Reconstruction of ovarian follicular-like structure by recellularization of a cell-free human ovarian scaffold with mouse fetal ovarian cells.

The present study assessed the supportive roles of the decellularized human ovarian tissue in homing of mouse fetal ovarian cells into the scaffold as well as the formation of the follicular-like structure. The human ovarian cortical tissues were decellularized by three freeze-thaw cycles and then, treated with Triton X-100 for 15 h and 0.5% sodium dodecyl sulfate for 72 h. After isolation and preparation of mouse fetal ovarian cells (19 dpc) they were seeded into the decellularized scaffolds and cultured for 7 days, then using a light microscope, laser confocal scanning microscope, and scanning electron microscope these scaffolds were studied. Analysis of gene expression related to oocyte and follicular cells such as Ddx4, Nobox, Gdf9, and Connexin37 was assessed by real-time RT-PCR and the DDX4 and GDF9 proteins were detected by immunohistochemistry. The result showed that the human ovarian tissue was decellularized properly and the tissue elements and integrity were well preserved. After 7 days of in vitro culture, the fetal ovarian cells attached and penetrated into different sites and depths of the scaffold. The formed organoid within the scaffold showed large round, small polyhedral, and elongated spindle cells similar to the follicle structure. The molecular analysis and immunohistochemistry were confirmed an increase in the expression of genes and proteins related to oocyte and follicular cells in these reconstructed structures. In conclusion, the recellularization of human ovarian scaffolds by mouse fetal ovarian cells could support the follicular-like structure formation and it provides an in vitro model for follicle reconstitution and offers an alternative approach for clinical usage.

Evaluation of Vascular Endothelial Growth Factor Gene Expression in Recellularized Liver Tissue by Mouse Embryo Fibroblast.

Background: The aim of the present study was to evaluate alterations in the vegf gene expression as an angiogenic factor in mouse embryo fibroblasts seeded on the decellularized liver fragments. Methods: Liver tissue samples (n = 10) collected from adult male mice were randomly divided into decellularized and native control groups. Tissues were decellularized by treating with 1% Triton X-100 and 0.1% SDS for 24 hours and assessed by H&E staining and SEM. Then DNA content analysis and toxicity tests were performed. By centrifugation, DiI-labeled mouse embryo fibroblasts were seeded on each scaffold and cultured for one week. The recellularized scaffolds were studied by H&E staining, SEM, and LSCM. After RNA extraction and cDNA synthesis, the expression of the vegf gene in these samples was investigated using real-time RT-PCR. Results: Our observations showed that the decellularized tissues had morphology and porous structure similar to the control group, and their DNA content significantly reduced (p < 0.05) and reached to 4.12% of the control group. The MTT test indicated no significant cellular toxicity for the decellularized scaffolds. Light microscopy, SEM, and LSCM observations confirmed the attachment and penetration of embryonic fibroblast cells on the surface and into different depths of the scaffolds. There was no statistically significant difference in terms of vegf gene expression in the cultured cells in the presence and absence of a scaffold. Conclusion: The reconstructed scaffold had no effect on vegf gene expression. Decellularized mouse liver tissue recellularized by embryonic fibroblasts could have an application in regenerative medicine.

Expression of Apoptosis-Related Genes in Cultured Ovarian Tissue of Mouse in Presence of Coenzyme Q10.

Objective: Oxidative stress affects the development of ovarian follicles during in vitro culture thus applying an antioxidant is necessary. The aim of this study was to investigate the effect of coenzyme Q10 (CoQ10) on the expression of apoptosis-related genes of mouse ovaries during in vitro culture. Materials and methods: The immature mouse ovaries were cultured in the presence of 50 µM CoQ10 for 7 days. Histological examinations were performed and the 17 beta-estradiol concentration was measured on the seventh day of culture. The relative expression of Caspase 3, Bax, Bad, and Bcl 2 genes were investigated by real-time RT-PCR. Results: The rates of normal follicles in the presence of CoQ10 was significantly increased compared to the control group. Also, in CoQ10-trated group a significant increase in the level of 17 beta-estradiol was seen compared to the control group. The mRNA expression of anti-apoptotic gene Bcl2 was significantly increased while the expression of pro-apoptotic genes (Caspase3, Bax and Bad) significantly declined in CoQ10 treated group compared to those of control group. Conclusion: The supplementation of the ovarian culture media with CoQ10 improved the follicular development through alteration in expression of apoptosis-related genes and stimulated the production of estradiol.

