory, locomotor, and sensorimotor performances were significantly improved in rats that received BMSC-CM after SCI. CONCLUSIONS: Intrathecal administration of BMSC-CM improved recovery in a rat model of SCI.

Human Menstrual Blood Stem Cell-Derived Granulosa Cells Participate in Ovarian Follicle Formation in a Rat Model of Premature Ovarian Failure In Vivo.

We recently reported the application of human menstrual blood stem cells' (HuMenSCs) transplantation as a treatment modality in a rat model of premature ovarian failure (POF). We continued to investigate further in this respect. Female rats were injected intraperitoneally with 36 mg/kg busulfan. HuMenSCs were obtained, grown, and analyzed for immunophenotypic features at passage three. The cells were labeled with CM-Dil and infused into the rats. There were four groups: normal, negative control, treatment, and Sham. One month after treatment, the ovaries were collected and weighed. Histological sections were prepared from the ovary and HuMenSCs were tracking. Subsequently, we examined the changes of expression of Bax and B cell lymphoma 2 (Bcl2) genes by real-time polymerase chain reaction assay. One month after HuMenSCs transplantation, these cells were located in the ovarian interstitium and granulosa cells (GCs). The number of TUNEL-positive cells significantly decreased in the treatment group. Also the expression level of Bax genes, unlike Bcl2 gene, significantly decreased compared with negative and sham groups. In our study, HuMenSCs were tracked in ovarian tissues within 2 months after transplantation, and they differentiated into GCs. Therefore, the use of these cells can be a practical and low-cost method for the treatment of POF patients.

Proliferation and differentiation of mouse spermatogonial stem cells on a three-dimensional surface composed of PCL/gel nanofibers / Proliferación y diferenciación de células madre espermatogónicas de ratón en una superficie tridimensional compuesta de nanofibras PCL / gel

Spermatogonial stem cells (SSCs) have self-renewal and differentiation capacity essential for sperm production throughout the male reproductive life. The electrospun polycaprolactone/gelatin (PCL/Gel) nanofibrous scaffold mimics important features of the extracellular matrix (ECM), which can provide a promising technique for the proliferation and differentiation of SSCs in vitro. The goal of the present study was to investigate the effects of PCL/Gel nanofibrous scaffold on the propagation and differentiation of neonate mouse SSCs (mSSCs). mSSCs were enzymatically isolated, and the cells were purified by differential plating method and seeded on scaffold. After 2 weeks, viability, colony number and diameter, and expression of specific spermatogonial cell genes were investigated. After mSSCs propagation, the cells were cultivated in a differentiation medium on the scaffold for another 2 weeks, and differentiating cells were analyzed by real-time PCR. The number of mSSC colony (P<0.01) and expression levels of specific spermatogonial genes Plzf and Inga6 (P<0.01) and also differentiation genes c-Kit, Tp1 and Ptm1 (P<0.05) were higher in scaffold group compared with control during the culture period. We conclude that mSSCs can be expanded and can differentiate toward spermatid cells on PCL/Gel nanofibrous scaffold with improved developmental parameters.

Las células madre espermatogónicas (CME) tienen capacidad de auto renovación y diferenciación esenciales para la producción de esperma a lo largo de la vida reproductiva masculina. El «scaffold¼ nanofibroso de policaprolactona / gelatina (PCL / Gel) electrohilado imita características importantes de la matriz extracelular (MEC), que puede proporcionar una técnica prometedora para la proliferación y diferenciación de CME in vitro. El objetivo del presente estudio fue investigar los efectos del «scaffold¼ nanofibroso PCL / Gel en la propagación y diferenciación de CME de ratones neonatos (mSSC). Los mSSC se aislaron enzimáticamente y las células se purificaron mediante un método de siembra diferencial y se sembraron en un «scaffold¼. Después de 2 semanas, se investigaron la viabilidad, el número y el diámetro de las colonias y la expresión de genes específicos de células espermatogónicas. Después de la propagación de mSSC, las células se cultivaron en un medio de diferenciación en el «scaffold¼ durante otras 2 semanas, y las células se analizaron mediante PCR en tiempo real. El número de colonias mSSC (P <0,01) y los niveles de expresión de los genes espermatogónicos específicos Plzf e Inga6 (P <0,01) y también los genes de diferenciación c-Kit, Tp1 y Ptm1 (P <0,05) fueron mayores en el grupo de «scaffold¼ en comparación con el control durante el período de cultivo. Concluimos que los mSSC pueden expandirse y diferenciarse en células espermátidas en un «scaffold¼ de nanofibras PCL / Gel con parámetros de desarrollo mejorados.

Colonization of Mouse Spermatogonial Cells in Modified Soft Agar Culture System Utilizing Nanofibrous Scaffold: A New Approach.

