MiRNAs secreted by human blastocysts could be potential gene expression regulators during implantation.

BACKGROUND: Micro RNAs (miRNAs) are small non-coding RNAs known as essential regulators of cell-cell communication. Recent studies have revealed that miRNAs are secreted by a blastocyst in culture media. We hypothesized that endometrial epithelial cells take up embryo-derived miRNAs as well as other soluble factors and regulate their receptivity-related gene expression. METHODS AND RESULTS: Blastocyst culture media (BCM) were collected from the individually cultured embryos, while human endometrial epithelial cells (HEECs) were collected from healthy fertile volunteers. To evaluate the effect of BCM on the endometrial receptivity gene expression, HEECs were co-cultured with implanted BCM, non-implanted BCM, and a control culture medium. After determining altered gene expression in the HEECs, the miRNAs-related genes through bioinformatics databases were identified and evaluated in the BCM. Co-culture of primary HEECs with BCM significantly stimulated the expression levels of VEGFA, HBEGF, HOXA10, and LIF in the implanted group compared with non-implanted and control groups. The fold changes of miR-195 significantly diminished in the implanted BCM group compared with the non-implanted BCM group. Reduced fold changes of miR-29b, 145 and increased miR-223 were also observed in the implanted BCM group compared with the non-implanted ones. CONCLUSION: miRNAs could function as potential gene expression regulators during implantation. These molecules are secreted by human blastocyst, taken up by endometrial epithelial cells, and cause a change in the endometrial function. We found that BCMs can be effective in implantation process by stimulating related receptivity gene expression.

GM-CSF (granulocyte-macrophage colony-stimulating factor) treatment improves sperm parameters in men with oligoasthenoteratospermia via PI3K/AKT pathway.

Poor sperm quality in oligoasthenoteratospermia patients negatively affects assisted reproductive technology outcomes. Therefore, the development of sperm media is necessary to improve sperm parameters. This study investigated the effect of GM-CSF via PI3K/AKT pathway on sperm quality in OAT patients. Semen samples were collected from 20 OAT patients, and each sample was divided into two groups: Experiment and Control. In the experimental group, the samples were incubated with medium containing GM-CSF, and control samples were incubated without GM-CSF. Sperm parameters, mitochondrial membrane potential, acrosome reaction and DFI were studied; in addition, gene expression of PI3KR1, PI3KCA, GLUT1, GLUT3 and AKT1 was analysed, evaluation of PAKT/TAKT, and expression of GLUT 1, 3 was examined; subsequent fertilization rate and embryo quality were assessed. Our data showed that GM-CSF supplementation could significantly increase motility, mitochondrial activity, gene expression of PI3KCA, AKT1, the protein level of PAKT/TAKT and expression of GLUT 1, 3 while it decreases DNA fragmentation. The fertilization rate and embryo quality significantly improved in the treatment group. LY294002 had adverse effects on sperm motility and the PAKT/TAKT ratio. GM-CSF can improve in vitro sperm quality and could be a suitable supplement to sperm media for OAT patients.

Sperm-oviduct interaction: Differential gene expression of growth factors induced by sperm DNA fragmentation.

The present study investigated the effects of DNA fragmentation of spermatozoa on the growth factors expression by a human oviduct epithelial cell line (OE-E6/E7). Two separate groups were examined in this study. The cell line was cultured in the presence of spermatozoa with normal DNA fragmentation index (DFI) or abnormal DFI. Total RNA from the cell line in each group was isolated, and relative expression of objective genes was analysed using PCR array. Also, the concentration of VEGF, BMP-2, BMP-7 and MSTN in the supernatant of cell culture was analysed by the ELISA method. The PCR array analysis revealed that most of the growth factors had been upregulated in the abnormal group. However, the differences between groups were statistically significant (p < 0.05) for five genes, including VEGF-A, BMP-2, BMP-6, BMP-7 and OSM. Furthermore, MSTN was the only gene that down-regulated significantly under the influence of the spermatozoa with abnormal DFI. Moreover, the results of ELISA analysis were in agreement with the data of the PCR array. It has been concluded that DNA fragmentation in human spermatozoa can probably change regular events throughout the oviducts. Consequently, the genes of interest may change sperm function and probably its fate in the female reproductive tract.