Co-Culture of Mouse Blastocysts on A Human Recellularized Endometrial Scaffold: An In Vitro Model for Future Implantation Studies.

OBJECTIVE: This study evaluates the interaction of mouse blastocysts as a surrogate embryo on a recellularized endometrial scaffold by seeding human endometrial mesenchymal cells (hEMCs). MATERIALS AND METHODS: In this experimental study, prepared decellularized human endometrial tissues were characterized by morphological staining, DNA content analysis, and scanning electron microscopic (SEM) analysis. The scaffolds were subsequently recellularized by hEMCs. After seven days of cultivation, the mouse blastocysts were co-cultured on the recellularized scaffolds for 48 hours. Embryo attachment and implantation within these scaffolds were evaluated at the morphological, ultrastructural, molecular, and hormonal levels. RESULTS: There was no morphological evidence of cells and nuclei in the decellularized scaffold. DNA content significantly decreased by 89.92% compared to the control group (P<0.05). Both decellularized and native tissues had similar patterns of collagen bundles and elastin fibers, and glycosaminoglycan (GAGs) distribution in the stroma. After recellularization, the hEMCs attached to the scaffold surface and penetrated different parts of these scaffolds. In the co-cultured group, the embryo attached to the surface of the scaffold after 24 hours and penetrated the recellularized endometrial tissue after 48 hours. We observed multi-layered organoid-like structures formed by hEMC proliferation. The relative expressions of epithelial-related genes, ZO-1 and COL4A1, and SSP1, MMP2, and PRL, as decidualizationrelated genes, were significantly higher in the recellularized group on day 9 in the presence of the embryo compared to the other groups (P<0.05). Beta human chorionic gonadotropin (ß-hCG) and prolactin were statistically increased in the recellularized group on day 9 group (P<0.05). CONCLUSION: hEMCs and mouse embryo co-cultured on a decellularized endometrial scaffold provides an alternative model to study embryo implantation and the earlier stage of embryo development.

Differentiation of human endometrial mesenchymal cells to epithelial and stromal cells by seeding on the decellularized endometrial scaffold.

This study aimed to construct the endometrial-like structure by co-culturing of human mesenchymal endometrial cells and uterine smooth muscle cells in the decellularized scaffold. After decellularization of the human endometrium, cell seeding was performed by centrifugation of human mesenchymal endometrial cells with different speeds and times in 15 experimental subgroups. Analysis of residual cell count in suspension was done in all subgroups and the method with the lower number of suspended cells was selected for subsequent study. Then, the human endometrial mesenchymal cells and the myometrial muscle cells were seeded on the decellularized tissue and cultured for 1 wk; then, differentiation of the seeded cells was assessed by morphological and gene expression analysis. The cell seeding method by centrifuging at 6020 g for 2 min showed the highest number of seeded cells and the lowest number of residual cells in suspension. In the recellularized scaffold, the endometrial-like was seen with some protrusions on their surface and the stromal cells had shown spindle and polyhedral morphology. The myometrial cells almost were homed at the periphery of the scaffold and mesenchymal cells penetrated in deeper parts similar to their arrangement in the native uterus. The more expression of endometrial-related genes such as SPP1, MMP2, ZO-1, LAMA2, and COL4A1 and low-level expression of the OCT4 gene as a pluripotency marker confirmed the differentiation of seeded cells. Endometrial-like structures were formed by the co-culturing of human endometrial mesenchymal cells and smooth muscle cells on the decellularized endometrium.

