Publications by authors named "Mojdeh Salehnia"

79 Publications

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

Cell J 2021 Jul 26;23(2):154-163. Epub 2021 May 26.

Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran. Email:

Objective: This study evaluated a novel 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.
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http://dx.doi.org/10.22074/cellj.2021.6979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8181319PMC
July 2021

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

Prog Biomater 2021 May 21. Epub 2021 May 21.

Anatomy Department, Faculty of Medical Sciences, Tarbiat Modares University, 14115-111, Tehran, Iran.

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.
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http://dx.doi.org/10.1007/s40204-021-00156-5DOI Listing
May 2021

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

Avicenna J Med Biotechnol 2021 Apr-Jun;13(2):81-86

Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.

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 maturation in the absence of SS, and in experimental group 2, they were matured in the presence of 10 of SS up to 16 . 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 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 . 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 gene expression in both groups compared to the group with obtained oocytes (p<0.05). Moreover, there was a significant increase in 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 expression in MII oocytes which can affect the mitochondrial biogenesis of oocytes.
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http://dx.doi.org/10.18502/ajmb.v13i2.5526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8112142PMC
May 2021

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

Int J Reprod Biomed 2021 Apr 22;19(4):361-370. Epub 2021 Apr 22.

Anatomy Department, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.

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.
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http://dx.doi.org/10.18502/ijrm.v19i4.9062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106821PMC
April 2021

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

J Reprod Infertil 2021 Jan-Mar;22(1):23-31

Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.

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.
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http://dx.doi.org/10.18502/jri.v22i1.4992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7903669PMC
March 2021

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

Vet Res Forum 2020 15;11(4):377-383. Epub 2020 Dec 15.

Prevention of Metabolic Research Disorder Center, Research Institute for Endocrine Disorder, Shahid Beheshti University of Medical Science, Tehran, Iran.

maturation (IVM) of oocytes is widely used in assisted reproduction technologies. The present study aimed to improve the 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 group. After 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 and SS supplemented groups rather than the control one, however, no significant difference was seen between these rates of the experimental and 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.
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http://dx.doi.org/10.30466/vrf.2018.93471.2255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904118PMC
December 2020

Supplementation of Culture Media with Lysophosphatidic Acid Improves The Follicular Development of Human Ovarian Tissue after Xenotransplantaion into The Back Muscle of γ-Irradiated Mice.

Cell J 2020 Oct 15;22(3):358-366. Epub 2019 Dec 15.

Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran. Electronic Address:

Objective: The aim of the present study was to evaluate the effects of lysophosphatidic acid (LPA) supplementation of human ovarian tissue culture media on tissue survival, follicular development and expression of apoptotic genes following xenotransplantation.

Materials And Methods: In this experimental study, human ovarian tissue was collected from eight normal female to male transsexual individuals and cut into small fragments. These fragments were vitrified-warmed and cultured for 24 hours in the presence or absence of LPA, then xenografted into back muscles of γ-irradiated mice. Two weeks post-transplantation the morphology of the recovered tissues were evaluated by hematoxylin and eosin staining. The expression of genes related to apoptosis ( and ) were analyzed by real time revers transcription polymerase chain reaction (RT-PCR) and detection of BAX protein was done by immunohistochemical staining.

Results: The percent of normal and growing follicles were significantly increased in both grafted groups in comparison to the non-grafted groups, however, these rates were higher in the LPA-treated group than the non-treated group (P<0.05). There was a higher expression of the anti-apoptotic gene, BCL2, but a lower expression of the pro-apoptotic gene, BAX, and a significant lower BAX/ BCL2 ratio in the LPA-treated group in comparison with non-treated control group (P<0.05). No immunostaining positive cells for BAX were observed in the follicles and oocytes in both transplanted ovarian groups.

Conclusion: Supplementation of human ovarian tissue culture medium with LPA improves follicular survival and development by promoting an anti-apoptotic balance in transcription of and genes.
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http://dx.doi.org/10.22074/cellj.2020.6752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6947004PMC
October 2020

Folliculogenesis-Associated Genes Expression in Human Vitrified Ovarian Tissue after Xenotransplantation in γ-Irradiated Mice.

Cell J 2020 Oct 15;22(3):350-357. Epub 2019 Dec 15.

Department of Anatomical Sciences, Faculty of Medicine, Tarbiat Modares University, Tehran, Iran. Electronic Address:

Objective: Autograft transplantation of vitrified cortical ovarian tissue is an acceptable clinical technique for fertility preservation in women. Xenograft transplantation into animal models could be useful for evaluating the safety of human vitrified ovarian tissue. This study targeted to evaluate impact of vitrification on expression of the genes associated with folliculogenesis after xenograft transplantation of human vitrified ovarian tissue to γ-irradiated mice.

Materials And Methods: In this experimental study, ovarian biopsies were gathered from six transsexual persons. The cortical section of ovarian biopsies was separated and chopped into small pieces. These pieces were randomly divided into vitrified and non-vitrified groups. In each group some pieces were considered as non-transplanted tissues and the others were transplanted to γ-irradiated female National Medical Research Institute (NMRI) mice. Before and after two weeks of xenograft transplantation, histological assessment and evaluation of the expression of folliculogenesisassociated genes ( and ) were performed in both vitrified and non-vitrified groups.