Microfluidic chips as a method for sperm selection improve fertilization rate in couples with fertilization failure.

PURPOSE: Sperm quality plays a vital role in successful fertilization and pregnancy. Patients with fertilization failure (total failure or low-fertilization rate) despite having normal semen parameters are a challenging group whose sperm cannot fertilize the oocyte via the intracytoplasmic sperm injection (ICSI) technique. Microfluidics is offered as a new method for proper sperm sorting. METHODS: This study aimed to evaluate sperm parameters, DNA fragmentation index (DFI), expression of phospholipase C zeta 1 (PLCZ1), and transition nuclear proteins 1 (TNP1) mRNAs in sperm selected by microfluidic sperm sorting (MSS) chip compared with conventional density gradient centrifugation technique in patients with fertilization failure following ICSI. Subsequence fertilization rate and embryo quality were assayed. RESULTS: Normal morphology and total motility were significantly higher, and DFI was significantly lower in sperm selected by the MSS chip in fertilization failure and control groups. The RT-PCR results demonstrated a significant increase in the expression of PLCZ1 and TNP1 genes in sperm of both groups selected by MSS chips compared to the DGC method. In addition, with the selected sperm by MSS chip, an increase in fertilization rate and improvement of embryo quality was obtained. CONCLUSION: The present study findings show that sperm sorting by the microfluidic method improves fertilization rate in patients with poor fertilization outcomes following ICSI.

Functional differences of Toll-like receptor 4 in osteogenesis, adipogenesis and chondrogenesis in human bone marrow-derived mesenchymal stem cells.

Multipotent human bone marrow-derived mesenchymal stem cells (hMSCs) are promising candidates for bone and cartilage regeneration. Toll-like receptor 4 (TLR4) is expressed by hMSCs and is a receptor for both exogenous and endogenous danger signals. TLRs have been shown to possess functional differences based on the species (human or mouse) they are isolated from therefore, the effects of knockdown of TLR4 were evaluated in humans during the differentiation of MSCs into bone, fat and chondrocyte cells in vitro. We investigated the expression profile of TLR4 during the differentiation of hMSCs into three different lineages on days 7, 14 and 21 and assessed the differentiation potential of the cells in the presence of lipopolysaccharide (LPS, as an exogenous agonist) and fibronectin fragment III-1c (FnIII-1c, as an endogenous agonist). TLR4 expression increased following the induction of hMSC differentiation into all three lineages. Alkaline phosphatase activity revealed that FnIII-1c accelerated calcium deposition on day 7, whereas LPS increased calcium deposition on day 14. Chondrogenesis increased in the presence of LPS; however, FnIII-1c acted as a reducer in the late stage. TLR4 silencing led to decreased osteogenesis and increased adipogenesis. Furthermore, Wnt5a expression was inversely related to chondrogenesis during the late stage of differentiation. We suggest that understanding the functionality of TLR4 (in the presence of pathogen or stress signal) during the differentiation of hMSCs into three lineages would be useful for MSC-based treatments.

Comparing various protocols of human and bovine ovarian tissue decellularization to prepare extracellular matrix-alginate scaffold for better follicle development in vitro.

BACKGROUND: Nowadays, the number of cancer survivors is significantly increasing as a result of efficient chemo/radio therapeutic treatments. Female cancer survivors may suffer from decreased fertility. In this regard, different fertility preservation techniques were developed. Artificial ovary is one of these methods suggested by several scientific groups. Decellularized ovarian cortex has been introduced as a scaffold in the field of human fertility preservation. This study was carried out to compare decellularization of the ovarian scaffold by various protocols and evaluate the follicle survival in extracellular matrix (ECM)-alginate scaffold. RESULTS: The micrographs of H&E and DAPI staining confirmed successful decellularization of the ovarian cortex in all experimental groups, but residual DNA content in SDS-Triton group was significantly higher than other groups (P < 0.05). SEM images demonstrated that complex fiber network and porosity structure were maintained in all groups. Furthermore, elastin and collagen fibers were observed in all groups after decellularization process. MTT test revealed higher cytobiocompatibility of the SDS-Triton-Ammonium and SDS-Triton decellularized scaffolds compared with SDS groups. Compared to the transferred follicles into the sodium alginate (81%), 85.9% of the transferred follicles into the decellularized scaffold were viable after 7 days of cultivation (P = 0.04). CONCLUSION: Although all the decellularization procedures was effective in removal of cells from ovarian cortex, SDS-Triton-Ammonium group showed less residual DNA content with higher cytobiocompatibility for follicles when compared with other groups. In addition, the scaffold made from ovarian tissues decellularized using SDS-Triton-Ammonium and sodium alginate is suggested as a potential 3D substrate for in vitro culture of follicles for fertility preservation.

