ABSTRACT
Objective: This experimental study aimed to evaluate the effects of 17beta-estradiol [E2] and progesterone [P4] on the interaction between mouse embryo and human endometrial mesenchymal stromal cells, and gene expressions related to implantation [alphaV and beta3 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 alphaV and beta3 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
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 alphaV and beta3 integrins, LIFR, and on the morphology and ultrastructure of endometrial mesenchymal stromal cells
ABSTRACT
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 Mito Tracker 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
ABSTRACT
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 and 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]. 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
ABSTRACT
Background: The aim of the present study was to evaluate the effectiveness of human ovarian vitrification protocol followed with in vitro 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-beta 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 in vitro culture, thus this method could be useful for fertility preservation; however, additional studies are needed to improve the culture condition
ABSTRACT
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 gamma irradiated mice as model A; or they were isolated and cultured up to fourth passages. 2×106 cultured stromal cells were transplanted into ? 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-beta 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-beta 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
ABSTRACT
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 LIFtreatedgroup [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 LIFtreated 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
ABSTRACT
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 superovulated 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
ABSTRACT
Objective: This study aimed to evaluate the expression of the genes related to folliculo-genesis 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 [alpha-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
ABSTRACT
Objective: This study was designed to evaluate the effects of vitrification and in vitro 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 alpha-MEM medium for 2 weeks] subgroups. The normality of follicles was assessed by morphological observation under a light microscope using hematoxylin and eosin [H and E] staining. Expression levels of factor in the germ line alpha [FIGLA], KIT ligand [KL], growth differentiation factor 9 [GDF-9] and follicle stimulating hormone receptor [FSHR] 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 FIGLA gene was significantly decreased, KL gene was not changed, GDF-9 and FSHR genes was significantly increased [P<0.05]
Conclusion: Human ovarian vitrification following in vitro culture has no impairing effects on follicle normality and development and expression of related-genes. However, in vitro culture condition has deleterious effects on normality of follicles
Subject(s)
Humans , Women , Young Adult , Adult , Gene Expression Regulation , Stem Cell Factor/genetics , Basic Helix-Loop-Helix Transcription Factors/genetics , Receptors, FSH/genetics , Fertility Preservation/methods , Tissue Culture TechniquesABSTRACT
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 investi-gate whether two step IVM manner change reactive oxygen species [ROS] and total antioxidant 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
ABSTRACT
Background: The aim of this study was to investigate the percentage of the stem cells population in human endometrial tissue sections and cultured cells at fourth passage
Methods: Human endometrial specimens were divided into two parts, one part for morphological studies and the other part for in vitro culture. Full thickness of human normal endometrial sections and cultured endometrial cells at fourth passage were analyzed via immunohistochemistry for CD146 and some stemness markers such as Oct4, Nanog, SoX[2], and Klf4 and the expression of typical mesenchymal stem cell markers CD90, CD105
Results: 11.88 +/- 1.29% of human endometrial cells within tissue sections expressed CD146 marker vs. 28 +/- 2.3% of cultured cells, CD90 and CD105 were expressed by functionalis stroma [85 +/- 2.4 and 89 +/- 3.2%] than basalis stroma [16 +/- 1.4 and 17 +/- 1.9%], respectively [P<0.05]. Oct4 and Nanog-expressing cells comprise 1.43 +/- 0.08 and 0.54 +/- 0.01% of endometrial stromal cells in endometrial sections vs. 12 +/- 3.1% and 8 +/- 2.9% of cultured cells, respectively. They reside near the glands in the basal layer of endometrium. SoX[2] and Klf4 were not commonly expressed in tissue samples and cultured cells. CD9 and EpCAM were expressed by epithelial cells of the endometrium, rather than by stroma or perivascular cells
Conclusion: The human endometrial stem cells and pluripotency markers may be localized more in basalis layer of endometrium. The immunostaining observations of endometrial cells at fourth passage were correlated with the immunohistochemistry data
ABSTRACT
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 CD146P + P 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 CD146P+P cells and it has important implications for medical sciences and cell therapies