Results: Percentage of the normal follicles and expression of the all examined genes from transplanted and nontransplanted tissue were similar in both vitrified and non-vitrified groups (P>0.05). After transplantation, the normal follicle rate was significantly decreased and among the folliculogenesis-associated genes, expression of gene was significantly increased, rather than before transplantation in vitrified and non-vitrified tissues (P<0.05).

Conclusion: The vitrification method using dimethyl solphoxide and ethylene glycol (EG) had no remarkable effect on the normal follicular rate and expression of folliculogenesis-associated genes after two weeks human ovarian tissue xenografting. In addition, transplantation process can cause a significant decrease in normal follicular rate and expression of gene.
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http://dx.doi.org/10.22074/cellj.2020.6553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6947005PMC
October 2020

Analysis of Apoptosis in Cultured Human Vitrified Ovarian Tissue in the Presence of Leukemia Inhibitory Factor.

J Reprod Infertil 2018 Oct-Dec;19(4):193-202

Department of Anatomical Sciences, Medical Sciences Faculty, Tarbiat Modares University, Tehran, Iran.

Background: For improving the human ovarian tissue culture, this study was designed to assess the incidence of apoptosis in this tissue following vitrification and culture in the presence of leukemia inhibitory factor (LIF) as an anti-apoptotic factor.

Methods: After collecting the ovarian tissue samples they were divided into non-vitrified and vitrified groups and cultured for 14 days in the presence and absence of LIF then morphological, ultrastructural and steroidogenesis studies, TUNEL and caspase-3/7 assays, and apoptosis analysis by real time RT-PCR were done in all groups. The data were analyzed by independent t-tests and the real time RT-PCR results were compared by one-way ANOVA (p-values of <0.05 were considered significant).

Results: No significant difference was observed between non-vitrified and vitrified groups in normality rate of follicles, the levels of hormones, TUNEL positive cells and caspase-3/7 activity. But in all LIF-treated groups, the levels of 17-β estradiol and progesterone were higher and TUNEL signals and caspase-3/7 activity were lower than non-LIF treated groups. The expression of Fas and FasL genes was higher in vitrified group in comparison with non-vitrified group but the expression of other genes was not significantly different. In LIF-treated groups, the expression of pro-apoptotic genes was significantly lower and the expression of anti-apoptotic genes was higher than non-LIF treated group.

Conclusion: The vitrification of human ovarian tissue did not increase the incidence of apoptosis at the morphological and molecular levels during long term culture and LIF improves the survival and development of cultured follicles.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328982PMC
February 2019

The mitochondrial DNA copy number, cytochrome c oxidase activity and reactive oxygen species level in metaphase II oocytes obtained from in vitro culture of cryopreserved ovarian tissue in comparison with in vivo-obtained oocyte.

J Obstet Gynaecol Res 2018 Oct 6;44(10):1937-1946. Epub 2018 Aug 6.

Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran.

Aim: To evaluate the mitochondrial DNA (mtDNA) copy number, reactive oxygen species (ROS) level and intensity of mitochondrial enzyme activity in metaphase II oocytes derived from vitrified cultured immature mouse ovarian tissue in comparison with nonvitrified group and in vivo-obtained oocytes.

Methods: Vitrified and nonvitrified ovaries from neonate female mice were cultured for 7 days. Then, preantral follicles were isolated and cultured in a three-dimensional culture system. Follicular development and oocyte maturation were evaluated and compared in both groups. Some of the collected metaphase II oocytes derived from in vitro and in vivo conditions were inseminated with capacitated spermatozoa, and then, the fertilization and embryo developmental rates were assessed. In the other series of oocytes, mtDNA copy number, distribution and enzyme activity and ROS level were analyzed.

Results: The embryo development, mtDNA copy number and mitochondrial enzyme activity in collected metaphase II oocytes from two in vitro-cultured groups were significantly lower, and the ROS level was higher than those of the in vivo group (P < 0.05), but there was no significant difference between vitrified and nonvitrified groups.

Conclusion: This study showed that a two-step in vitro culture of mouse ovarian tissue decreased the mtDNA copy number and cytochrome c oxidase activity of metaphase II oocytes through an increase in their ROS level in comparison with in vivo-obtained oocytes. Thus, the in vitro culture methods should be improved.
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http://dx.doi.org/10.1111/jog.13747DOI Listing
October 2018

Reactive oxygen species level, mitochondrial transcription factor A gene expression and succinate dehydrogenase activity in metaphase II oocytes derived from cultured vitrified mouse ovaries.

Vet Res Forum 2018 15;9(2):145-152. Epub 2018 Jun 15.

Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.