HOX cluster and their cofactors showed an altered expression pattern in eutopic and ectopic endometriosis tissues.

Endometriosis is major gynecological disease that affects over 10% of women worldwide and 30%-50% of these women have pelvic pain, abnormal uterine bleeding and infertility. The cause of endometriosis is unknown and there is no definite cure mainly because of our limited knowledge about its pathophysiology at the cellular and molecular levels. Therefore, demystifying the molecular mechanisms that underlie endometriosis is essential to develop advanced therapies for this disease. In this regard, HOX genes are remarkable because of their critical role in endometrial development and receptivity during implantation, which is attributed to their ability to mediate some of the sex steroid functions during the reproductive period. Access to the expression profiles of these genes would provide the necessary information to uncover new genes for endometriosis and assist with disease diagnosis and treatment. In this study we demonstrate an altered expression pattern for the HOX clusters (A-D) and their cofactors in both eutopic and ectopic conditions compared to control tissue biopsies. Remarkably, most of the intensive changes occurred in eutopic samples from endometriosis patients compared to control tissue biopsies. Pathway analysis revealed the involvement of differentially expressed genes in cancer that correlate with an association between endometriosis and cancer. Our results suggest critical roles for the HOX cluster and their cofactors in endometriosis pathophysiology.

Microarray analysis identification of key pathways and interaction network of differential gene expressions during osteogenic differentiation.

BACKGROUND: Adult bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stem cells that can differentiate into three lineages. They are suitable sources for cell-based therapy and regenerative medicine applications. This study aims to evaluate the hub genes and key pathways of differentially expressed genes (DEGs) related to osteogenesis by bioinformatics analysis in three different days. The DEGs were derived from the three different days compared with day 0. RESULTS: Gene expression profiles of GSE37558 were obtained from the Gene Expression Omnibus (GEO) database. A total of 4076 DEGs were acquired on days 8, 12, and 25. Gene ontology (GO) enrichment analysis showed that the non-canonical Wnt signaling pathway and lipopolysaccharide (LPS)-mediated signaling pathway were commonly upregulated DEGs for all 3 days. KEGG pathway analysis indicated that the PI3K-Akt and focal adhesion were also commonly upregulated DEGs for all 3 days. Ten hub genes were identified by CytoHubba on days 8, 12, and 25. Then, we focused on the association of these hub genes with the Wnt pathways that had been enriched from the protein-protein interaction (PPI) by the Cytoscape plugin MCODE. CONCLUSIONS: These findings suggested further insights into the roles of the PI3K/AKT and Wnt pathways and their association with osteogenesis. In addition, the stem cell microenvironment via growth factors, extracellular matrix (ECM), IGF1, IGF2, LPS, and Wnt most likely affect osteogenesis by PI3K/AKT.

The role of sperm in inducing genomic changes in the implantation: An experimental study.

Endometrial receptivity and implantation are important topics in reproductive sciences. No evidence was found to support sperm involvement in endometrial receptivity and its associated factors. This study aimed to explore the effect of the normal human spermatozoa-endometrium cell interaction in regulating genes in the endometrial receptivity pathway. Semen samples were collected from a healthy and fertile man; then, they were incubated with endometrial cells for 24 hr and considered as the sperm group. A group was cultured without spermatozoa and considered as a control group. About 24 hr later, cells were collected from the bottom of the culture dish. The expressions of the VEGF, FGF2, HBEGF, LIFR, EGF, LIF, MUC1, HOXA10, CSF and PGR genes were evaluated in the two groups. Statistical analysis was performed using an independent sample test. Compared with the control group, in the sperm group, the mRNA levels of PGR (p = .0451), VEGF (p = .0101), HBEGF (p = .0163), EFG (p = .0339), FGF2 (p = .012), LIF (p = .0324), LIFR (p = .0321) and HOXA10 (p = .0098) were significantly upregulated. The results showed that there is a need for the interaction between spermatozoa and endometrium for implantation and can be used for preparing uterine in in vitro fertilisation cycles.