Comparison of mouse ovarian follicular development and gene expression in the presence of ovarian tissue extract and sodium selenite: An experimental study.

Background: Ovarian tissue extract (OTE) and sodium selenite (SS) enhance the growth and maturation of preantral follicles in a dose-dependent manner. Objective: The present study was designed to bring more information regarding the mechanism of OTE and SS on the mRNA expression of follicle-stimulating hormone receptors (FSHR) and the proliferation cell nuclear antigens (PCNA) of in vitro matured isolated follicles. Materials and Methods: The tissue extract was prepared from adult ovaries. The preantral follicles (n = 266) were isolated from 12-16-day-old mice and cultured in the control, experimental I (10 ng/ml SS), and experimental II (OTE) groups for 12 days. The follicular diameter, survival, and maturation rates, also, the production of 17-ß-estradiol and progesterone, and the follicular expression of PCNA and FSH receptor genes were analyzed. Results: The survival rate of follicles in the SS-treated group (84.58%) was significantly higher than that OTE (75.63%; p = 0.023) and control (69.38%; p = 0.032) groups. The mean diameter of culture follicles in experimental group I (403.8 µm) and experimental group II (383.97 µm) increased significantly in comparison with the control group (342.05 µm; p = 0.032). The developmental rate of follicles, percentages of antrum formation, released metaphase II oocytes (p = 0.027; p = 0.019 respectively), production of hormones and the expression of 2 studied genes were significantly increased in both experimental groups in compare with control group (p = 0.021; p = 0.023 respectively). Conclusion: The OTE and SS have a positive effect on development of mouse preantral follicles via over-expression of FSHR and PCNA genes.

Left Ventricular Geometry and Angiogenesis Improvement in Rat Chronic Ischemic Cardiomyopathy following Injection of Encapsulated Mesenchymal Stem Cells.

OBJECTIVE: Injection of hydrogel and cells into myocardial infarction (MI) patients is one of the emerging treatment techniques, however, it has some limitations such as a lack of electromechanical properties and neovascularization. We investigated the therapeutic potential of new electroactive hydrogel [reduced graphene oxide (rGO)/Alginate (ALG)] encapsulated human bone marrow mesenchymal stem cells (BMSCs). MATERIALS AND METHODS: The experimental study involved ligating the left anterior descending coronary artery (LAD) in rat models of chronic ischemic cardiomyopathy. Echocardiograms were analyzed at 4 and 8 weeks after MI treatment. In the eighth week after injection in the heart, the rats were sacrificed. Histological and immunohistochemical analyses were performed using Hematoxylin and Eosin (H and E) staining, Masson's trichrome staining and anti-CD31 antibody to analyze tissue structure and detect neovascularization. RESULTS: In comparison to the control and other treatment groups, MSCs encapsulated in rGO-ALG showed significant improvements in fractional shortening (FS), ejection fraction (EF), wall thickness and internal diameters (P<0.05). The morphological observation showed several small blood vessels formed around the transplantation site in all treated groups especially in the MSC-ALG-rGO group 8 weeks after the transplantation. Also, Masson's trichrome staining indicated an increased amount of collagen fibers in rGO-ALG-MSC. Microvessel density was significantly higher using MSC-ALG-rGO compared to controls (P<0.01). CONCLUSION: This study demonstrates that intramyocardial injection of rGO/ALG, a bio-electroactive hydrogel, is safe for increasing LV function, neovascularization, and adjusting electrical characteristics following MI. The results confirm ALG promising capability as a natural therapeutic for cardiac regeneration.

Analysis of decellularized mouse liver fragment and its recellularization with human endometrial mesenchymal cells as a candidate for clinical usage.