BACKGROUND: Spermatogonial stem cells (SSCs) are considered in fertility management approaches of prepubertal boys facing cancer therapies. However, in vitro propagation has become an important issue due to a small number of SSCs in testicular tissue. The present study aimed to investigate a modified soft agar culture system by using a nanofibrous scaffold as a new approach to mimic in vivo conditions of SSCs development. MATERIALS AND METHODS: The SSCs were isolated from neonate mouse testes, cultured on polycaprolactone scaffold, and covered by a layer of soft agar for 2 weeks. Then, the number and diameter of colonies formed in experimental groups were measured and spermatogonial markers (i.e., Plzf, Gfrα1, Id4, and c-Kit) in SSCs colonies were evaluated by a real-time polymerase chain reaction and immunostaining. RESULTS: Our results indicated that the colonization rate of SSCs was significantly higher in the present modified soft agar culture system (P<0.05). Only Plzf indicated a significant increased at the levels (P<0.05), the gene expression levels of Id4, Plzf, and Gfrα1 were higher in the present culture system. In addition, the expression of the c-Kit gene as a differentiating spermatogonia marker was higher in presence of scaffold and soft agar compared with the amount of other experimental groups (P<0.05). CONCLUSION: The culture system by using nanofibrous scaffold and soft agar as a new culture method suggests the potential of this approach in SSCs enrichment and differentiation strategies for male infertility treatments, as well as in vitro spermatogenesis.

The effects of human menstrual blood stem cells-derived granulosa cells on ovarian follicle formation in a rat model of premature ovarian failure.

Many studies have reported that human endometrial mesenchymal stem cells (HuMenSCs) are capable of repairing damaged tissues. The aim of the present study was to investigate the effects of HuMenSCs transplantation as a treatment modality in premature ovarian failure (POF) associated with chemotherapy-induced ovarian damage. HuMenSCs were isolated from menstrual blood samples of five women. After the in vitro culture of HuMenSCs, purity of the cells was assessed by cytometry using CD44, CD90, CD34, and CD45 FITC conjugate antibody. Twenty-four female Wistar rats were randomly divided into four groups: negative control, positive control, sham, and treatment groups. The rat models of POF used in our study were established by injecting busulfan intraperitoneally into the rats during the first estrus cycle. HuMenSCs were transplanted by injection via the tail vein into the POF-induced rats. Four weeks after POF induction, ovaries were collected and the levels of Amh, Fst, and Fshr expression in the granulosa cell (GC) layer, as well as plasma estradiol (E2) and progesterone (P4) levels were evaluated. Moreover, migration and localization of DiI-labeled HuMenSCs were detected, and the labeled cells were found to be localized in GCs layer of immature follicles. In addition to DiI-labelled HuMenSCs tracking, increased levels of expression of Amh and Fshr and Fst, and the high plasma levels of E2 and P4 confirmed that HuMenSC transplantation had a significant effect on follicle formation and ovulation in the treatment group compared with the negative control (POF) group.

In vitro effects of melatonin on colonization of neonate mouse spermatogonial stem cells.

We have recently reported that antioxidant supplements enhance the efficacy of cryopreserved spermatogonial stem cells. Melatonin is considered a free radical scavenger which has direct and indirect antioxidant effects in in vitro and in vivo microenvironments. Due to the anti-apoptotic properties of melatonin, researchers have proposed that melatonin may improve the efficiency of spermatogonial stem cell (SSC) transplantation. However, the appropriate methodology which facilitates SSC proliferation remains to be determined. Identification of a proper propagation system is essential for the future application of SSCs in the field of infertility. The aim of the present study was to investigate the effects of melatonin on the colonization of SSCs. SSCs were isolated from the testes of three to six day old mice, and their purities were assessed by cytometry using Plzf antibody. Isolated testicular cells were cultured in the absence or presence of melatonin extract for two weeks. Suppression of differentiation and maintenance of spermatogonial stem cells was confirmed by alkaline phosphatase staining and immunocytochemistry using Plzf antibody. The number and diameter of the colonies of SSCs were assessed during the 7th and 14th days of culture, and the expression of Id4, Plzf, and C-kit were evaluated using real-time PCR at the end of the culture period. The survival rate of the cultured cells in the presence of melatonin was significantly higher than the control group. The number and diameter of colonies also increased in the cells treated with melatonin. The results of our study suggest that culture of SSCs with 100 µM melatonin supplementation can increase SSCs proliferation significantly.

Comparison of Human Amniotic, Chorionic, and Umbilical Cord Multipotent Mesenchymal Stem Cells Regarding Their Capacity for Differentiation Toward Female Germ Cells.

Placenta harbors a plentiful source of various cells with stem cells or stem-like cell properties, which can be used in therapeutic procedures and research. Mesenchymal stem cells (MSCs) have attracted much attention due to their specific differentiation potential and tolerogenic properties. MSCs have been isolated from different parts of placenta; however, in this study, we isolated MSCs from amnion and chorion membrane, as well as umbilical cord (Wharton's jelly [WJ]) and compared their capacity regarding differentiation toward female germ cells under influence of 10 ng/mL BMP4. All placenta samples were collected from delivering mothers by normal cesarean section and cells were isolated by different methods. Results showed that all isolated cells were mostly positive for the MSC markers CD73, CD166, and CD105, and minimally reacted with CD34 and CD45 (hematopoietic markers). After differentiation induction using third passage cultured cells, immunocytochemistry staining showed that cells were positive for germline cell-related genes Ssea4, Oct4, and Ddx4, and oocyte-related gene Gdf9. RT-qPCR results indicated that human chorion MSCs (hCMSCs) had a greater potential to be differentiated into female germline cells. Moreover, the results of this study indicate that human umbilical cord MSCs originated from either male or female umbilical cord have the same differentiation potential into female germline cells. We recommend that for presumptive application of MSCs for infertility treatment and research, hUMSCs are best candidates due to their higher differentiation potential, ease of proliferation and expansion, and low immunogenicity.