Kidney Mesenchymal Stem Cell Differentiation: Effect of Scaffold and Basic Fibroblast Growth Factor.

Background: Chronic kidney disease (CKD) poses a global health challenge, and it needs alternative therapeutic approaches for patients with end-stage renal disease (ESRD). Although organ transplantation is effective, it faces challenges such as declining quality of life, immunological responses, transplant rejection, and donor shortages. Tissue engineering, by using suitable scaffolds, cells, and growth factors, emerges as a promising treatment option for kidney regeneration. Experiment: We precisely decellularized scaffold, derived from rat kidneys while maintaining its native three-dimensional (3D) architecture. The efficiency of decellularization was evaluated through histological examinations, including hematoxylin and eosin, periodic acid-Schiff, and DAPI staining, as well as scanning electron microscopy. The scaffolds were then recellularized with kidney mesenchymal stem cells (kMSCs), and their adhesion, proliferation, and differentiation were assessed over 1, 2, and 3 weeks. The expression of specific renal markers, including Wt-1, ZO-1, AQP-1, and ANG-1, was examined through quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in monolayer and 3D cultures. Results: The infiltration rate of cells into the scaffold increased in a time-dependent manner, and the expression of specific renal markers significantly increased, demonstrating successful differentiation of kMSCs within the scaffold. The application of basic fibroblast growth factor (bFGF) could intensify the expression of kidney-specific genes. Conclusions: The study highlighted the importance of preserving the 3D architecture of the scaffold during decellularization to achieve optimal cellular responses. Moreover, the capacity of mesenchymal stem cells in recellularized scaffolds facilitated tissue regeneration.

The Effect of Normal Follicular Fluid on the Differentiation of PCOS Ovarian Stem Cells into Oocyte-Like Cells.

Background: Polycystic ovarian syndrome (PCOS) is one of the causes of infertility for which treatment methods do not have a high rate of pregnancy. In this study, the stem cells in the follicular fluid (FF) of patients were grown in the normal FF, and their differentiation into oocytes was evaluated. Materials and Methods: The FF of PCOS patients was centrifuged, and their cells were cultured with and without 20% normal FF for 2 weeks. The cells were evaluated for their morphology by inverted microscope and for markers of stem cells (NANOG and OCT4) and oocytes (zona pellucida (ZP) 2 and ZP3) by RT-PCR and immunocytochemistry. The amount of steroids was measured by enzyme-linked immunosorbent assay (ELISA). Results: The cells were all round on day 0. After that, they had a heterogeneous morphology (fibroblast-like cells, epithelial-like cells, and round oocyte-like cells). In the first week, NANOG and OCT4 genes in the study group were less expressed than those in the control group (P < 0.0001) (~0.5-fold), while ZP2 and Z3 genes were more expressed (P < 0.0001) (~2-fold). In the second week, stem cell genes were more expressed in the control group (~2 fold), and oocyte genes were more expressed in the study group (P < 0.0001) (~2.5-3.11 fold). These results were also confirmed by immunocytochemistry. The amount of steroids was much higher in the study group (three times and five times in two weeks) (P < 0.0001). Conclusions: Stem cells can be obtained from the FF of PCOS, and normal FF has a positive effect on the growth and maturation of oocyte-like cells in vitro.

Utilizing Calcium Alginate for the Assessment of Bone Morphogenetic Protein 15 Induction Effect on the Differentiation of Mesenchymal Stem Cell Derived from Human Follicular Fluid to Oocyte-Like Structure.

BACKGROUND: Follicular fluid (FF)-derived mesenchymal stem cells (MSCs) are possible new source of cells in the study of oogenesis and regenerative medicine. Several biomaterials have been used as scaffolds to mimic ovarian tissue stroma. Using good matrix is essential for increasing the cell survival rate, proliferation, and differentiation. However, no study has been performed to investigate the effects of BMP15 and calcium alginate hydrogel on the differentiation potential of FF-derived MSCs to oocyte-like structures (OLSs). MATERIALS AND METHODS: In this work, FF MSCs, which were collected from women in routine in vitro fertilization procedure, were capsulated with 0.5% calcium alginate, and then the encapsulated cells were cultured in medium containing BMP15 for 2 weeks. Trypan blue staining was carried out to determine cell viability. Real-time polymerase chain reaction (PCR) and immunofluorescence (ICC) staining method were performed to characterize the expression of OCT4, Nanog, ZP2, and ZP3 genes and protein. The encapsulation process did not change the morphology and viability of the encapsulated cells. RESULTS: Reverse-transcription-PCR and ICC showed that MSCs expressed germ line stem cell markers such as OCT4 and Nanog. After 4 days of culture, OLSs formed and expressed zona pellucida markers. OLSs at least reached 180-230 µm in diameter in the control and BMP15-treated groups. Finally, a reduction in the expression pattern of pluripotency and ZP markers was detected in the encapsulated cells cultured in the BMP15-supplemented medium. CONCLUSION: The three-dimensional alginate culture system seems to be a promising method of getting in vitro differentiation and development of ovarian cells, which could mimic the native ovarian condition.