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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 mm[2]] 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 and 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
Subject(s)
Animals, Laboratory , Cryopreservation , Testis , Transplantation , Spermatogenesis , MiceABSTRACT
Background: Bone morphogenetic protein 15 [BMP15] is a growth factor derived from oocyte and is essential for in vivo ovarian follicular growth and in this study, its effects on the improvement of growth and development of follicles during in vitro 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-beta 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 in vitro development of small size mouse follicles to antral stage
ABSTRACT
Objective: Endometriosis is a common disease in which the endometrial stroma and glands grow abnormally outside the uterine cavity. The establishment of animal models can be effective for determining the etiology of endometriosis. In this study we compare two endometriosis models using endometrial fragments and isolated cultured endometrial cells
Methods: We obtained endometrial tissues that were in the proliferative or secretory phases from women who underwent hysteroscopies for benign reasons at Imam Khomeini Hospital [Tehran]. Following confirmation of the tissue's normality, we cut the tissues into 2 mm cube pieces. The remainder of endometrial tissues were used for isolation and cultured to the fourth passage. In the first model of endometriosis, the endometrial tissue fragments and in the second model, 2×106 isolated endometrial cells were subcutaneously transplanted into gamma irradiated mice. The mice were kept under controlled, sterile conditions for 20 days. The mice sera were collected before and after transplantation for assessment of 17beta estradiol. The ectopic tissues in both models were assessed for morphological staining using hematoxylin and eosin, as well as periodic acid Schiff [PAS] for gland secretion. The gland sections per mm2 were analyzed
Results: At 20 days after tissue transplantation, we observed endometrial-like glands in the subcutaneous tissue of both endometriosis models. The number of gland sections was 57.55 +/- 17.18/mm2 for the first model and 271.57 +/- 77.98/mm2 for the second model. This result was significantly higher in the second model when compared to the first model. Gland secretion was positive for PAS. The level of 17beta estradiol was higher in both models compared to the control group. This level was significantly higher in the second model compared to the first [P
Conclusion: The endometriosis model that used cultured endometrial cells showed more efficiency in morphology, gland formation and level of17beta estradiol
ABSTRACT
Objective: This study evaluates the effect of a mouse fallopian tube cell co-culture on the proliferation of human endometrial stromal cells
Methods: We used hysteroscopy to collect the endometrial cells from the uterus. The cells were isolated with collagenase type 3 and passed through 150 microm and 40 microm filters, respectively, after which the isolated cells were cultured in DMEM/F12 medium. At the end of the fourth subculture we used flow cytometry to evaluate the percentage of CD90 positive cells. The endometrial cells were co-cultured with mitomycin C-treated cells from the mouse fallopian tube as the experimental group. Those cultured without the feeder layer were considered the control group. The proliferation rate of cells in both groups were compared by the MTT assay
Results: The endometrial cells adhered to the floor of the plate after 24 hours. After 72 hours, they had a spindle shape which was similar to fibroblasts. The rate of CD90 positive cells at the fourth passage was 94.26 +/- 0.08%. The proliferation rate of the coculture experimental group was 1.1 +/- 0.02 and for the control group, it was 1.17 +/- 0.17 which was not significantly different
Conclusion: Co-culture of mouse fallopian tube cells with endometrial stem cells did not affect the proliferation rate of endometrial stem cells
ABSTRACT
Objective: This study aimed to evaluate the effect of vitrification on the morphology of human ovarian tissue in a stimulated group compared to a non-stimulated group
Methods: Ovarian cortex biopsies collected from stimulated and non-stimulated groups were transported to the laboratory in L-15 medium. Biopsies were cut into small pieces and divided randomly into the vitrified and non-vitrified subgroups. The vitrified-warmed and fresh samples were fixed using Bouin's solution, then passaged, sectioned and stained with hematoxylin and eosin and Masson's trichrome. The follicles at different developmental stages were counted and evaluated
Results: Morphological observations showed that the follicles and stromal tissue had well preserved normal structures after vitrification and warming. The percentage of normal follicles in the non-stimulated non-vitrified group was 89.22%; for the non-stimulated vitrified group, it was 84.60%. In previous groups the proportion of primordial follicles were 68.7 +/- 1.60% and 67.80 +/- 3.71%, primary follicles were 28.60 +/- 1.72% and 29.40 +/- 3.51%, and secondary follicles were 3.60 +/- 0.66% and 2.70 +/- 1.20%, respectively. The percentage of normal follicles in the stimulated non-vitrified group was 88.18%; for the stimulated vitrified group, it was 84.19%. In stimulated non-vitrified and stimulated vitrified groups the proportion of primordial follicles were 49.70 +/- 4.13% and 49.34 +/- 2.86%, primary follicles were 44.50 +/- 3.83% and 44.72 +/- 2.68%, and secondary follicles were 5.60 +/- 0.72% and 5.91 +/- 0.77%, respectively. There was no significant difference between the vitrified and non-vitrified and stimulated and non-stimulated groups