The aim of this study was to evaluate the effects of ovarian tissue vitrification and two-step culture on the metaphase II (MII) oocyte reactive oxygen species (ROS) level, mitochondrial transcription factor A TFAM) expression and succinate dehydrogenase (SDH) activity. After collection of neonatal mouse ovaries, 45 ovaries were vitrified and the others (n = 45) were considered as control. All ovaries were cultured for seven days, and their isolated preantral follicles were cultured in three-dimensional culture system. After 12 days, the follicular development and oocyte maturation were evaluated and compared in vitrified and non-vitrified ovaries. The collected MII oocytes were inseminated with capacitated spermatozoa. Then, the fertilization, embryonic development, ROS level, TFAM gene expression and SDH activity of oocytes were assessed and compared. There was no significant difference between morphology and percentage of normal follicles between vitrified and non-vitrified ovaries at the beginning of culture. The follicular development and hormone level in the vitrified group was significantly lower than non-vitrified group and the ROS concentration in the vitrified group was significantly higher than non-vitrified group after one-week culture. After follicular culture, there was no significant difference in follicular development, oocyte maturation, fertilization rate, TFAM gene expression, ROS level and mitochondrial SDH activity between the groups. This study showed that ovarian tissue vitrification influences the follicular development through increase in ROS level during culture but these harmful effects may be recovered during the follicular culture period. Thus, vitrification and ovarian culture method should be improved.
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http://dx.doi.org/10.30466/VRF.2018.30824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047572PMC
June 2018

The Effects of Lysophosphatidic Acid on The Incidence of Cell Death in Cultured Vitrified and Non-Vitrified Mouse Ovarian Tissue: Separation of Necrosis and Apoptosis Border.

Cell J 2018 Oct 15;20(3):403-411. Epub 2018 May 15.

Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran.

Objective: The aim of the present study was to examine whether lysophosphatidic acid (LPA) could decrease cell death and improve in vitro culture (IVC) conditions in cultured vitrified mouse ovarian tissue.

Materials And Methods: In this experimental study, we collected and randomly divided 7-day-old mouse ovarian tissues into vitrified and non-vitrified groups. The ovaries were cultured in the presence and absence of LPA for one week. Morphology and follicular development were evaluated by hematoxylin and eosin (H&E) and Masson's trichrome (MTC) staining. The incidence of cell death was assessed by flow cytometry using annexin V/propidium iodide (PI) and a caspase-3/7 assay in all studied groups.

Results: The vitrified groups had a significantly decreased follicle developmental rate compared to the non-vitrified groups (P<0.05). Overall, qualitative and quantitative results showed prominent follicular degeneration in the vitrified groups compared with the respective non-vitrified groups. Both LPA treated groups had a significantly higher proportion of preantral follicles compared to the non-LPA treated groups (P<0.05). Flow cytometry analysis results showed significantly greater early and late apoptotic cells in all groups (17.83 ± 8.80%) compared to necrotic cells (7.97 ± 0.92%, P<0.05). The percentage of these cells significantly increased in the vitrified groups compared with non-vitrified groups. LPA treated groups had a lower percentage of these cells compared to non-LPA treated groups (P<0.05). The lower enzyme activity was observed in non-vitrified (especially in the LPA+ groups) cultured ovaries compared to the vitrified group (P<0.05).

Conclusion: Both vitrification and IVC adversely affected cell survival and caused cell death. We postulated that LPA supplementation of culture medium could improve the developmental rate of follicles and act as an anti-cell death factor in non-vitrified and vitrified ovarian tissues. It could be used for in vitro maturation of ovarian tissue.
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http://dx.doi.org/10.22074/cellj.2018.5180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6005000PMC
October 2018

The Effects of Sodium Selenite on Mitochondrial DNA Copy Number and Reactive Oxygen Species Levels of In Vitro Matured Mouse Oocytes.

Cell J 2018 Oct 15;20(3):396-402. Epub 2018 May 15.

Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran. Electronic Address:

Objective: The aim of present study is to determine the effects of supplementation of oocyte maturation medium with sodium selenite (SS) on oocyte mitochondrial DNA (mtDNA) copy number and reactive oxygen species (ROS) levels.

Materials And Methods: In this experimental study, germinal vesicle (GV), metaphase I (MI), and metaphase II (MII) stage oocytes were recovered from 6-8 week old female mice after superovulation. Some of the GV oocytes were cultured and matured in the presence and absence of SS. Then in vivo and in vitro matured (IVM) oocytes were subjected to mitochondria staining by MitoTracker green, ROS analysis, and mtDNA copy number determination using absolute real-time polymerase chain reaction (PCR).

Results: The maturation rate of GV oocytes to the MII stage significantly increased in the SS supplemented group (79.25%) compared to the control group (72.46%, P<0.05). The intensity of mitochondrial staining was not different among the studied groups, whereas the mitochondria distribution in the cytoplasm of the IVM oocytes showed some aggregation pattern. The in vivo obtained MII oocytes had lower ROS levels and higher mtDNA copy numbers than IVM-MII oocytes (P<0.05). The SS supplemented group had significantly lower ROS levels and higher mtDNA copy numbers than the non-treated group (P<0.05).

Conclusion: SS increased oocyte mtDNA copy number by decreasing oxidative stress. SS had an association with better oocyte developmental competence.
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http://dx.doi.org/10.22074/cellj.2018.5430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004999PMC
October 2018

Morphological, Ultrastructural, and Molecular Aspects of In Vitro Mouse Embryo Implantation on Human Endometrial Mesenchymal Stromal Cells in The Presence of Steroid Hormones as An Implantation Model.

Cell J 2018 Oct 15;20(3):369-376. Epub 2018 May 15.

Reproductive Health Research Center, Tehran University of Medical Sciences, Tehran, Iran. Electronic

Objective: This experimental study aimed to evaluate the effects of 17β-estradiol (E2) and progesterone (P4) on the interaction between mouse embryo and human endometrial mesenchymal stromal cells, and gene expressions related to implantation [αV and β3 integrins, interleukin-1 receptor (IL-1R), and leukemia inhibitory factor receptor (LIFR)] using an in vitro twodimensional model.

