فهرست مطالب fardin amidi

Drosophila melanogaster flies are smooth, low upkeep and safe model organisms, they can be effortless ly used in different fields of life sciences like genomics, biotechnology, genetics, disease model, and Wolbachia-based approaches to fight vectors and the pathogens they transmit.

Fruit fly specimens were collected in 25 districts (14 provinces) of Iran and their morphological recognition was proven by molecular analysis based on sequence homology of mitochondrial COI barcode region. Essential in formation and specific requirements were provided for laboratory rearing of D. melanogaster.

Drosophila melanogaster colonies were found in 23 out of 25 districts. Also, five related species coincident with D. melanogaster were reported in this study including D. ananassae/D. parapallidosa, D. hydei, D. repleta, Zapri onus indianus (Diptera: Drosophilidae), and Megaselia scalaris (Diptera: Phoridae). The Iranian D. melanogaster mo lecular signature and their rearing techniques have been described here. The complete life cycle, from (egg to adult), takes approximately 8 days at 25 °C. Some biological points have been presented with highlighting capturing, rearing, culturing, and embryo collection along with primitive recognition and segregation between females and males have been presented. A recipe for culture media and the quantity of various ingredients have been provided.

This is the first report on the D. repleta and D. ananassae/D. parapallidosa species for the country. Re sults of this study provide efficient and effective rearing procedures which are requirement for both small-scale for fa cilitating entomological research and large-scale use in justifiable vector control management such as disease model or Dengue control.

Astaxanthin (ASX) is a lipid-soluble keto-carotenoid with several biological effects. These effects may benefit polycystic ovarian syndrome (PCOS) patients. Imbalanced apoptosis/anti-apoptosis signaling has been considered the major pathogenesis of PCOS. In a randomized clinical trial, we tested the impact of ASX on the apoptotic pathway in PCOS granulosa cells (GCs). The present study hypothesizes that ASX may improve apoptosis in PCOS patients. This trial recruited patients with confirmed PCOS. A total of 58 patients were randomly assigned to take ASX (12 mg) or placebo for 8 weeks. Aspirated follicular fluid (FF) and blood samples were taken from both groups to measure BAX and BCL2 protein expression. Following FF aspiration, GCs from both groups were obtained; Real-Time PCR and Western blotting were used to evaluate the apoptotic pathway’s gene and protein expression levels in GCs.BAXBCL2 In GCs analysis, ASX reduced DR5 gene and protein expression after 8 weeks compared to placebo(p<0.05). Also, Caspase8 (p>0.05) and BAX (p<0.05) gene expression declined, although the difference was not statistically significant for Caspase8. Besides,ASX treatment contributed to an elevated BCL2 gene expression in GCs(p<0.05). In FF and serum analysis, a statistically significant increase was found in BCL2 concentration in the ASX group (p<0.05). Moreover, a reduction in BAX level was confirmed in both FF and serum of the ASX group; however, this change was not significant in the serum (p>0.05). It seems that ASX consumption among women with PCOS improved serum and FF levels of apoptotic factors and modulated genes and protein expression of the apoptosis pathway in GCs. Nevertheless, further investigations are needed to reveal the potential role of this compound in PCOS treatment.

Polycystic ovary syndrome (PCOS) is the most prevalent endocrine disorder of women in reproductive age with significant effects on reproductive and metabolic functions. Many molecular players may be involved in PCOS pathology; however, miRNAs possess great ability in gene expression control in normal ovarian function and folliculogenesis. We appraised the relative expression of miR-146a, miR-222, miR-9, and miR-224 in serum and follicular fluid (FF) of PCOS patients compared to control subjects. PCOS (n = 35) and control (n = 30) subjects were recruited in the study during their enrolment in IVF cycles. Serum and FF of human subjects were collected and stored. Total RNA was isolated from samples and cDNA was synthesized using miRNA-specific stem-loop RT primers. Quantitative real-time PCR was used to evaluate the expression of miRNAs relative to U6 expression. The predictive value of miRNAs’ expression for discrimination of PCOS patients from control subjects was evaluated by receiver-operating characteristic (ROC) curve analysis. miR-224 was not detected in serum and FF samples. Significantly, higher levels of miR-146a and miR-9 in serum of PCOS group were detected. In contrast, relative expression of miR-146a and miR-9 significantly decreased in FF. In PCOS group, relative expression of all detected miRNAs was elevated in serum in comparison to FF, whereas in control group no change was noticed. Combination of FF miRNAs showed improved predictive value with area under the ROC curve (AUC) of 0.84, 93.8% sensitivity, and 83.3% specificity. Contradicting alternations of miRNAs in serum and FF are indicative of different sources of miRNAs in body fluids. Presumptive target genes of studied miRNAs in signaling pathways may show the potential role of these miRNA in folliculogenesis.