Decellularized tissue has been used as a natural extracellular matrix (ECM) or bioactive biomaterial for tissue engineering. The present study aims to compare and analyze different decellularization protocols for mouse liver fragments and cell seeding and attachment in the created scaffold using human endometrial mesenchymal cells (hEMCs).After collecting and dissecting the mouse liver into small fragments, they were decellularized by Triton X-100 and six concentrations of sodium dodecyl sulfate (SDS; 0.025, 0.05, 0.1, 0.25, 0.5, and 1%) at different exposure times. The morphology and DNA content of decellularized tissues were studied, and the group with better morphology and lower DNA content was selected for additional assessments. Masson's tri-chrome and periodic acid Schiff staining were performed to evaluate ECM materials. Raman confocal spectroscopy analysis was used to quantify the amount of collagen type I, III and IV, glycosaminoglycans and elastin. Scanning electron microscopy and MTT assay were applied to assess the ultrastructure and porosity and cytotoxicity of decellularized scaffolds, respectively. In the final step, hEMCs were seeded on the decellularized scaffold and cultured for one week, and finally the cell attachment and homing were studied morphologically.The treated group with 0.1% SDS for 24 h showed a well preserved ECM morphology similar to native control and showing the minimum level of DNA. Raman spectroscopy results demonstrated that the amount of collagen type I and IV was not significantly changed in this group compared to the control, but a significant reduction in collagen III and elastin protein levels was seen (P < 0.001). The micrographs showed a porous ECM in decellularized sample similar to the native control with the range of 2.25 µm to 7.86 µm. After cell seeding, the infiltration and migration of cells in different areas of the scaffold were seen. In conclusion, this combined protocol for mouse liver decellularization is effective and its recellularization with hEMCs could be suitable for clinical applications in the future.

Lysophosphatidic acid supports the development of vitrified ovarian follicles by decreasing the incidence of cell death: An experimental study.

Background: Lysophosphatidic acid (LPA) contributes to follicular activation, oocyte maturation, in vitro fertilization, and embryo implantation. Objective: This study was designed to evaluate the effects of LPA to improve the development of isolated follicles derived from whole mouse cultured vitrified ovaries. Materials and Methods: In this experimental study, first, the 1-wk-old mouse ovaries in the non-vitrified and vitrified groups were cultured in the presence of 20 µM of LPA for 1 wk. Then, their isolated preantral follicles were cultured individually for 12 days in the presence or absence of 40 µM of LPA. The following evaluations were done for the cultured follicles: a viability test using Calcein AM staining, flow cytometry using annexin V/Pi, and analysis of the expression of genes by real-time reverse transcription polymerase chain reaction. The maturation rates of the oocytes were compared among groups and some of the released metaphase II oocytes were subjected to in vitro fertilization. Results: In all LPA treated groups, the rates of survival and follicular development were higher, and the incidence of cell death and expression of pro-apoptotic genes were lower, than in the non-LPA supplemented groups (p = 0.035). There was no significant difference between the vitrified and non-vitrified groups regarding follicular or oocyte development, but the expression of Bad and LPA receptors genes was significantly altered in the vitrified LPA supplemented group in comparison with the non-vitrified LPA supplemented group (p = 0.028). Conclusion: LPA improved the survival and developmental potential of the isolated follicles. Despite some alterations in the expression of apoptosis-related genes in the vitrified ovaries, LPA had positive effects on the survival and development of these follicles.

Fabrication and characterization of PHEMA-gelatin scaffold enriched with graphene oxide for bone tissue engineering.