Materials And Methods: In this experimental study, the endometrial stromal cells were isolated enzymatically and mechanically, and cultured to the fourth passage. Next, their immunophenotype was confirmed by flow cytometric analysis as mesenchymal stromal cells. The cells were cultured as either the experimental group in the presence of E2 (0.3 nmol) and P4 (63.5 nmol) or control group without any hormone treatment. Mouse blastocysts were co-cultured with endometrial mesenchymal stromal cells in both groups for 48 hours. Their interaction was assessed under an inverted microscope and scanning electron microscopy (SEM). Expressions of αV and β3 integrins, LIFR, and IL-1R genes were analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR).

Results: Similar observations were seen in both groups by light microscopy and SEM. We observed the presence of pinopode-like structures and cell secretions on the apical surfaces of endometrial mesenchymal stromal cells in both groups. The trophoblastic cells expanded and interacted with the mesenchymal monolayer cells. At the molecular level, expression of IL-1R significantly increased in the hormonal treated group compared to the control (P≤0.05). Expressions of the other genes did not differ.

Conclusion: This study has shown that co-culture of endometrial mesenchymal stromal cells with mouse embryo in media that contained E2 (0.3 nmol) and P4 (63.5 nmol) could effectively increase the expression of IL-1R, which is involved in embryo implantation. However, there were no significant effects on expressions of αV and β3 integrins, LIFR, and on the morphology and ultrastructure of endometrial mesenchymal stromal cells.
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http://dx.doi.org/10.22074/cellj.2018.5221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004996PMC
October 2018

Effect of lysophosphatidic acid on the follicular development and the expression of lysophosphatidic acid receptor genes during in vitro culture of mouse ovary.

Vet Res Forum 2018 15;9(1):59-66. Epub 2018 Mar 15.

Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran.

Lysophosphatidic acid (LPA) known as a serum-derived growth factor, is involved in several cell physiological functions in the female reproductive system including: oocyte maturation, fertilization and embryo implantation by its transmembrane G protein-coupled receptors. The aim of the present study was to examine the effect of LPA on follicular development of mouse ovarian tissue. Neonatal mouse ovarian tissues were cultured in five different concentrations of LPA (0, 5, 10, 20 and 40 µM). The developmental competence and the function of cultured ovarian tissue were assessed by morphological study using hematoxylin and eosin staining and hormonal analysis. The expression of LPA receptor (LPAR 1-4) genes were analyzed by real-time RT-PCR. The proportion of preantral follicles and the level of E hormone were significantly higher in the 20 µM LPA-treated group than those in the other treatment groups. There was a significant difference in the expression of LPAR 1-4 genes in 20 µM LPA treated group in comparison with 0 µM LPA (control group) treated and non-cultured groups. In addition, the expression of LPAR1 gene was higher than other receptor genes in all studied groups. In conclusion supplementation of the media with 20 µM LPA, could improve the survival and developmental potential of follicles and it had positive effects on cell function and stimulation of E synthesis in mouse whole ovarian tissues.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5913562PMC
March 2018

Vitrification and in vitro culture had no adverse effect on the follicular development and gene expression of stimulated human ovarian tissue.

J Obstet Gynaecol Res 2018 Mar 5;44(3):474-487. Epub 2018 Jan 5.

Reproductive Health Research Center, Tehran University of Medical Sciences, Tehran, Iran.

Aim: The study assesses the effect of the vitrification procedure on the integrity, morphology, follicular development and gene expression of stimulated human ovarian tissue after warming and two weeks of in vitro culture.

Methods: Ovarian specimens were divided into non-vitrified and vitrified groups and were cultured for two weeks. Morphological analysis and immunohistochemistry were performed. The 17-β estradiol and anti-Müllerian hormone levels in collected media were assessed. Gene expression was analyzed using real-time reverse transcription polymerase chain reaction.

Results: The morphology and immunohistochemistry of bcl-2-like protein 4 and B-cell lymphoma 2 of human stimulated ovarian tissue were similar in both groups. There was no significant difference in the percentage of normal follicles between the groups before and after in vitro culture. In spite of an increase in the percentage of growing follicles in cultured tissues compared to the non-cultured groups, the rate of normal follicles was significantly decreased in both cultured groups (P < 0.05). Gene expression was no different in vitrified tissues compared to the control; however, the expression of growth differentiation factor 9 and follicle stimulating hormone receptor genes were increased and factor in germ line alpha and kit ligand genes were decreased during in vitro culture (P < 0.05). In the two cultured groups, the level of 17-β estradiol was increased (P < 0.05), but the anti-Müllerian hormone concentration was not statistically altered.

Conclusions: These results showed that the integrity of stimulated human ovarian tissue after vitrification/warming was well preserved; however, the in vitro culture condition needs improvement.
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http://dx.doi.org/10.1111/jog.13530DOI Listing
March 2018

Vitrification of Mouse MII Oocyte Decreases the Mitochondrial DNA Copy Number, TFAM Gene Expression and Mitochondrial Enzyme Activity.

J Reprod Infertil 2017 Oct-Dec;18(4):343-351

Department of Biotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.

Background: The objective of this study was determination of the changes in the reactive oxygen species (ROS) level, mitochondrial DNA (mtDNA) copy number and enzyme activity and transcription factor A (TFAM) gene expression in oocytes after vitrification.