The current research was established to make a comparison between the delayed-start GnRH antagonist and flare-up GnRH agonist protocols in poor response patients.

The present study is a randomized, prospective, controlled trial that was performed on 150 women who referred to two distinct in vitro fertilization (IVF) centers in Iran. Patients were randomly assigned to two experimental groups, as one group was treated with the delayed-start GnRH antagonist protocol (delayed-start group), while another group was treated with the flare-up protocol (flare-up group).

The serum concentrations of estradiol and progesterone, along with the thickness of endometrial tissue and the number of follicles ≥13 mm was significantly increased in the delayed-start group compared with the flare-up group. Also, the number of total oocytes, retrieved mature oocytes, total embryos, fertilized oocytes, as well as the quality of embryos were markedly higher in the delayed-start group when compared with the flare-up group. No statistically significant difference was found in the rates of fertilization, implantation, and pregnancy between the two experimental groups.

According to the above evidence, it seems that the effect of delayed-start protocol on ovarian responsiveness was more pronounced during controlled ovarian stimulation in comparison with the flare-up protocol and the delayed start protocol probably lead to better implantation and pregnancy rates in comparison with the flare up agonist protocol cycle in poor responders.

The process of implantation, a critical phenomenon in early development of embryo, is result of regulated reciprocal interaction between receptive endometrium and competent embryo. This dialogue is mediated by cell surface molecules, components of extracellular matrix and both embryo and endometrium secreted substances such as cytokines, growth factors, hormones and extracellular vesicles. Intensive recent studies introduce secreted microRNAs (miRNAs) as novel mediators in intercellular communication. MiRNAs are endogenous small single-stranded non-coding RNAs which involved in regulating post-transcriptionally target genes and influencing various intracellular processes. These molecules can be secreted and delivered into recipient cells where they affect host cellular events through target gene suppressing. Inthis regard, we reviewed the role of miRNAs in trophoblast cell differentiation, blastocyst secreted miRNAs in interaction with endometrium and regulation of implantation. In infertility treatment through assisted reproductive techniques, clinician needs to non-invasive strategies for exploring the criteria to select the best in vitro-produced embryo for transfer into uterine and achieving pregnancy. Here, we also reviewed potential role of miRNAs as biomarkers for selecting of competent embryos to transfer into uterine during infertility treatment such as in vitro fertilization and intracytoplasmic sperm injection.

Sulforaphane (SFN) is a natural free radical scavenger that can reduce oxidative stress (OS) through mediating nuclear factor (erythroid-derived 2)-like 2 (NF-E2-related factor 2 or NRF2)/antioxidant response element (ARE) signaling pathway and the downstream antioxidant enzymes. Here, we intended to study the role of SFN in OSinduced human granulosa cells (GCs) by investigating the intracellular levels of reactive oxygen species (ROS), cell death, and NRF2-ARE pathway.

This experimental study was conducted on GCs of 12 healthy women who had normal menstrual cycles with no history of polycystic ovary syndrome (PCOS), endometriosis, menstrual disorders, hyperprolactinemia, or hormonal therapy. After isolation of GCs, the MTT assay was performed to explore GCs viability after treatment with SFN in the presence or absence of H2 O2 . Flow cytometry was utilized to determine the intracellular ROS production and the apoptosis rate. Evaluation of the mRNA and protein expression levels of NRF2 and phase II enzymes including superoxide dismutase (SOD) and catalase (CAT) was performed by quantitative real-time polymerase chain reaction (PCR) and western blotting. Finally, the data were analyzed by SPSS software using One-way ANOVA and the suitable post-hoc test. Significance level was considered as P<0.05.

Pretreatment of GCs with SFN attenuated intracellular ROS production and apoptosis rate in the H2 O2 -exposed cells. Moreover, SFN treatment increased the mRNA expression level of NRF2, SOD, and CAT. Higher expression of NRF2 and SOD was also observed at the protein level.