BACKGROUND: Growing investigations demonstrate that graphene oxide (GO) has an undeniable impact on repairing damaged bone tissue. Moreover, it has been stated in the literatures that poly(2-hydroxyethyl methacrylate) (PHEMA) and gelatin could provide a biocompatible structure. METHODS: In this research, we fabricated a scaffold using freeze-drying method comprised of PHEMA and gelatin, combined with GO. The validation of the successful fabrication of the scaffolds was performed utilizing Fourier-transform infrared spectroscopy (FTIR) and X-ray diffraction assay (XRD). The microstructure of the scaffolds was observed using scanning electron microscopy (SEM). The structural properties of the scaffolds including mechanical strength, hydrophilicity, electrical conductivity, and degradation rate were also evaluated. Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) were used to evaluate the cytotoxicity of the prepared scaffolds. The osteogenic potential of the GO-containing scaffolds was studied by measuring the alkaline phosphatase (ALP) activity after 7, 14, and 21 days cell culturing. RESULTS: SEM assay showed a porous interconnected scaffold with approximate pore size of 50-300 µm, appropriate for bone regeneration. The increase in GO concentration from 0.25 to 0.75% w/v exhibited a significant improvement in scaffolds compressive modulus from 9.03 ± 0.36 to 42.82 ± 1.63 MPa. Conventional four-probe analysis confirmed the electrical conductivity of the scaffolds in the semiconductor range. The degradation rate of the samples appeared to be in compliance with bone healing process. The scaffolds exhibited no cytotoxicity using MTT assay against hBM-MSCs. ALP analysis indicated that the PHEMA-Gel-GO scaffolds could efficiently cause the differentiation of hBM-MSCs into osteoblasts after 21 days, even without the addition of the osteogenic differentiation medium. CONCLUSION: Based on the results of this research, it can be stated that the PHEMA-Gel-GO composition is a promising platform for bone tissue engineering.

Lysophosphatidic Acid Alters The Expression of Apoptosis Related Genes and miR-22 in Cultured and Autotransplanted Ovaries.

OBJECTIVE: The aim of this study was to evaluate the effect of lysophosphatidic acid (LPA) on the follicular development, incidence of cell death, and expressions of apoptosis related genes and miR-22 in transplanted ovaries. MATERIALS AND METHODS: In this experimental study, three-week-old mice ovaries were cultured for 24 hours in the presence and absence of LPA, and we assessed cell survival and normal follicular rates in some of the cultured ovaries. The remaining cultured ovaries were autotransplanted in the presence and absence of LPA as four experimental groups (LPA-/LPA-, LPA-/LPA+, LPA+/LPA-, LPA+/LPA+). The follicular development, immunohistochemistry for BAX, and expressions of genes related to apoptosis and miR-22 by real time reverse transcription polymerase chain reaction (RTPCR) were studied at the first oestrous cycles in the recovered ovaries. Sera 17-ß-oestradiol (E2) and progesterone (P4) levels were also assessed. RESULTS: Both cell survival and normal follicular rates were significantly higher in cultured ovaries in the presence of LPA after 24 hours (P<0.05). There was an increase in follicular development in comparison with the intact control group in the four transplanted groups (P<0.05). The LPA+/LPA- group had significantly higher follicular development, a decline in BAX positive cells, and a decrease in pro-apoptotic gene expressions in parallel with enhanced expression of anti-apoptotic and miR-22 genes and higher levels of hormones compared with the non-treated and intact control groups (P<0.05). CONCLUSION: LPA, as a survival factor, improves follicular development in transplanted ovaries by providing a balance between the anti- and pro-apoptotic genes in association with an increase in miR-22 expression.

Effect of Lysophosphatidic Acid on the Vascular Endothelial Growth Factor Expression in Autotransplanted Mouse Ovaries Encapsulated in Sodium Alginate.

Objective: The aim of this study was to evaluate the effect of lysophosphatidic acid (LPA) supplementation during in vitro culture and transplantation of mouse ovaries on the follicular development and expression of vascular endothelial growth factor (VEGF) as an angiogenesis factor at the mRNA and protein levels. Materials and methods: Three weeks old mice ovaries were cultured in the presence and absence of LPA for 24 hours, then they were capsulated in sodium alginate in the presence and absence of LPA as four experimental groups. After transplantation the vaginal smears were performed daily to evaluate the initiation of the estrous cycle. The morphology and follicular distribution were analyzed at the first and fourth estrous cycles using hematoxylin and eosin staining. Then in the groups that showed higher and lower follicular development the immunohistochemistry assay was conducted to identify VEGF protein expression, and the real time RT-PCR was done to analyze the expression of Vegf gene at the first estrus cycle. Results: The large size follicles and also the corpus luteum were prominent in all transplanted groups at fourth estrus cycle in comparison with intact control groups. The statistically lowest percentage of small size follicles and the highest percentages of large size follicles were seen in LPA+/LPA- group (p<0.05). The expression ratio of Vegf to ß-actin was significantly higher in this group in comparison with non-LPA treated and intact control groups (p <0.05). Conclusion: LPA as an angiogenesis factor increases the follicular development in transplanted ovaries but it causes early discharge of ovarian reserve.