Notch signaling pathway in cumulus cells reflecting zygote and embryo quality in polycystic ovary syndrome.

PURPOSE: The present study aimed to explore the associations between the expression pattern of molecules in the Notch pathway in the cumulus cells of polycystic ovary syndrome (PCOS) patients and the quality of zygotes and embryos. METHODS: A total of 200 cumulus complexes surrounding mature oocytes were obtained from 40 patients with and without PCOS undergoing intracytoplasmic sperm injection (ICSI). The expressions of Notch-1, Notch-2, and Notch-3 genes were examined by Reverse Transcription Q-PCR assay. Moreover, immunocytochemistry was performed for the expressions of Jagged-1 and Jagged-2 proteins. The correlations between the Notch receptors and their ligand expressions and the qualities of the zygote and embryo were investigated. RESULTS: The expression levels of Notch-2, Notch-3, Jagged-1, and Jagged-2 were significantly lower in patients with PCOS than in normal women (p < 0.05), while Notch-1 showed no meaningful difference between the groups. A positive correlation was found between Notch-1 and embryo quality. Furthermore, only Notch-2 and Jagged-2 marginally correlated with zygote quality. CONCLUSION: The data of the present study indicated that evaluating the molecules in the Notch pathway in PCOS patients' cumulus cells provides a novel approach to predict the zygote and embryo quality. However, further studies on a larger population are needed to validate this finding.

Proteome analysis of endometrial tissue from patients with PCOS reveals proteins predicted to impact the disease.

Polycystic ovary syndrome (PCOS) is a complex disease that causes an ovulatory infertility in approximately 10% of reproductive-age women. We searched for candidate proteins that might contribute to endometrial receptivity defects in PCOS patients, and result in adverse reproductive outcomes. Shotgun proteomics approach was used to investigate the proteome profile of the endometrium at the luteal phase in PCOS patients compared to healthy fertile individuals. Biological process and pathway analyses were conducted to categorize the proteins with differential expressions. Confirmation was performed for a number of proteins via immunoblotting in new samples. 150 proteins with higher abundance, and 46 proteins with lower abundance were identified in the endometrial tissue from PCOS patients compared to healthy fertile individuals. The proteins with higher abundance were enriched in protein degradation, cell cycle, and signaling cascades. Proteins with lower abundance in PCOS patients were enriched in extracellular matrix (ECM) composition and function, as well as the salvage pathway of purine biosynthesis. Metabolism was the most affected biological process with over 100 up-regulated, and approximately 30 down-regulated proteins. Our results indicate significant imbalances in metabolism, proteasome, cell cycle, ECM related proteins, and signaling cascades in endometrial tissue of PCOS, which may contribute to poor reproductive outcomes in these patients. We postulate that the endometria in PCOS patients may not be well-differentiated and synchronized for implantation. Possible roles of the above-mentioned pathways that underlie implantation failure in PCOS will be discussed. Our findings need to be confirmed in larger populations.

Three-dimensional decellularized amnion membrane scaffold promotes the efficiency of male germ cells generation from human induced pluripotent stem cells.

Cells grow differently in conventional 2D cell culture than when they grow in the physiological microenvironment. In this study, we developed a 3D cell culture model for generating male germ cells from human iPSCs using a human decellularized amnion membrane (DAM) scaffold. To this end, human iPSCs were generated using retroviral vectors and characterized for pluripotency properties by immunofluorescence assay, flow cytometry, ALP staining, cytogenetic assay, and differentiation capacity. The iPSCs were used for investigating male germ cells differentiation efficiency in both conventional 2D culture and 3D-DAM scaffold. The expression of male germ cell markers was evaluated at day 21 of differentiation using immunofluorescence assay, flow-cytometry, and RT-qPCR. The results indicated a successful reprogramming of human foreskin fibroblast cells into pluripotent iPSCs. The reprogrammed cells were positive for pluripotency markers and differentiated into the three germ layers. During male germ cell differentiation, the cells tend to aggregate and form colony-like structures in both 2D and 3D conditions. However, significant expression of VASA, DAZL, PLZF, STELLA, and NANOS3 markers and more efficient haploid male germ cell production were observed in the 3D condition when compared to the 2D model. Considering the effect of the 3D-DAM scaffold in prompting male germ cell-specific markers and increased efficiency of germ cell differentiation in 3D culture, it appears that DAM scaffold is a useful tool for in vitro studies of human germ cell development and ultimately future clinical application.