Our study demonstrated that SFN protects human GCs against H2 O2 induced-OS by reducing the intracellular ROS production and the following apoptosis through a mechanism by which NRF2 increases the antioxidant enzymes such as SOD and CAT. This result may have a potential application in assisted reproduction cycles by improving the quality of GCs and the embedded oocyte, especially in PCOS patients.

Astaxanthin (AST) has been introduced as a radical scavenger and an anti-apoptotic factor that acts via regulating the nuclear factor-E2-related factor 2 (NRF2) and related factors. Here, we intended to examine the effect of AST on granulosa cells (GCs) against oxidative stress by examining NRF2 and downstream phase II antioxidant enzymes.

In this experimental study, we used cultured human primary GCs for the study. First, we performed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test to evaluate cells viability after treatment with hydrogen peroxide (H2 O2 ) and AST. The apoptosis rate and ROS levels were measured by flow cytometry. To determine NRF2 and phase II enzymes expression, we performed real-time polymerase chain reaction (PCR). Finally, we used western blot to measure the protein levels of NRF2 and Kelch-like ECsH-associated protein 1 (KEAP1). Enzyme activity analysis was also performed to detect NRF2 activity.

This study showed that AST suppressed ROS generation (P<0.01) and cell death (P<0.05) in GCs induced by oxidative stress. AST also elevated gene and protein expression and nuclear localization of NRF2 and had an inhibitory effect on the protein levels of KEAP1 (P<0.05). Furthermore, when we used trigonelline (Trig) as a known inhibitor of NRF2, it attenuated the protective effects of AST by decreasing NRF2 activity and gene expression of phase II enzymes (P<0.05).

Our results presented the protective role of AST against oxidative stress in GCs which was mediated through up-regulating the phase II enzymes as a result of NRF2 activation. Our study may help in improving in vitro fertilization (IVF) outcomes and treatment of infertility.

Pregnancy with the help of a third party, including the use of sperm, oocyte, embryo, and uterus, can be considered as an option for some infertile couples. Due to the important role of health professionals in infertility treatments, their attitudes are of particular importance in the acceptance or rejection of fertility suggestions involving the help of a third party. This study aimed to determine the attitudes of medical students at medical universities in Tehran toward third-party reproduction.

This descriptive cross-sectional study was carried out at the Medical University of Tehran in 2018. Medical students (n=187) filled out the questionnaire, which consisted of two parts: the demographic characteristics of the research subjects and the questionnaire consisted of 76 questions about attitudes toward third-party reproduction. The content and face validity of the questionnaire were determined, and test-retest reliability of the questionnaire was established (0.89).

According to gender, participants’ attitudes toward childbearing, the importance of genetic dependency between parents and children, law issues, anonymity in donation programs, parental affection, the importance of the recipient's and donor's characteristics, surrogacy, gamete, and embryo donation were all statistically non-significant (Pvalue>0.05). According to age and also to year of entering the university, participants’ attitude only toward childbearing was statistically significant (Pvalue= 0.018 and 0.01, respectively).

Since medical school students may set on the road to a specialty associated with infertility and its ramifications, it’s better to educate our soon-to-be health system professionals on all necessary aspects of infertility and third-party reproduction.

Mitochondrion is the main indicator of oocyte quality and one of the components of oocyte, which is sensitive to oxidative damage during the maturation process. Mitoquinone mesylate (MitoQ) is a strong antioxidant targeting mitochondria as well as anti-apoptotic agent. However, the effect of MitoQ on the quality of oocytes during in vitro maturation (IVM) is still unknown.

This study investigated the possible effects of MitoQ on maturation and developmental competency in mice oocytes.

The oocytes were collected at germinal vesicle stage from 6-8-week old female NMRI mice and then cultured in TCM-199 medium supplemented with 0, 0.01, 0.02 and 0.04 μM MitoQ. The sham group was treated with DMSO (0.01% v.v). Then intracellular Glutathione (GSH), reactive oxygen species (ROS) levels, mitochondria membrane potential (ΔΨm), as well as in vitro fertilization (IVF) rate in the 18-20 h matured oocytes and metaphase II (MII) oocytes (in vivo-control), were assessed.