Effect of vitamin D on apoptotic marker, reactive oxygen species and human sperm parameters during the process of cryopreservation.

OBJECTIVES: Sperm cryopreservation plays an undeniable role in assisted reproductive technology. However, this process significantly reduces the motility, viability, morphology and nuclear integrity of sperm. Reasons of these changes were oxidative stress and apoptosis. The aim of this study was to evaluate the influence of vitamin D on the survival and integrity of fertile sperm after cryopreservation. MATERIALS AND METHODS: Semen sample of 18 males with normal parameters was used. After swimming up, each sample was divided into two parts. 20 µmol vitamin D was added to one part as experimental group and the other part was left untreated as control group. The samples in all groups were frozen for 14 days. Post-thawing, the groups were evaluated for sperm motility, and viability using eosin staining, morphology using the Diff-Quick staining and apoptosis by TUNEL, Annexin-V and caspase-3 activity assay. By using nitrobluetetraxolium test and thiobarbituric acid, the reactive oxygen species (ROS) and lipid peroxidation of sperms were measured, respectively. RESULTS: In comparison with control groups, motile and viable sperm concentration was substantially higher in treated groups (P-value<0.05); however, morphological analysis did not show any remarkable changes. Also, ROS and lipid peroxidation values were dramatically reduced by vitamin D (P-value<0.05). TUNEL and Annexin assay for apoptosis were considerably lower in treated groups (P-value<0.05), but caspase activity assay revealed no significant difference between groups. CONCLUSION: The results have shown that the addition of vitamin D to a freezing medium leads to higher quality and function of human sperm.

A Case of Mayer-Rokitansky-Küster-Hauser Syndrome with a Fused Pancake-shaped Pelvic Kidney.

Mayer-Rokitansky-Küster-Hauser (MRKH) Syndrome is a female reproductive system disorder. It is characterized by a defect in the Müllerian ducts development, and it causes the absence of the uterus in variable degrees in upper vaginal hypoplasia. In addition, it is often associated with the unilateral renal dysplasia. Müllerian agenesis affects 1 in 4500 newborn girls and is considered as a sporadic anomaly. Women with MRKH Syndrome have a normal female chromosome pattern 46, XX with normal ovarian function. The presence of bilateral kidney agenesis with a pelvic pancake-shaped kidney is a rare condition, and a few cases have been reported in medical journals. This case study focuses on a case of MRKH Syndrome with bilateral renal agenesis and a pancake-shaped kidney.

Effects of Mono-(2-Ethylhexyl) Phthalate and Di-(2-Ethylhexyl) Phthalate Administrations on Oocyte Meiotic Maturation, Apoptosis and Gene Quantification in Mouse Model.