Epigenetic role of the nuclear factor NF-Y on ID gene family in endometrial tissues of women with endometriosis: a case control study.

BACKGROUND: A predominant difference between endometrial and normal cells is higher proliferation rate in the former cells which is benign. The genes of inhibitor of differentiation (ID) family play a major role in cell proliferation regulation which might be targeted by the nuclear transcription factor Y (NF-Y) for subsequent epigenetic modifications through the CCAAT box regulatory region. The present study was designed to investigate the epigenetic role of NF-Y on ID gene family in endometrial tissue of patients with endometriosis. MATERIALS & METHODS: In this case-control study, 20 patients with endometriosis and 20 normal women were examined for the relative expression of the NF-YA, NF-YB, NF-YC and ID genes by real-time PCR during the proliferative phase. The occupancy of NF-Y on CCAAT box region of ID genes was investigated using chromatin immunoprecipitation (ChIP) followed by real-time PCR. RESULTS: The NF-YA was over-expressed in eutopic endometrium during the proliferative phase. Although the expression level of NF-YB and NF-YC were unchanged in eutopic samples, they were remarkably higher in ectopic group (P<0.05). The ID2 and ID3 genes were up-regulated in ectopic and eutopic tissues, however ID1 and ID4 genes were down-regulated in these samples (P<0.05). The ChIP analysis revealed significant enrichment of NF-Y on regulatory regions of ID2,3 genes in eutopic group, but reduced binding level of NF-Y to the ID1,3 promoters in ectopic specimens (P<0.05). CONCLUSION: The ability of NF-Y to regulate ID genes via CCAAT box region suggests the possible role of NF-Y transcription factor in epigenetic changes in endometrial tissues which may open novel avenues in finding new therapeutic strategies.

Distinct changes in the proteome profile of endometrial tissues in polycystic ovary syndrome compared with healthy fertile women.

RESEARCH QUESTION: What is the molecular basis of infertility related to uterine dysfunction in women with polycystic ovary syndrome (PCOS)? DESIGN: In this study, differences in protein expression between PCOS and normal endometrium were identified using a proteomic approach based on two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS). The proteome of endometrium were analysed during the proliferative (on day 2 or 3 before ovulation, n = 6) and luteal phases (on day 3-5 after ovulation, n = 6) from healthy women and PCOS patients (12-14 days after spontaneous bleeding, n = 12). The differentially expressed proteins were categorized based on the biological process using the DAVID bioinformatics resources. RESULTS: Over 803 reproducible protein spots were detected on gels, and 150 protein spots showed different intensities between PCOS and normal women during the proliferative and luteal phases. MS analysis detected 70 proteins out of 150 spots. For four of the 70 proteins, 14-3-3 protein, annexin A5, SERPINA1 and cathepsin D, 2-DE results were validated and localized by Western blot and immunohistochemistry, respectively, and their gene expression profiles were confirmed by real-time quantitative PCR. The obtained results corresponded to the proteomic analysis. The differentially expressed proteins identified are known to be involved in apoptosis, oxidative stress, inflammation and the cytoskeleton. CONCLUSIONS: The processes related to the differentially expressed proteins play important roles in fecundity and fecundability. The present study may reveal the cause of various endometrial aberrations as a limiting factor for achieving pregnancy in PCOS women.

Evaluation of toll-like receptor 4 expression in human bone marrow mesenchymal stem cells by lipopolysaccharides from Shigella.