Conclusion: Vitrification had no harmful effect on the morphology of stimulated human ovarian tissue and stroma and ovarain tissue structure was similar to the non-stimulated group. This method could be a good alternative for fertility preservation
ABSTRACT
Objective: The present study compared the efficiency of three mouse ovarian tissue culture methods - hanging drop, under mineral oil, and on the insert with regards to improving in vitro ovarian follicular development
Methods: Ovaries from 7-day-old old female NMRI mice sacrificed by cervical dislocation were collected and cultured in alpha-MEM medium supplemented with 5% fetal bovine serum for 7 days in 3 groups [hanging drop, under mineral oil and on the insert]. We evaluated and compared the morphology and surface area of the ovaries and percentage of normal follicles in all groups. After the 7-day culture, the ovaries cultured on the insert showed better growth compared to the other groups. Their preantral follicles were isolated and cultured for 12 days. We assessed the follicular diameter, survival and maturation rate of these oocytes at the end of the last culture period
Results: The percentages of normal follicles in cultured ovaries were 73.85 +/- 2.49% [insert], 51.63 +/- 3.93% [hanging drop], and 40.52 +/- 5.86% [mineral oil] after the 7-day culture. The percentage of preantral follicles significantly increased from 2.1 +/- 0.44 to 24.5 +/- 2.4 in the group cultured on insert [P<0.05], however there was no significant difference in the other groups [P>0.05]. There were significantly increased surface areas of the ovarian tissues after the 7-day culture in all groups [P<0.05]. Ovaries cultured on the insert had a diameter of isolated follicles after 12 days of culture of 410 +/- 7.07 microm and the MII rate was 30.26%
Conclusion: The ovarian growth and morphology were well preserved in tissues cultured on the insert compared to the other culture methods
ABSTRACT
Cadmium chloride which is potentially toxic is currently used in industry. The toxic effects of cadmium on testes have been reported to range from apoptosis to necrosis, with different effects on fertility. This research aimed to study the effect of different doses of cadmium on testicular tissues at acute stage by light and electron microscopy. Cadmium chloride was injected into mature Balb/c mice intraperitoneally in 7 doses. Five mice were studied in each group. After 48 hr, the testes were extracted and prepared for light microscopy. Then two concentrations [15 and 25 micro M/kg] of them were selected for electron microscopic study based on histological changes. The cellular changes of luminal epithelium of seminiferous tubules were studied under an electron microscope. Histological and ultrastructural changes were reported. The absence of sperm in the tubules was observed at 20 micro M/kg concentration. At 25 micro M/kg, histological destruction and epithelial damages were observed. Histological changes were not remarkable at 5 and 10 micro M/kg. However, ultrastructural changes of seminiferous tubules at 20 micro M/kg included spermatogonial cell death. At 25 micro M/kg, vacuolation of Sertoli cells and death of spermatids as well as spermatocytes were observed. Cell death in the tubules was limited to germ cells. However, Sertoli cells exhibited architectural alterations without any cell death. Both apoptosis and necrosis occurred in testicular tissue by exposure to cadmium in a concentration-dependent manner. Gonadal cells were sensitive to cadmium administration. Supportive cells such as Sertoli cells in seminiferous tubules did not exhibit sensitivity to cadmium
Subject(s)
Animals, Laboratory , Testis/drug effects , Apoptosis , Necrosis , Cell Death , Mice, Inbred BALB CABSTRACT
Ovarian tissue cryopreservation is an alternative strategy to preserve the fertility of women predicted to undergo premature ovarian failure. This study was designed to evaluate the expression of folliculogenesis-related genes, including factor in the germline alpha [FIGLA], growth differentiation factor-9 [GDF-9], follicle-stimulating hormone receptor [FSHR], and KIT LIGAND after vitrification/warming of human ovarian tissue. Human ovarian tissue samples were collected from five transsexual women. In the laboratory, the ovarian medullary part was removed by a surgical blade, and the cortical tissue was cut into small pieces. Some pieces were vitrified and warmed and the others were considered as non-vitrified group [control]. Follicular normality was assessed with morphological observation by a light microscope, and the expression of FIGLA, KIT LIGAND, GDF- 9, and FSHR genes was examined using real-time RT-PCR in both the vitrified and non-vitrified groups. Overall, 85% of the follicles preserved their normal morphologic feature after warming. The percentage of normal follicles and the expression of FIGLA, KIT LIGAND, GDF-9, and FSHR genes were similar in both vitrified and non-vitrified groups [P > 0.05]. Vitrification/warming of human ovarian tissue had no remarkable effect on the expression of folliculogenesis-related genes