The results showed that between three dose of MitoQ, the 0.02 μM significantly increased nuclear maturation rate, GSH level, fertilization rate and blastulation (92.6, 231.7, 90.19 and 81.66%, respectively) than the in vitro-control (71.14,152, 78.84 and 73.50%, respectively) and more comparable to that of the in vivo matured oocytes (100, 243.5, 92.10 and 83%, respectively). Also, the mitochondria membrane potential in the 0.02 μM MitoQ was significantly higher compared with those in the other groups (4.4). However, the intracellular ROS level in 0.02 μM MitoQ was significantly decreased(38.72%) compared to in vitro-control (82.2%) and was similar to the in vivo-control (33.5%).

The results indicated that supplementation of IVM medium with MitoQ (specially 0.02 μM) enhance maturationand fertilization rate. In conclusion, MitoQ might be considered as a novel component that could be added to IVM media.

This article describes the steps of creating an active learning space based on John Kotter's Leadership Change Model and evaluating the consequences of creating this learning space in the Faculty of Medicine of Tehran University of Medical Sciences.

This action research study was conducted from 2017 to 2019 after obtaining permission from Tehran University of Medical Sciences. The leadership of this change was guided by the John Kotter model and the evaluation of the consequences of creating the active learning space using a valid and reliable researcher-made questionnaire in collaboration with 265 basic science students through available sampling and 33 faculty members who had class in the Active learning space.

The change leadership process was successful and resulted in the creating the active physical space. Based on the findings of this study, 129 students (48.76%) agreed and strongly agreed that learning in this area had a greater impact on deepening their learning. The results showed that 30 (90.91%) of faculty members believed that they spend more time in creating collaboration and teamwork activities.

It seems that changing physical space has facilitated group discussion and interaction. In this study, we describe the leadership experience of this change and evaluate the opinions of students and faculty about active teaching in the classroom. The application of this study is for managers and leaders in education in medical universities seeking to change curricula, as attention to physical change alongside curricula is generally neglected. Keywords: Active Learning, Student-centered, Physical Space, Change Leadership, John Kotter Model

In this study, a dispersive liquid–liquid microextraction method using an extraction solvent lighter than water has been developed for the extraction and preconcentration of cholecalciferol and calcifediol from plasma samples followed by high performance liquid chromatography determination. Initially, acetonitrile and sodium chloride (NaCl) are added into the plasma as an extraction solvent and a salting–out agent, respectively. After manual shaking, the mixture is centrifuged. In the presence of sodium chloride, a two–phase system is formed. Then a portion of the upper phase is removed and mixed with n–hexane at µL–level and rapidly injected into distilled water by a syringe. In this process, the analytes are extracted into the fine droplets of n–hexane (as an extraction solvent). Under optimal conditions, enrichment factor was obtained 92 and 94 for calcifediol and cholecalciferol, respectively. The intra– (n=6) and inter–day (n=4) precisions were less than or equal to 8.1% at a concentration of 10 ng mL–1 of each analyte. Finally, this method was applied to the analysis of the analytes in human plasma samples.

  Colon tumor is generated and maintained by a small subset of chemo-resistant cancer cells known as Cancer Stem-like Cells (CSCs) that are able to self-renew and differentiate into various cell types within the cancer milieu. CSCs are identified through expression of CD133 that is the most important surface marker of these cells. Epithelial Cell Adhesion Molecule (EpCAM) is another colon CSCs marker. Other markers that are probably involved in colon tumorigenesis are Leucine-rich repeat-containing G-protein-coupled Receptor 5 (LGR5), B cell-specific Moloney murine leukemia virus insertion site 1 (BMI1) and Ten-Eleven Translocations (TETs). Here, mRNA expression rates of LGR5, BMI1 and TETs were surveyed by real-time PCR. After collection and digestion, colon samples were used to isolate CD133 and EpCAM positive CSCs through evaluation of AC133 EpCAM by Magnetic Activated Cell Sorting (MACS) and flow cytometry. Real-time PCR was carried out for assessing expressions of LGR5, BMI1 and TETs. High expressions for LGR5, BMI1, TET1 and TET2 in the CD133 and EpCAM positive CSCs (p≤0.05 vs. non-CSCs) were found. TET3, however, showed no significant changes for mRNA expression in the CSCs. In conclusion, high mRNA expressions for LGR5, BMI1, TET1 and TET2 in the CD133 and EpCAM positive CSCs may be a useful criterion for better identification of the cells involved in colon cancer in order to specify therapeutic targets against this type of cancer.