Lipopolysaccharides (LPS) from gram negative bacteria stimulate toll-like receptor 4 (TLR4) expression in immune cells. Recent reports state that bone marrow-derived cells such as mesenchymal stem cells (MSCs) also express TLR proteins. Numerous researches have studied the effect of a number of LPSs on TLR4 expression, but no data exists on the effect of LPSs from different strains of one bacterial genus on TLR4 expression. In this study, we investigate the effects of various concentrations of LPS from different Shigella strains on TLR4 expression in human bone marrow (hBM)-MSCs. At the mRNA level, we have found that untreated hBM-MSCs (control) did not express TLR4 compared to the experimental groups. Cells treated with LPS from Shigella flexneri had the highest expression of TLR4, whereas cells treated with LPS from Shigella sonnei had the lowest expression. We observed that LPSs had a dose-dependent effect on TLR4 expression in all of the treatment groups. ELISA findings for interleukin-6 secretion have confirmed mRNA expression results for all treatment groups. Hence, LPS from S. flexneri can be considered as an optimum LPS to stimulate the immune system for vaccine production against shigellosis. Also, TLR activation in hBM-MSCs can modulate their function such as homing.

The effects of letrozole and clomiphene citrate on ligands expression of Wnt3, Wnt7a, and Wnt8b in proliferative endometrium of women with Polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS) is a common endocrinologic disorder in women of reproductive age characterized by polycystic ovaries, oligo/anovulation, and hyperandrogenism. Not only anovulation but also endometrial dysfunction can reduce fertility in PCOS patients. Wnt pathway is responsible for endometrial proliferation which be strongly regulated by estradiol. To determine the effects of clomiphene citrate (CC) and letrozole, we measured the expression of some main ligands of Wnt/ß-catenin signaling including Wnt7a, Wnt3, and Wnt8b in the endometrial samples taken from PCOS women on day 12 of the menses who received 100 mg CC or 5 mg letrozole as well as from women without treatment. Significantly, the mean estrogen and progesterone concentration were lower and higher, respectively, in letrozole than CC. The mean endometrial thickness (ET) was significantly greater in letrozole compared to CC. Assessment of the mRNA and protein expression of Wnt7a, Wnt3, and Wnt8b showed significantly lower expression in CC than the letrozole and control groups. Collectively, letrozole provided a better molecular response in the endometrium of PCOS patients during the proliferative phase, similar to natural cycles, compared to CC. CC decreased the ligands expression of Wnt3, Wnt7a, and Wnt8b, resulting in endometrial dysfunction.

Apoptotic biomarkers in cumulus cells in relation to embryo quality in polycystic ovary syndrome.

PURPOSE: To investigate associations between gene expression pattern of apoptotic biomarkers in cumulus cells of polycystic ovary syndrome (PCOS) patients and the quality of oocytes and embryos. METHODS: 40 intracytoplasmic sperm injection patients, of whom 20 were PCOS and 20 were healthy women, were included in this study. Serum hormone levels were measured using Radioimmunoassay for each patient. The expression of survivin, caspase-3, and caspase-7 in 200 cumulus complexes surrounding mature oocytes (100 in PCOS versus 100 in control groups) collected individually at pick up was examined by real-time polymerase chain reaction (real-time PCR). RESULTS: The expression levels of survivin were significantly lower in PCOS patients than those of normal women while caspase-3 and caspase-7 expression levels were higher in PCOS patients (p < 0.05). There was a statistically significant correlation between the levels of these genes and embryo quality. CONCLUSIONS: This study reveals that the measurement of survivin, caspase-3, caspase-7 levels in cumulus cells of PCOS patients could be used as genetic biomarkers for oocyte and embryo selection under an ART program. However, further prospective studies are required to elucidate this issue.

Pluripotency and differentiation of cells from human testicular sperm extraction: An investigation of cell stemness.