Methods: The oocytes at metaphase II (MII) stage (n=320) were collected from super-ovulated adult female mice (n=40). These oocytes were divided into vitrified and non-vitrified groups (n=160 in each group). After vitrification of oocytes, ROS level, mtDNA copy number; TFAM gene expression and mitochondrial enzymes activity (cytochrome C oxidase and succinate dehydrogenase) were assessed and compared with non-vitrified group. Visualization of the mitochondria was done using Mitotracker green staining under confocal microscope. Data were compared by independent T-test. Values of p<0.05 were considered as statistically significant.

Results: The survival rate of oocytes after vitrification and warming was 96.05%. The intensity of cytochrome C oxidase activity, mtDNA copy number and TFAM gene expression in non-vitrified oocytes were significantly lower and the level of ROS was higher in vitrified oocytes in comparison with non-vitrified group (p<0.05). But the intensity of succinate dehydrogenase activity was not significantly different between the two groups. The pattern of mitochondrial distribution in two groups of study was similar but the intensity of Mitotracker green in non-vitrified oocytes was significantly higher than vitrified oocytes (p<0.05).

Conclusion: This study showed that vitrification of mouse MII oocytes reduced the mtDNA copy number and mitochondrial cytochrome C oxidase activity by increasing ROS level, thus the subsequent embryo development may be affected.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5691250PMC
December 2017

Morphological and Molecular Aspects of In Vitro Culture of Preantral Follicles Derived from Vitrified Ovarian.

Cell J 2017 Oct 19;19(3):332-342. Epub 2017 Aug 19.

Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran.

Objective: This study aimed to evaluate the expression of the genes related to folliculogenesis after vitrification of mouse ovarian tissues using a two-step in vitro culture.

Materials And Methods: In this experimental study, vitrified and non-vitrified ovaries from 7- day old (neonate) female mice were cultured using alpha-Minimum Essential Medium (α-MEM) supplemented with 5% fetal bovine serum (FBS) for 7 days. Morphology, surface area of ovaries and percentage of normal follicles were evaluated and compared in both groups. After one-week culture, in non-vitrified group, preantral follicles of cultured ovaries were isolated and cultured in a three-dimensional alginate culture system for 12 days. Then, the collected metaphase (M) II oocytes were inseminated with capacitated spermatozoa derived from 7-8-week old (adult) male NMRI mice. Follicular diameter, oocyte maturation, fertilization, embryo development and the expression of genes related to follicular development (Pcna, Fshr and Cyp17a1,) using real time reverse transcription-polymerase chain reaction (RT-PCR) were assessed at the end of last culture period in both groups.

Results: The ovarian area in vitrified group (162468.20 703.78) was less than non-vitrified group (297211.40 6671.71), while the percentage of preantral follicles in vitrified group (18.40%) was significantly lower than those of non-vitrified group (24.50%) on day 7 of culture (P>0.05). There were no significant differences between the two groups in terms of follicular diameter, expression of genes related to development of follicles, oocyte maturation, fertilization, as well as embryo development (P>0.05).

Conclusion: The results of this study showed that vitrification of ovarian tissue following in vitro culture had negative impact on the survival and development of follicles within the tissue. However, no significant alterations were observed in development, gene expression and hormonal production of in vitro culture of isolated follicles derived from vitrified ovarian tissues as compared to the non-vitrified samples.
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http://dx.doi.org/10.22074/cellj.2017.4264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570399PMC
October 2017

Leukemia inhibitory factor increases the proliferation of human endometrial stromal cells and expression of genes related to pluripotency.

Int J Reprod Biomed 2017 Apr;15(4):209-216

Anatomy Department, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.

Background: Concerning the low population of human endometrial mesenchymal cells within the tissue and their potential application in the clinic and tissue engineering, some researches have been focused on their in vitro expansion.

Objective: The aim of this study was to evaluate the effect of leukemia inhibitory factor (LIF) as a proliferative factor on the expansion and proliferation of human endometrial stromal cells.

Materials And Methods: In this experimental study, the isolated and cultured human endometrial stromal cells from women at ovulatory phase aged 20-35 years, after fourth passage were divided into control and LIF-treated groups. In the experimental group, the endometrial cells were treated by 10 ng/ml LIF in culture media and the cultured cells without adding LIF considered as control group. Both groups were evaluated and compared for proliferation rate using MTT assay, for CD90 marker by flow cytometric analysis and for the expression of Oct4, Nanog, PCNA and LIFr genes using real-time RT-PCR.

Results: The proliferation rate of control and LIF-treated groups were 1.17±0.17 and 1.61±0.06 respectively and there was a significant increase in endometrial stromal cell proliferation following in vitro treatment by LIF compared to control group (p=0.049). The rate of CD90 positive cells was significantly increased in LIF-treated group (98.96±0.37%) compared to control group (94.26±0.08%) (p=0.0498). Also, the expression ratio of all studied genes was significantly increased in the LIF-treated group compared to control group (p=0.0479).

Conclusion: The present study showed that LIF has a great impact on proliferation, survival, and maintenance of pluripotency of human endometrial stromal cells and it could be applicable in cell therapies.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555038PMC
April 2017

Retraction Note: Assessment of the influence of whole body vibration on Cochlear function.

J Occup Med Toxicol 2017 29;12:17. Epub 2017 Jun 29.