Characteristics of a decellularized human ovarian tissue created by combined protocols and its interaction with human endometrial mesenchymal cells.

The present study makes assessments by analyzing the efficacy of combined decellularization protocol for human ovarian fragments. Tissues were decellularized by freeze-thaw cycles, and treated with Triton X-100 and four concentrations (0.1, 0.5, 1 and 1.5%) of sodium dodecyl sulfate (SDS) at two exposure times. The morphology and DNA content of decellularized tissues were analyzed, and the group with better morphology and lower DNA content was selected for further assessments. The Acridine orange, Masson's trichrome, Alcian blue, and Periodic Acid-Schiff staining were used for extracellular matrix (ECM) evaluation. The amount of collagen types I and IV, glycosaminoglycans (GAGs), and elastin was quantified by Raman spectroscopy. The fine structure of the scaffold by scanning electron microscopy was studied. The endometrial mesenchymal cells were seeded onto decellularized scaffold by centrifugal method and cultured for 7 days. After 72 h the treated group with 0.5% SDS showed well-preserved ECM morphology with the minimum level of DNA (2.23% ± 0.08). Raman spectroscopy analysis confirmed that, the amount of ECM components was not significantly decreased in the decellularized group (P < 0.001) in comparison with native control. The electron micrographs demonstrated that the porosity and structure of ECM fibers in the decellularized group was similar to native ovary. The endometrial mesenchymal cells were attached and penetrated into the decellularized scaffold. In conclusion this combined protocol was an effective method to decellularize human ovarian tissue with high preservation of ECM contents, and human endometrial mesenchymal cells which successfully interacted with this created scaffold.

In Vitro Implantation Model Using Human Endometrial SUSD2+ Mesenchymal Stem Cells and Myometrial Smooth Muscle Cells.

OBJECTIVE: This study evaluated a novel in vitro implantation model using human endometrial mesenchymal stem cells (EMSCs), SUSD2+, and myometrial smooth muscle cells (SMCs) that were co-cultured with mouse blastocysts as the surrogate embryo. MATERIALS AND METHODS: In this experimental study, SUSD2+ MSCs were isolated from human endometrial cell suspensions (ECS) at the fourth passage by magnetic-activated cell sorting. The ECS and SUSD2+ cells were separately co-cultured with human myometrial muscle cells for five days. After collection of mouse blastocysts, the embryos were placed on top of the co-cultured cells for 48 hours. The interaction between the embryo and the cultured cells was assessed morphologically at the histological and ultrastructural levels, and by expression profiles of genes related to implantation. RESULTS: Photomicrographs showed that trophoblastic cells grew around the embryonic cells and attached to theECS and SUSD2+ cells. Ultrastructural observations revealed pinopode and microvilli-like structures on the surfaces of both the ECS and SUSD2+ cells. Morphologically, the embryos developed to the egg-cylinder stage in both groups. Gene expression analysis showed no significant differences between the two groups in the presence of an embryo, but an increased expression of αV was detected in SUSD2+ cells compared to ECS cells in the absence of an embryo. CONCLUSION: This study showed that SUSD2+ cells co-cultured with SMCs could interact with mouse embryos. The co-cultured cells could potentially be used as an implantation model.

Follicular development and the expression of BAX and vascular endothelial growth factor in transplanted ovaries in uni- and bilateral ovariectomized mice: An experimental study.