Fertility in men mainly depends on the number, quality, motility, and morphology of the sperms, and disruption of each of these factors leads to infertility. A large number of couples suffer from infertility problems. Among the various therapies, medicinal herbs are used in many countries to treat male infertility. Current systematic review was conducted to study the effects of garlic on male fertility. The information of this systematic review was collected by searching the key words: treatment, fertility, infertility, male, herbal medicine, garlic, Allium sativum, medicinal plant, sperm, sex hormones, testis and spermatogenesis in international databases such as: Web of Science (ISI), PubMed, Scopus and Embase until March 2018. This study was conducted in accordance with the PRISMA statement for systematic reviews and meta- analysis. and the SYRCLE risk of bias tool was used for qualitative assessment. A total of 18 experimental studies were included in the study. Thirteen studies evaluated garlic and 5 studies compared garlic effect with adriamycin, titanium dioxide, furan, vitamin E, N-acetylcysteine and cadmium. All studies were conducted in in vivo condition. The results of the studies indicated the potential effect of garlic on enhancing fertility and spermatogenesis, increasing the level of testosterone and improving the testicular structure. Garlic can increase fertility probably due to its antioxidant properties. However, more clinical trials are recommended.

Polycystic ovarian syndrome (PCOS) is a metabolic and endocrine disorder which is characterized by hyperandrogenism, anovulation or oligomenor-rhea and polycystic ovarian morphology. It is believed that modulation in metabolism of granulosa cells of PCOS patients may lead to infertility. One of the metabolic modulators is FNDC5 and its cleaved form, irisin. The axis of PGC1α- FNDC5 pathway is one of the main factors affecting cellular energy balance the purpose of this study was to evaluate this pathway in granulosa cells derived from PCOS mice model in comparison with control group.
In the present study, PCOS mouse model was developed by injection of dehydroepiandrosterone (DHEA) hormone in 20 mice for a period of 20 days. Also, 20 uninjected mice were used as the control. Meanwhile, a vehicle group consisted of mice which received daily subcutaneous sesame oil injection (n=20). Relative expressions of PGC1α and FNDC5 in granulosa cells were evaluated by RT-qPCR. Analysis of gene expressions was calculated by the ΔΔCT method and the relative levels of mRNA were normalized to GAPDH transcript levels. Differences in genes expression among three groups were assessed using one-way ANOVA, Tukey's Post Hoc test.
Our results showed that expression of FNDC5 was significantly reduced in granulosa cells of DHEA-induced PCOS mice compared with control and vehicle groups (p Down regulation of FNDC5 transcript level may contribute in metabolic disturbance of granulosa cells derived from PCOS ovary apart from PGC1α levels which remained unchanged.

In vitro spermatogenesis has a long research history beginning in the early 20th century. This organ culture method was therefore abandoned, and alternative cell culture methods were chosen by many researchers. Here, whether Tnp1, Tekt1, and Plzf, which play a crucial role in spermatogenesis, can be expressed during testis organ culture was assessed.
Testes of 10 mouse pups were first removed, and the testis tissue was then separated into smaller pieces of seminiferous tubules. The size of the pieces was arbitrary; approximately 1 mg in weight or 1 mm3 in size when compacted. Afterwards, the testis tissue fragments (1–3) were transferred to the hexahedrons, incubated in a culture incubator and cultured for 12 weeks. Histological assessment and molecular evaluation were carried out at the end of the study.
The results showed that the expression of Tekt1 as a mitotic gene in mouse pups decreased significantly (p ≤ 0.05) in comparison to adult mouse testis. Meanwhile, the expression of Tnp1 as a meiotic gene increased significantly (p ≤ 0.05) as compared to neonate mouse testis at the beginning of the culture. The expression of Plzf showed no significant difference during the 12 weeks of culture (p ≥ 0.05). Based on histological study, different types of spermatocytes and post-meiotic stages of germ cells could not be detected.
This kind of three-dimensional culture can induce expression of post-meiotic gene, Tnp1, but only at the molecular level and not beyond meiosis.