Human male germ-line stem cells (hmGSCs) and human testis-derived embryonic stem cell-like (htESC-like) cells are claimed to be in vitro pluripotent counterparts of spermatogonial stem cells (SSCs), but the origin and pluripotency of human testis-derived cell cultures are still under debate. The aim of this study was to generate putative pluripotent stem cells in vitro from human testicular sperm-extracted (TESE) samples of infertile men, and to assess their pluripotency and capacity to differentiate. TESE samples were minced, enzymatically disaggregated and dispersed into single-cell or cluster suspensions, and then cultured. Initially, cell clusters resembled those described for hmGSCs and htESC-like cells, and were positive for markers such as OCT4/POU5F1, NANOG, and TRA-2-54. Prolonged propagation of cell clusters expressing pluripotency markers did not thrive; instead, the cells that emerged possessed characteristics of mesenchymal stromal cells (MSCs) such as STRO-1, CD105/EGLN1, CD13/ANPEP, SOX9, vimentin, and fibronectin. KIT, SOX2, and CD44 were not expressed by these MSCs. The multipotential differentiation capacity of these cells was confirmed using Oil Red-O and Alizarin Red staining after induction with specific culture conditions. It is therefore concluded that pluripotent stem cells could not be derived using the conditions previously reported to be successful for TESE samples.

Epigenetic alterations of CYP19A1 gene in Cumulus cells and its relevance to infertility in endometriosis.

PURPOSE: The purpose of the present study was to investigate the epigenetic mechanisms responsible for the aberrant aromatase expression (CYP19A1) in Cumulus Cells (CCs) of infertile endometriosis patients. METHOD: Cumulus cells were obtained from 24 infertile patients with and without endometriosis who underwent ovarian stimulation for intracytoplasmic sperm injection. Expression of CYP19A1 gene was quantified using Reverse Transcription Q-PCR. DNA methylation, histone modifications, and binding of Estrogen Receptor, ERß to regulatory DNA sequences of CYP19A1 gene were evaluated by Chromatin ImmunoPrecipitation (ChIP) assay. RESULTS: CYP19A1 gene expression in CCs of endometriosis patients was significantly lower than the control group (P = 0.04). Higher incorporation of MeCP2 (as a marker of DNA methylation) on PII and PI.4 promoters, and hypoacetylation at H3K9 in PII and hypermethylation at H3K9 in PI.4 were observed in CYP19A1 gene in endometriosis patients (P < 0.05). Moreover, a decreased level of ERß binding to PII and an increased level of its binding to PI.3 and PI.4 promoters of CYP19A1 were observed in endometriosis patients when compared to control. CONCLUSION: Significant reduction of CYP19A1 gene expression in CCs of endometriosis patients may be the result of epigenetic alterations in its regulatory regions, either by DNA methylation or histone modifications. These epigenetic changes along with differential binding of ERß (as a transcription factor) in CYP19A1 promoters may impair follicular steroidogenesis, leading to poor Oocyte and embryo condition in endometriosis patients.

Alteration of spermatogenesis following spermatogonial stem cells transplantation in testicular torsion-detorsion mice.

PURPOSE: Testicular ischemia is the main consequence of testicular torsion, in both clinical and experimental aspects. Preservation and auto-transplantation of spermatogonial stem cells (SSCs) could be a new treatment for infertility in testicular ischemia following testicular torsion. METHODS: To apply the idea in this study, animals were randomly divided into four groups of control, sham, with torsion, and with torsion followed by transplantation (TT). Isolated SSCs from neonatal mice were cultured and identified by flow cytometry (C-KIT(-), INTEGRIN ß1 (+)) and RT-PCR (Reverse transcription polymerase chain reaction) for specific spermatogonial cell markers (Oct4, Gfrα-1, Plzf, Vasa, Itgα 6 , and Itgß 1 ). SSCs were transplanted upon a 2-h testicular torsion in the TT group. Cultured cells were transplanted into ischemia reperfusion testicle 2 weeks post-testicular torsion. Eight weeks after SSCs transplantation, the SSCs-transplanted testes and epididymis were removed for sperm analysis, weight and histopathological evaluation, and pre- and post-meiotic gene expression assessment by qRT-PCR. RESULTS: Our findings indicated that all evaluated parameters (epididymal sperm profile, Johnsen score, Plzf, Gfrα-1, Scp-1, Tekt-1 expressions, and histopathological profile) were significantly decreased following testicular torsion (group 3) when compared to the control group (p ≤ 0.05). However, all abovementioned parameters showed a significant increase/improvement in torsion-transplantation group compared to torsion group. However, these parameters in the TT group were significantly lower in the sham and control groups (p ≤ 0.05). CONCLUSION: SSCs transplantation could up-regulate the expression of pre- and post-meiotic genes in testicular ischemia, which resulted in improvement of both testicular function and structure after testicular torsion.