Department of Audiology, School of Rehabilitation, Iran University of Medical Sciences (IUMS), Tehran, Iran.

[This retracts the article DOI: 10.1186/1745-6673-7-12.].
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http://dx.doi.org/10.1186/s12995-017-0162-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5492715PMC
June 2017

In-vitro construction of endometrial-like epithelium using CD146 mesenchymal cells derived from human endometrium.

Reprod Biomed Online 2017 Sep 15;35(3):241-252. Epub 2017 Jun 15.

Midwifery Department, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.

Endometrial CD146 cells were purified, using magnetic activated cell sorting, and then embedded and cultured in a collagen-matrigel scaffold on top of myometrial smooth muscle cells for 10 days. At the end of culture period, the differentiation and formation of the epithelial-like cells were confirmed by morphological and ultrastructural evaluations, and analysis by reverse transcription polymerase chain reaction of the specific expression of genes: osteopontin (SPP1), matrix metalloproteinase 2, zonula occludens 1, laminin alpha 2 and collagen type IV; and by western blotting of CD9 protein. The results showed that the human endometrial mesenchymal CD146 cells were able to produce endometrial glandular tube-like structures in vitro. Ultrastructural observation revealed some projections on the apical surfaces, appearance of basal lamina-like structures on the basal surface, and tight junctions and desmosomes on the lateral surfaces of the epithelial-like cells. The expression of studied genes at RNA level and CD9 at protein level confirmed the formation of endometrial epithelial-like cells. This culture system may have potential applications in cell therapy and in studies on human embryo implantation.
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http://dx.doi.org/10.1016/j.rbmo.2017.05.020DOI Listing
September 2017

Short Term Culture of Vitrified Human Ovarian Cortical Tissue to Assess the Cryopreservation Outcome: Molecular and Morphological Analysis.

J Reprod Infertil 2017 Jan-Mar;18(1):162-171

Reproductive Health Research Center, Tehran University of Medical Sciences, Tehran, Iran.

Background: The aim of the present study was to evaluate the effectiveness of human ovarian vitrification protocol followed with culture at the morphological and molecular levels.

Methods: Ovarian tissues were obtained from 10 normal transsexual women and cut into small pieces and were divided into non-vitrified and vitrified groups and some of the tissues fragments in both groups were randomly cultured for two weeks. The morphological study using hematoxylin and eosin and Masson's trichrome staining was done. The analysis of mean follicular density, 17-β estradiol (E2) and anti mullerian hormone (AMH), and real-time RT-PCR was down for the evaluation of expression of genes related to folliculogenesis. Data were compared by paired-samples and independent-samples T test. Values of p<0.05 were considered statistically significant.

Results: The proportion of normal follicles did not show significant difference between vitrified and non-vitrified groups before and after culture but these rates and the mean follicle density significantly decreased in both cultured tissues (p<0.05). The expression of genes was similar in vitrified and non-vitrified groups but in cultured tissues the expression of GDF9 and FSHR genes increased and the expression of FIGLA and KIT-L genes decreased (p<0.05). An increase in E2 and AMH concentration was observed after 14 days of culture in both groups.

Conclusion: In conclusion, the present study indicated that the follicular development and gene expression in vitrified ovarian tissue was not altered before and after culture, thus this method could be useful for fertility preservation; however, additional studies are needed to improve the culture condition.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5359853PMC
April 2017

Improved Isolation, Proliferation, and Differentiation Capacity of Mouse Ovarian Putative Stem Cells.

Cell Reprogram 2017 04;19(2):132-144

1 Department of Anatomy, School of Medicine, Tehran University of Medical Sciences , Tehran, Iran .

The recent discovery of ovarian stem cells in postnatal mammalian ovaries, also referred to as putative stem cells (PSCs), and their roles in mammalian fertility has challenged the long-existing theory that women are endowed with a certain number of germ cells. The rare amount of PSCs is the major limitation for utilizing them through different applications. Therefore, this study was conducted in six phases to find a way to increase the number of Fragilis- and mouse vasa homolog (MVH)-positive sorted cells from 14-day-old NMRI strain mice. Results showed that there is a population of Fragilis- and MVH-positive cells with pluripotent stem cell characteristics, which can be isolated and expanded for months in vitro. PSCs increase their proliferation capacity under the influence of some mitogenic agents, and our results showed that different doses of stem cell factor (SCF) induce PSC proliferation with the maximum increase observed at 50 ng/mL. SCF was also able to increase the number of Fragilis- and MVH-positive cells after sorting by magnetic-activated cell sorting and enhance colony formation efficiency in sorted cells. Differentiation capacity assay indicated that there is a basic level of spontaneous differentiation toward oocyte-like cells during 3 days of culture. However, relative gene expression was significantly higher in the follicle-stimulating hormone-treated groups, especially in the Fragilis- sorted PSCs. We suggest that higher number of PSCs provides us either a greater source of energy that can be injected into energy-impaired oocytes in women with a history of repeat IVF failure or a good source for research.
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http://dx.doi.org/10.1089/cell.2016.0054DOI Listing
April 2017

Expression of Folliculogenesis-Related Genes in Vitrified Human Ovarian Tissue after Two Weeks Culture.

Cell J 2017 Apr-Jun;19(1):18-26. Epub 2016 Dec 21.