Oocyte cryopreservation is an essential part of the assisted reproductive technology (ART), which was recently introduced into clinical practice. This study aimed to evaluate the effects of two vitrification systems-Cryotop and Open Pulled Straw (OPS)-on mature oocytes gene expressions.
In this experimental study, the survival rate of metaphase II (MII) mouse oocytes were assessed after cryopreservation by vitrification via i. OPS or ii. Cryotop. Then we compared the fertilization rate of oocytes produced via these two methods. In the second experiment, we determined the effects of the two vitrification methods on the expression of Hspa1a, mn-Sod, and ß-actin genes in vitrified-warmed oocytes. Denuded MII oocytes were vitrified in two concentrations of vitrification solution (VS1 and VS2) by Cryotop and straw. We then compared the results using the two vitrification methods with fresh control oocytes.
mn-Sod expression increased in the vitrified-warmed group both in OPS and Cryotop compared with the con- trols. We only detected Hspa1a in VS1 and control groups using Cryotop. The survival rate of the oocytes was 91.2% (VS1) and 89.2% (VS2) in the Cryotop groups (P=0.902) and 85.5% (VS1) and 83.6% (VS2) in the OPS groups (P=0.905). There were no significant differences between the Cryotop and the OPS groups (P=0.927). The survival rate in the Cryotop or the OPS groups was, nevertheless, significantly lower than the control group (P Our results indicated that Cryotop vitrification increases both cooling and warming rates, but both Cryo- top and OPS techniques have the same effect on the mouse oocytes after vitrification.

Contrary to a common belief, most mammalian females lose the ability of Germ Cell (GC) renewal and oogenesis during fetal life. Although, it has been claimed that germ line stem cells preserve oogenesis in postnatal mouse ovaries, that postnatal oogenesis keeps producing functional and sufficient GCs in the case of infertility (caused by different reasons) is doubtful. On the other hand, there are many studies showing derivation of primordial GCs and late GCs from Embryonic Stem Cells (ESCs) in vitro. This study aimed to clarify the role of ESC-derived GCs in oogenesis.
Mouse ESCs via Embryoid Body (EB) formation were differentiated into GC lineage by adding Bone Morphogenetic Protein 4 (BMP4) and Retinoic Acid (RA) to the culture medium. Expression of GC markers was characterized by using Reverse Transcription Polymerase Chain Reaction (RT-PCR) and immunohistochemistry. Several 6- to 10-week-old female mice, sterilized using chemical agents, were injected with ESCs-derived GCs thorough their tail veins. To track the transplanted cells, their ovaries were immunohistochemically stained after two months.
Expression of GC specific markers such as mouse vasa homologue (Mvh) and Deleted in Azoospermia-Like (DAZL) indicated that GCs were successfully developed from ESCs. Interestingly, there was no evidence of homing of GCs in the transplanted ovaries after transplantation of ESCs-derived GCs.
Our findings do not suggest any contribution of ESC-derived GCs within the sterilized mice ovaries.

Teratoasthenozoospermia (TA) is a severe form of male infertility with no clear etiology.

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.

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.

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).

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.

Tissue engineering is a new approach to reconstruction and/or regeneration of lost or damaged tissue. The purpose of this study was to fabricate the polycaprolactone (PCL) random nanofiber scaffold as well as evaluation of the cell viability, adhesion, and proliferation of rat nestin‑positive hair follicle stem cells (HFSCs) in the graft material using electrospun PCL nanofiber scaffold in regeneration medicine.

The bulge HFSCs was isolated from rat whiskers and cultivated in Dulbecco’s modified Eagle’s medium/F12. To evaluate the biological nature of the bulge stem cells, flow cytometry using nestin, CD34 and K15 antibodies was performed. Electrospinning was used for the production of PCL nanofiber scaffolds. Furthermore, scanning electron microscopy (SEM) for HFSCs attachment, infiltration, and morphology, 3‑(4, 5‑di‑methylthiazol‑2‑yl)‑2, 5‑diphenyltetrazolium bromide (MTT) assay for cell viability and cytotoxicity, tensile strength of the scaffolds mesh, and histology analysis were used.

Flow cytometry showed that HFSCs were nestin and CD34 positive but K15 negative. The results of the MTT assay showed cell viability and cell proliferation of the HFSCs on PCL nanofiber scaffolds. SEM microscopy photographs indicated that HFSCs are attached and spread on PCL nanofiber scaffolds. Furthermore, tensile strength of the scaffolds mesh was measured.

The results of this study revealed that modified PCL nanofiber scaffolds are suitable for HFSCs seeding, attachment, and proliferation. Furthermore, HFSCs are attached and proliferated on PCL nanofiber scaffolds.