OBJECTIVE: Phthalates, which are commonly used to render plastics into soft and flexible materials, have also been determined as developmental and reproductive toxicants in human and animals. The purpose of this study was to evaluate the effect of mono-(2- ethylhexyl) phthalate (MEHP) and di-(2-ethylhexyl) phthalate (DEHP) oral administrations on maturation of mouse oocytes, apoptosis and gene transcription levels. MATERIALS AND METHODS: In this experimental study, immature oocytes recovered from Naval Medical Research Institute (NMRI) mouse strain (6-8 weeks), were divided into seven different experimental and control groups. Control group oocytes were retrieved from mice that received only normal saline. The experimental groups I, II or III oocytes were retrieved from mice treated with 50, 100 or 200 µl DEHP (2.56 µM) solution, respectively. The experimental groups IV, V or VI oocytes were retrieved from mouse exposed to 50, 100 or 200 µl MEHP (2.56 µM) solution, respectively. Fertilization and embryonic development were carried out in OMM and T6 medium. Apoptosis was assessed by annexin V-FITC/Dead Cell Apoptosis Kit, with PI staining. In addition, the mRNA levels of Pou5f1, Ccna1 and Asah1 were examined in oocytes. Finally, mouse embryo at early blastocyst stage was stained with acridine-orange (AO) and ethidium-bromide (EB), in order to access their viability. RESULTS: The proportion of oocytes that progressed up to metaphase II (MII) and 2-cells embryo formation stage was significantly decreased by exposure to MEHP or DEHP, in a dose-dependent manner. Annexin V and PI positive oocytes showed greater quantity in the treated mice than control. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) revealed that expression levels of Pou5f1, Asah1 and Ccna1 were significantly lower in the treated mouse oocytes than control. The total cell count for blastocyst developed from the treated mouse oocytes was lower than the controls. CONCLUSION: These results indicate that oral administration of MEHP and DEHP could negatively affect mouse oocyte meiotic maturation and development in vivo, suggesting that phthalates could be risk factors for mammalians' reproductive health. Additionally, phthalate-induced changes in Pou5f1, Asah1 and Ccna1 transcription level could explain in part, the reduced developmental ability of mouse-treated oocytes.

Nifedipine compared to magnesium sulfate for treating preterm labor: A randomized clinical trial.

BACKGROUND: Preterm labor is the leading cause of infant morbidity and mortality so it may be necessary to administer tocolytics for treatment of it. OBJECTIVE: The aim of this study was to compare the efficacy and safety of magnesium sulfate and nifedipine in the management of preterm labor. MATERIALS AND METHODS: 100 women with documented preterm labor were randomly assigned to receive magnesium sulfate (n=50) and nifedipine (n=50) as tocolytic therapy. Before tocolysis, patient did not receive any sedation. After tocolysis, if patient continued to have contractions, they received other tocolytic agents. The main outcome variables examined were days gain in utero, success rate and side effects of tocolysis. RESULTS: Both drugs were equally effective in prevention of labor and delaying delivery >7 days, 56% vs. 64% in the nifedipine and magnesium sulfate groups, and the days gain in utero was no statistically different in two groups. 6% of nifedipine group and 2% of magnesium sulfate group required drug discontinuation due to severe symptoms. There were also no significant differences in maternal characteristics between two groups. The total success rate and side effects were similar in two groups. CONCLUSION: Oral nifedipine could be a suitable alternative for magnesium sulfate with the same efficacy and side effects in the management of preterm labor. Registration ID in IRCT: IRCT2013090914603N1.

Protective effects of co-administration of gallic Acid and cyclosporine on rat myocardial morphology against ischemia/reperfusion.

BACKGROUND: Irreversible myocardial ischemic injury begins 20 minutes after the onset of coronary occlusion. Then the infarcted cells show signs of necrosis and death. OBJECTIVES: This study investigated the effects of co-administration of Gallic acid (antioxidant) with cyclosporine (mitochondrial permeability transition pore [mPTP] inhibitor) on myocardial morphology of rats during ischemia and reperfusion. MATERIALS AND METHODS: Fifty-four male Wistar rats (250-300 g), were randomly divided into 9 groups: sham, control (Ca received saline, 1 mL/kg, Cb: perfused with cyclosporine CsA 0.2 µM), 3 groups pretreated with Gallic acid in saline (G1a:7.5, G2a:15, and G3a: 30 mg/kg/day, and gavage daily for 10 days, n = 6), and the other three groups were pretreated with Gallic acid then perfused using CsA, (G1b:7.5, G2b:15, and G3b: 30 mg/kg/day) at the first 13 minutes of reperfusion period. After 10 days pretreatment, the rat hearts were isolated and transferred to Langendorff apparatus and exposed to 30 minutes ischemia following 60 minutes reperfusion. Afterward, the hearts were preserved in 10% formalin for histological studies at the end of the experiment. Finally, hematoxylin and eosin and Masson's trichrome staining techniques were used for evaluating the changes in myocardial architecture, degradation of myofibers, and collagen integrity. The differences were analyzed using Pearson test. RESULTS: Cell degenerative changes, pyknotic nuclei, contraction bands, edema, and loosening of collagen in between muscle fibers were observed during ischemia-reperfusion. Myocardial architecture and cellular morphology were recovered in co-administration groups, especially in (Gallic acid 15 mg/kg + CsA, P < 0.001). CONCLUSIONS: The results suggest the important role of the antioxidant system potentiation in the prevention of myocardial damage.