Department of Biostatistics, Faculty of Medicine, Tarbiat Modares University, Tehran, Iran.

Objective: This study was designed to evaluate the effects of vitrification and culture of human ovarian tissue on the expression of oocytic and follicular cell-related genes.

Materials And Methods: In this experimental study, ovarian tissue samples were obtained from eight transsexual women. Samples were cut into small fragments and were then assigned to vitrified and non-vitrified groups. In each group, some tissue fragments were divided into un-cultured and cultured (in α-MEM medium for 2 weeks) subgroups. The normality of follicles was assessed by morphological observation under a light microscope using hematoxylin and eosin (H&E) staining. Expression levels of factor in the germ line alpha (), KIT ligand (), growth differentiation factor 9 () and follicle stimulating hormone receptor () genes were quantified in both groups by real-time reverse transcriptase polymerase chain reaction (RT-PCR) at the beginning and the end of culture.

Results: The percentage of normal follicles was similar between non-cultured vitrified and non-vitrified groups (P>0.05), however, cultured tissues had significantly fewer normal follicles than non-cultured tissues in both vitrified and non-vitrified groups (P<0.05). In both cultured groups the rate of primary and secondary follicles was significantly higher than non-cultured tissues (P<0.05). The expression of all examined genes was not significantly altered in both non-cultured groups. Whiles, in comparison with cultured tissues non-cultured tissues, the expression of gene was significantly decreased, gene was not changed, and genes was significantly increased (P<0.05).

Conclusion: Human ovarian vitrification following culture has no impairing effects on follicle normality and development and expression of related-genes. However, culture condition has deleterious effects on normality of follicles.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5241514PMC
http://dx.doi.org/10.22074/cellj.2016.4890DOI Listing
December 2016

Evaluation of two endometriosis models by transplantation of human endometrial tissue fragments and human endometrial mesenchymal cells.

Int J Reprod Biomed 2017 Jan;15(1):21-32

Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.

Background: The animal models of endometriosis could be a valuable alternative tool for clarifying the etiology of endometriosis.

Objective: In this study two endometriosis models at the morphological and molecular levels was evaluated and compared.

Materials And Methods: The human endometrial tissues were cut into small fragments then they were randomly considered for transplantation into γ irradiated mice as model A; or they were isolated and cultured up to fourth passages. 2×10 cultured stromal cells were transplanted into γ irradiated mice subcutaneously as model B. twenty days later the ectopic tissues in both models were studied morphologically by Periodic acid-Schiff and hematoxylin and eosin staining. The expression of osteopontin (OPN) and matrix metalloproteinase 2 (MMP2) genes were also assessed using real time RT-PCR. 17-β estradiol levels of mice sera were compared before and after transplantation.

Results: The endometrial like glands and stromal cells were formed in the implanted subcutaneous tissue of both endometriosis models. The gland sections per cubic millimeter, the expression of OPN and MMP2 genes and the level of 17-β estradiol were higher in model B than model A (p=0.03).

Conclusion: Our observation demonstrated that endometrial mesenchymal stromal cells showed more efficiency to establish endometriosis model than human endometrial tissue fragments.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5340136PMC
January 2017

Short Term Organ Culture of Mouse Ovary in the Medium Supplemented with Bone Morphogenetic Protein 15 and Follicle Stimulating Hormone: A Morphological, Hormonal and Molecular Study.

J Reprod Infertil 2016 Oct-Dec;17(4):199-207

Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran.

Background: Bone morphogenetic protein 15 (BMP15) is a growth factor derived from oocyte and is essential for ovarian follicular growth and in this study, its effects on the improvement of growth and development of follicles during culture of neonatal mouse ovaries was investigated.

Methods: Two week old mice were cultured for 7 days in the basic culture media with or without follicle stimulating hormone (FSH) and BMP15 as four experimental groups; FSH/BMP15, FSH/BMP15, FSH/BMP15 and FSH/BMP15. The ovarian follicles at different developmental stages in paraffin embedding sections of cultured and non-cultured ovaries were counted and compared. The 17-β estradiol (E2) and progesterone (P4) levels were analyzed in collected culture media. The expression ratio of developmental genes (PCNA, BMPR-IB, BMPR-II, FSH-R, CYP17 and ZP3) to housekeeping gene (GAPDH) was analyzed by real time PCR (RT-PCR) in comparison with non-cultured control ovaries. The data was compared by independent t-test and one-way ANOVA (with Tukey's Post Hoc test). The p<0.05 was considered significant.

Results: The percentage of antral follicles, ovarian size, concentration of E2 and P4 and the expression ratio of PCNA and ZP3 genes in the ovaries cultured in medium supplemented with BMP15 and FSH increased significantly in comparison with other cultured groups (p<0.05). The BMPR-IB, BMPR-II and FSH-R mRNA level was significantly lower (p<0.05) and CYP 17 mRNA level did not change in the FSH/BMP15 group than other cultured groups.

Conclusion: This study demonstrated a favorable effect of BMP15 in combination with FSH on development of small size mouse follicles to antral stage.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5124338PMC
December 2016

Expression of pluripotent stem cell markers in mouse uterine tissue during estrous cycle.

Vet Res Forum 2016 15;7(3):181-188. Epub 2016 Sep 15.

Department of Cellular and Molecular Biology, School of Biology, Damghan University, Damghan, Iran; ; Institute of Biological Sciences, Damghan University, Damghan, Iran.