Cryopreservation of immature testicular tissue should be considered as an important factor for fertility preservation in young boys with cancer. The objective of this study is to investigate whether immature testicular tissue of mice can be successfully cryopreserved using a simple vitrification procedure to maintain testicular cell viability, proliferation, and differentiation capacity.
In this experimental study, immature mice testicular tissue fragments (0.5-1 mm²) were vitrified-warmed in order to assess the effect of vitrification on testicular tissue cell viability. Trypan blue staining was used to evaluate developmental capacity. Vitrified tissue (n=42) and fresh (control, n=42) were ectopically transplanted into the same strain of mature mice (n=14) with normal immunity. After 4 weeks, the graft recovery rate was determined. Hematoxylin and eosin (H&E) staining was used to evaluate germ cell differentiation, immunohistochemistry staining by proliferating cell nuclear antigen (PCNA) antibody, and terminal deoxynucleotidyl transferase (TdT) dUTP Nick- End Labeling (TUNEL) assay for proliferation and apoptosis frequency.
Vitrification did not affect the percentage of cell viability. Vascular anastomoses was seen at the graft site. The recovery rate of the vitrified graft did not significantly differ with the fresh graft. In the vitrified graft, germ cell differentiation developed up to the secondary spermatocyte, which was similar to fresh tissue. Proliferation and apoptosis in the vitrified tissue was comparable to the fresh graft.
Vitrification resulted in a success rates similar to fresh tissue (control) in maintaining testicular cell viability and tissue function. These data provided further evidence that vitrification could be considered an alternative for cryopreservation of immature testicular tissue.

Human sperm cryopreservation has proven to be very valuable. Cryopreservation has effects on function, morphology and percentage of fertilization capacity of human sperm. Also, it has been revealed that cryopreservation has a role in sperm DNA fragmentation and infertility. In this study, viability, motility, DNA fragmentation and for the first time, intracellular nitric oxide assessed before and after cryopreservation process of human semen samples in asthenozoospermic men. Semen samples were collected from 50 asthenozoosprmic men and divided into2 groups: 25 fresh semen samples as a control group, 25 frozen–thawed semen samples. Viability was assessed by eosin-negrosin staining. Motility was evaluated with a phase contrast microscope and intracellular nitric oxide was measured by flowcytometry. For evaluation of DNA fragmentation in sperm, apoptosis was assessed by flowcytometry. cryopreservation of asthenozoospermic semen samples decreased sperm viability and motility and increased the intracellular nitric oxide concentration and DNA damage significantly (p<0.001). cryopreservation process has detrimental effects on viability and motility, intracellular nitric oxide concentration and DNA integrity in human semen samples.

Mesenchymal stem cells (MSCs) derived from Wharton’s jelly (WJ-MSCs) are now much more appealing for cell-based infertility therapy. Hence, WJ-MSCs differentiation toward germ layer cells for cell therapy purposes is currently under intensive study. MSCs were isolated from human Wharton’s jelly and treated with BMP4, retinoic acid (RA) or co-cultured on human amniotic epithelial (HAE) and chorionic plate (HCP) placenta feeder cells. profile of POU5F1, Fragilis, Plzf, DDX4, Piwil2, Stra8, Dazl, β1- and α6-integrins (ITΒ1, ITA6) genes expression as germ cell markers were analyzed using RT-PCR and real-time PCR. Immunocytochemistry of surface markers were conducted. After 3 weeks treatment with different reagents and co-culture system, morphology of WJ-MSCs changed to shiny clusters and germ cell specific markers in mRNA were up-regulated in both placental feeder + RA and BMP4 + RA. Induction of hWJ-MSCs with BMP4 in presence of RA resulted in significant up-regulation (P≤0.05) of all germ cell specific genes (c-Kit; 2.84±0.59, DDX4; 1.69±0.39, Piwil2; 1.14±0.21, Dazl; 0.65±0.25, α6 integrin; 1.26±0.53, β1 integrins; 1.18±0.65) compared to control and placental feeder cells + RA. Our results indicated that HAE and HCP followed by RA treatment were involved in human germ cell development. We demonstrated that under the right conditions, hWJ-MSCs have the ability to differentiate to germ cells and this provides an excellent pattern to study infertility cause and treatment.