BACKGROUND: Several conflicting results have been reported on the survival and function of transplanted ovaries. OBJECTIVE: Evaluation of the follicular development and the expression of vascular endothelial growth factor (VEGF) and Bcl-2-associated X protein (BAX) in ovaries transplanted into uni- and bilaterally ovariectomized mice. MATERIALS AND METHODS: In this experimental study, 40 female NMRI mice (21-days-old, 12-15 gr) were ovariectomized uni- and bilaterally (n = 20/ group), while the 8-wk-old mice were considered as intact control group (n = 6). 5 weeks after transplantation at the proestrus stage, the morphology of recovered transplanted ovaries and the proportion of follicles were studied at different developmental stages. The apoptosis cell death by pro-apoptotic protein BAX and the expression of VEGF were evaluated using immunohistochemistry. RESULTS: In the bilaterally ovariectomized mice, among the 455 counted normal follicles, a lower rate of primordial and primary follicles and a higher rate of preantral and antral follicles were observed (p = 0.002). However, the percentages of preantral and antral follicles, and the corpus luteum were significantly lower in the intact control group (among the 508 counted normal follicles in this group) compared to other transplanted groups (p = 0.002). The number of BAX-positive cells in all groups was not significantly different. The VEGF expression was prominent in vessels of the corpus luteum, and also in the theca layer of large follicles of studied groups. CONCLUSION: Early discharge of ovarian reserve was prominent in the bilaterally ovariectomized group but the incidence of apoptotic cells and VEGF expression as angiogenic factor did not differ in both ovariectomized mice. Thus, unilaterally ovariectomy has less side effects on the ovarian reserve compared to bilateral ovariectomy.

The Effect of Sodium Selenite on Expression of Mitochondrial Transcription Factor A during In Vitro Maturation of Mouse Oocyte.

BACKGROUND: The aim of the present study was to investigate the effect of Sodium Selenite (SS) supplemented media on oocyte maturation, expression of mitochondrial transcription factor A (TFAM) and embryo quality. METHODS: Mouse Germinal Vesicle (GV) oocytes were collected after administration of Pregnant Mare Serum Gonadotropin (PMSG); in experimental group 1, oocytes were cultured and then subjected for in vitro maturation in the absence of SS, and in experimental group 2, they were matured in vitro in the presence of 10 ng/ml of SS up to 16 hr. The control group included MII oocytes obtained from the fallopian tubes after ovarian stimulation with PMSG, followed by human chorionic gonadotropin. Then, the expression of TFAM in MII oocytes in all three groups was investigated using real-time RT-PCR. The fertilization and embryo developmental rates were assessed, and finally the quality of the blastocysts was evaluated using propidium iodide staining. RESULTS: The oocyte maturation rate to MII stage in SS treated group was significantly higher than non-treated oocytes (75.65 vs. 68.17%, p<0.05). Also, the rates of fertilization, embryo development to blastocyst stage as well as the cell number of blastocyst in SS supplemented group were higher than other experimental group (p<0.05). There was a significant decrease in TFAM gene expression in both in vitro groups compared to the group with in vivo obtained oocytes (p<0.05). Moreover, there was a significant increase in TFAM gene expression in oocytes that matured in the presence of SS compared to that of the group without SS (p<0.05). CONCLUSION: Supplementation of oocyte maturation culture media with SS improved the development rate of oocytes and embryo and also enhanced TFAM expression in MII oocytes which can affect the mitochondrial biogenesis of oocytes.

An efficient protocol for decellularization of the human endometrial fragments for clinical usage.