It was assumed that uterine stem cells are responsible for the unique regenerative capacity of uterine. Therefore, the aim of the present study was to investigate the expression of the pluripotent stem cell markers in the mice uterine tissue during different stages of estrous cycles. Twelve virgin female NMRI mice (6 to 8 weeks old) were considered at proestrus, estrus, metestrus and diestrus according to the cell types observed in the vaginal smear and underwent hysterectomy operation. Quantitative real-time polymerase chain reaction (PCR) and immunohistochemical staining for pluripotent stem cell markers (SOX2, OCT4, KLF4, and NANOG) were performed. Immunofluorescence staining revealed that expression and localization of the pluripotency markers SOX2, OCT4, KLF4, and NANOG at the protein level were not different throughout estrous cycle. Also, mRNA of pluripotency markers was detected in all tested samples. However, there were no significant differences in their genes expression at each stage and during the estrous cycle. Different hormonal profile during the estrous cycle could not affect expression of pluripotent stem cell markers in uterine tissue.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5094165PMC
September 2016

The effect of stem cell factor on proliferation of human endometrial CD146(+) cells.

Int J Reprod Biomed 2016 Jul;14(7):437-42

Department of Midwifery, Faculty of Medical Sciences Tarbiat Modares University, Tehran, Iran.

Background: Stem cell factor (SCF) is a transcriptional factor which plays crucial roles in normal proliferation, differentiation and survival in a range of stem cells.

Objective: The aim of the present study was to examine the proliferation effect of different concentrations of SCF on expansion of human endometrial CD146(+) cells.

Materials And Methods: In this experimental study, total populations of isolated human endometrial suspensions after fourth passage were isolated by magnetic activated cell sorting (MACS) into CD146(+) cells. Human endometrial CD146(+) cells were karyotyped and tested for the effect of SCF on proliferation of CD146(+) cells, then different concentrations of 0, 12.5, 25, 50 and 100 ng/ml was carried out and mitogens-stimulated endometrial CD146(+) cells proliferation was assessed by MTT assay.

Results: Chromosomal analysis showed a normal metaphase spread and 46XX karyotype. The proliferation rate of endometrial CD146(+) cells in the presence of 0, 12.5, 25, 50 and 100 ng/ml SCF were 0.945±0.094, 0.962±0.151, 0.988±0.028, 1.679±0.012 and 1.129±0.145 respectively. There was a significant increase in stem/ stromal cell proliferation following in vitro treatment by 50 ng/ml than other concentrations of SCF (p=0.01).

Conclusion: The present study suggests that SCF could have effect on the proliferation and cell survival of human endometrial CD146(+) cells and it has important implications for medical sciences and cell therapies.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4971561PMC
July 2016

Developmental Potential of Vitrified Mouse Testicular Tissue after Ectopic Transplantation.

Cell J 2016 4;18(1):74-82. Epub 2016 Apr 4.

Department of Histology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran.

Objective: 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.

Materials And Methods: 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.

Results: 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.

Conclusion: 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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4819389PMC
http://dx.doi.org/10.22074/cellj.2016.3989DOI Listing
April 2016

Effect of In Vitro Maturation Technique and Alpha Lipoic Acid Supplementation on Oocyte Maturation Rate: Focus on Oxidative Status of Oocytes.

Int J Fertil Steril 2016 Jan-Mar;9(4):442-51. Epub 2015 Dec 23.

School of Biology, Damghan University, Damghan, Iran.

Background: Therapeutic potential of in vitro maturation (IVM) in infertility is growing with great promise. Although significant progress is obtained in recent years, existing IVM protocols are far from favorable results. The first aim of this study was to investigate whether two step IVM manner change reactive oxygen species (ROS) and total anti- oxidant capacity (TAC) levels. The second aim was to find the effect of alpha lipoic acid (ALA) supplementation on oocyte maturation rate and on ROS/TAC levels during IVM.

Materials And Methods: In this experimental study, mouse germinal vesicle (GV) oocytes divided into cumulus denuded oocytes (DOs) and cumulus oocyte complexes (COCs) groups. GVs were matured in vitro in the presence or absence of ALA only for 18 hours (control) or with pre-culture of forskolin plus cilostamide for an additional 18 hours. Matured oocytes obtained following 18 and 36 hours based on experimental design. In parallel, the ROS and TAC levels were measured at different time (0, 18 and 36 hours) by 2',7'-dichlorodihydrofluorescein (DCFH) probe and ferric reducing/antioxidant power (FRAP) assay, respectively.

Results: Maturation rate of COCs was significantly higher than DOs in control group (P<0.05), while there was no significant difference between COCs and DOs when were pre-cultured with forskolin plus cilostamide. ROS and TAC levels was increased and decreased respectively in DOs after 18 hours while in COCs did not change at 18 hours and showed a significant increase and decrease respectively at 36 hours (P<0.05). ROS and TAC levels in the presence of ALA were significantly decreased and increased respectively after 36 hours (P<0.05) whereas, maturation rates of COCs and DOs were similar to their corresponding control groups.

Conclusion: ALA decreased ROS and increased TAC but could not affect maturation rate of both COCs and DOs in one or two step IVM manner.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4793165PMC
http://dx.doi.org/10.22074/ijfs.2015.4601DOI Listing
March 2016