Althoughroutinely applied in assisted reproductive technology, human sperm cryopreservation is not a completely successful procedure. Adverse effects of cryopreservation on the fertilization capacity, motility, morphology, and viability of spermatozoa have been proven; cryopreservation has also shown a role in sperm DNA fragmentation and infertility. The post-thaw survival of spermatozoa improved after addition of supplementation of antioxidant molecules to freezing media. Nerve growth factor (NGF) as one of the prosurvival substances has gained great attention in recent years. The aim of this study was the usage of NGF as prosurvival factor after cryopreservation process of human semen samples to assess the motility and viability of sperm, nitric oxide (NO) concentration, and DNA fragmentation in normozoospermic men. Semen samples were collected from 25 normozoospermic men and were divided into fresh semen samples as control group, frozen–thawed semen samples without addition of exogenous NGF, and three groups of semen samples cryopreserved with addition of exogenous NGF (0.5, 1, and 5 ng/ml) in freezing medium. Viability was assessed by eosin-negrosin staining technique. Motility was evaluated with inverted microscope. NO concentration and apoptosis content were measured with flow cytometry. Results showed that exogenous NGF at 0.5 ng/ml could significantly (P-value <0.05) influence viability, motility, nitric oxide, and DNA fragmentation content. Exogenous NGF as cryoprotectant improved sperm viability and motility, increased intracellular NO concentration, and decreased apoptosis content in normal human spermatozoa.

The existence of female germ-line stem cells (FGSCs) has been the subject of a wide range of recent studies. Successful isolation and culture of FGSCs could facilitate studies on regenerative medicine and infertility treatments in the near future. Our aim in the present study was evaluation of the most commonly used techniques in enrichment of FGSCs and in establishment of the best procedure. In this experimental study, after digesting neonate ovary from C57Bl/6 mice, we performed 2 different isolation experiments: magnetic activated cell sorting (MACS) and pre-plating. MACS was applied using two different antibodies against mouse vasa homolog (MVH) and stage-specific embryonic antigen-1 (SSEA1) markers. After the cells were passaged and proliferated in vitro, colony-forming cells were characterized using reverse transcription-polymerase chain reaction (RT-PCR) (for analysis of expression of Oct4, Nanog, C-kit, Fragilis, Mvh, Dazl, Scp3 and Zp3), alkaline phosphatase (AP) activity test and immunocytochemistry. Data showed that colonies can be seen more frequently in pre-plating technique than that in MACS. Using the SSEA1 antibody with MACS, 1.98 ± 0.49% (Mean ± SDV) positive cells were yield as compared to the total cells sorted. The colonies formed after pre-plating expressed pluripotency and germ stem cell markers (Oct4, Nanog, C-kit, Fragilis, Mvh and Dazl) whereas did not express Zp3 and Scp3 at the mRNA level. Immunocytochemistry in these colonies further confirmed the presence of OCT4 and MVH proteins, and AP activity measured by AP-kit showed positive reaction. We established a simple and an efficient pre-plating technique to culture and to enrich FGSCs from neonatal mouse ovaries.

Mesenchymal Stem Cells (MSCs) are multipotent cells that can be collected from different sources. Under specific conditions, MSCs can be differentiated to tissue specific cells in vitro. Human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs) can easily be harvested and cultured in in vitro conditions. Production of germ cells from mesenchymal stem cells is a very interesting and promising area in the field of reproductive medicine. In the present study, the possible trans-differentiation of hUCMSCs into Primordial like Germ Cell (PGC) was performed in vitro under specific condition. Human umbilical cord mesenchymal stem cells were cultured and expanded in DMEM medium containing 10% FBS. The cultured cells were studied for differentiation ability to adipocytes and osteocytes. Furthermore, MSCs related markers were identified by flow cytometry method. For PGC differentiation, hUCMS cells were cultured in differentiation medium containing Bone Morphogenetic Protein 4 (BMP4) and it was followed by retinoic acid (RA). Real time PCR and immunocytochemistry analysis were performed to evaluate the expression of PGC specific genes and proteins, respectively. Our results showed that hUCMSCs cultured in the presence of BMP4 and RA are able to transdifferentiate in to PGC like cells in vitro. Real time PCR and immunocytochemistry results showed that differentiated cells expressed PGC specific markers after 14 days of culture. Based on these results, it was concluded that hUCMSC may be considered as a promising alternative cell source in reproductive medicine. More studies including laboratory and also animal models are needed to evaluate the functionality of differentiated PGCs before introducing them to clinical applications.