The present study was aimed to compare different decellularization protocols for human endometrial fragments. The freeze-thaw cycles in combination with treatment by Triton X-100 and four concentrations of sodium dodecyl sulfate (SDS; 0.1, 0.5, 1, and 1.5%) with two exposure times (24 and 72 h) were applied for tissues decellularization. After analysis the morphology and DNA content of tissues the group with better morphology and lower DNA content was selected for further assessments. The nucleus by Acridine orange and extracellular matrix (ECM) using Masson's trichrome, Alcian blue, and periodic acid-Schiff staining were studied. The amount of tissues collagen types I and IV, fibronectin, glycosaminoglycans (GAGs), and elastin was analyzed by Raman spectroscopy. The ultrastructure and porosity of decellularized scaffold were studied by scanning electron microscopy (SEM). The MTT assay was applied for assessments of cytotoxicity of scaffold. The treated group with 1% SDS for 72 h showed the morphology similar to native control in having the minimum level of DNA and well preserved ECM. Raman spectroscopy results demonstrated, the amount of collagen types I and IV, GAG, and fibronectin was not significantly different in decellularized scaffold compared with native group but the elastin protein level was significantly decreased (P < 0.001). SEM micrographs also showed a porous and fiber rich ECM in decellularized sample similar to the native control. This combined protocol for decellularization of human endometrial tissue is effective and it could be suitable for recellularization and clinical applications in the future.

The Effects of Ovarian Encapsulation on Morphology and Expression of Apoptosis-Related Genes in Vitrified Mouse Ovary.

BACKGROUND: The purpose of this study was to determine the effects of alginate hydrogel as a capsule to protect the ovary against possible detrimental effects of vitrification and warming on morphology and expression of apoptosis-related genes in the mouse ovary. METHODS: In this experimental study, the ovaries from twenty-five female 8-week-old mice were divided into five groups of non-vitrified ovaries, vitrified ovaries, ovaries that were encapsulated with concentrations of 0.5, 0.75 and 1% of alginate hydrogel. The morphological study was performed using hematoxylin and eosin staining. Expression levels of apoptosis-associated genes were quantified in each group by real-time RT-PCR. The one-way ANOVA and post hoc test were used to analyze the data and values of p<0.05 were considered statistically significant. RESULTS: The results of follicle count showed that the mean of total follicles in all groups was not significantly different. The average number of atretic follicles in vitrified and experimental groups significantly increased in comparison with the nonvitrified group (p=0.001). The results of the evaluation of apoptosis-related genes showed that the ratio of BAX/BCL-2 in experimental groups 1 and 2 was significantly higher than the vitrified group and experimental group 3 (p=0.000). The expression level of caspase 3 gene was not significantly different among all groups. CONCLUSION: Ovarian encapsulation with used concentrations of alginate hydrogel failed to improve the morphology and molecular aspects of follicles and it was not able to better preserve the intact follicles of vitrified ovaries. However, morphological and molecular findings appear to improve with increasing alginate hydrogel concentration.

The effect of sodium selenite on apoptotic gene expression and development of in vitro cultured mouse oocytes in comparison with in vivo obtained oocytes.

In vitro maturation (IVM) of oocytes is widely used in assisted reproduction technologies. The present study aimed to improve the in vitro oocyte maturation and its development through enriching the culture media with sodium selenite (SS). Moreover, the effects of SS on the expression of the oocytes apoptosis-related genes were assessed. In this study, male and female NMRI mice were used and after collecting their germinal vesicle (GV) oocytes, they were cultured with SS (experimental group) and without SS (control group). Collected metaphase II oocytes (MII) from the fallopian tube were considered as in vivo group. After in vitro culture, the oocytes were assessed in terms of nuclear maturation. The MII oocytes were inseminated and the development was examined until the blastocyst stage. Also, oocytes were subjected to the molecular analysis for evaluating the expression of BAX, BCL2, P53, and BAD genes using the real-time RT-PCR. The maturation rate was significantly increased in the SS supplemented group compared to the control one. The developmental rate of the embryos was significantly higher for both of the in vivo and SS supplemented groups rather than the control one, however, no significant difference was seen between these rates of the experimental and in vivo groups. Real-time RT-PCR did not show any significant differences in the expression of the apoptosis-related genes for all of the studied groups. The p53 gene was not expressed in any of groups. Sodium selenite improved the oocyte developmental competence but did not change the expression of the apoptosis-related genes in MII oocytes.