Background & Aims: Stem CELLS are undifferentiated CELLS and are found in different tissues. These CELLS have capacity of self-renewal and differentiation into other lineages. Granulosa CELLS (GCs) are the multipotent stem CELLS. In the present research we evaluated the differentiation potential of GCs into keratinocytes. Material & Methods: GCs were cultured after enzymatic isolation from ovarian follicle. Then, keratinocyte inductive medium was added and expression of keratin10 and keratin14 were investigated with western blotting technique. Results: The results of the flow cytometric analysis of the isolated CELLS indicated the high expression of mesenchymal stem cell specific antigens (p < 0. 05). Also, the results of the western blotting showed the expression of creatine 10 and creatine 14 proteins in all groups except for negative control (p< 0. 05). Conclusion: Human granulosa CELLS have a very high ability to differentiate into keratinocytic CELLS, and with further research, it is possible to provide a suitable substrate for the use of human granulosa CELLS to treat severe skin lesions.

Background: FOLLICULAR helper T lymphocyte (Tfh) promotes antibody production by B lymphocytes in various diseases, including Pulmonary Tuberculosis (PTB). Objective: To explore the potential role of Tfh CELLS and assess the expression level of PD-1, and IL-21 in PTB. Methods: 54 newly diagnosed smear-positive PTB, 27 people with latent tuberculosis (LTB) and 27 healthy controls (HC) were enrolled. The PTB group was further divided based on the range of lung field involved (focus number>=3, PTB-X3,<3, PTB-X2). After 6-month therapy, sputum smear (positive, PTB-SP,negative, PTBSN) or imaging examinations (lesion reduction significant, PTB-os,insignificant, PTB-s) were used to evaluate the conditions of PTB patients. Blood samples were collected from PTB group at month six. CD4+CXCR5+Tfh, and its subsets, CD4+CXCR5+PD-1+Tfh and CD4+CXCR5+ICOS+Tfh in peripheral blood mononuclear CELLS (PBMCs) were detected. Serum IL-21 concentrations were measured. Results: The frequencies of CD4+CXCR5+Tfh, CD4+CXCR5+ ICOS+Tfh and CD4+CXCR5+PD-1+Tfh were higher in PTB group than in HC. IL-21, IL-4 and IFNγ,concentrations were significantly higher in PTB group than in HC. The proportion of CD4+CXCR5+Tfh in PTB-X2 was lower than in PTB-X3 group. CD4+CXCR5+PD-1+Tfh proportion in PTB-X2 was lower than that in the PTB-X3. After treatment, CD4+CXCR5+Tfh proportion was significantly lower in the PTB-SN group. CD4+CXCR5+Tfh was lower in the PTB-os group than in the PTB-s group. However, the CD4+CXCR5+PD-1+Tfh and cytokine concentrations of IL-21 were not different. Conclusions: CD4+CXCR5+Tfh level might predict the sputum results, and lesion decrease rate while CD4+CXCR5+PD-1+Tfh subset and IL-21 were not associated with sputum results or lesion decrease after treatment.

Background and purpose: FOLLICULAR fluid (FF) is a rich source of compounds for the development of oocyte. Wharton's jelly mesenchymal stem CELLS (WJ-MSCs) have a same differentiation origin with primordial germ CELLS, therefore, WJ-MSCs could differentiate into oocyte-like CELLS (OLCs) in the presence of appropriate factors. The purpose of present study was to isolate WJ-MSCs and then investigate their differentiation capacity to OLCs by FF.Materials and methods: The fragments of Wharton's jelly were cultured in α-MEM supplemented with 10% FBS. WJ-MSC at the 3rd passage were studied for differentiation ability to adipocytes and osteocytes. Furthermore, WJ-MSCs related markers were assessed by flow cytometry. In the same passage, WJ-MSCs were induced to differentiate into oocyte-like CELLS by adding 10% human FF in α-MEM for 21 days.Results: WJ-MSCs could differentiate into adipocytes and osteocytes. Flow cytometry analysis indicated that WJ-MSCs do not express hematopoietic marker (CD34-CD45) and express MSCs markers (CD73, CD90 and CD105). WJ-MSCs which were under influence of FF differentiated into OLCs and immunocytochemistry analysis showed these CELLS have positive expression of ZP3, SYCP3 (oocyte’s markers) and VASA (germ cell’s marker).Conclusion: The present study demonstrated that WJ-MSCs could differentiate into OLCs (morphologically) under the influence of FF and also expressed ZP3, VASA, and SYCP3 markers positively. According to the capabilities of WJ-MSCs, these CELLS seem to be suitable for use in cell therapy projects to improve infertility treatments.

Background: One of the most annoying problems in IVF is poor ovarian response. It is anticipated that 5-18% of all IVF cycle are affected by poor response to ovarian hyperstimulation.Poor response to goandotropin may lead to decline in the pool of embryos for transfer or cryoperservation, and decrease pregnancy rates. Different mechanisms explain poor ovarian response for example decreased number of FSH receptors in granulosa CELLS, anti-FSH IgA and IgG potentially exerting a local FSH antagonizing effect in maturing follicles, the presence of a specific FSH receptor-binding inhibitor in the FOLLICULAR fluid, the higher FSH threshold to stimulation follicle development.As mentioned above, likely immune system is involved in the mechanism of poor responder and affects the steroidogenesis. First line of immune system is pattern recognition receptors (PRRs) as a compartment of innate immunity. The most important group of PRRs which also identified in female reproductive tract is Toll like receptors (TLRs) family. So far, TLR1-10 are characterized in human. TLR1, 2, 4, 5, 6 and TLR10 are expressed on the cell membrane and they recognize lipid and protein ligands. TLR3, 7, 8 and TLR9 can detect nu cleic acid from pathogens are found in endosomal compartment.In addition these receptors recognize a broad range of endogenous ligands including heat shock proteins, hyaloronan, host RNA, and reactive oxygen species (ROS).So TLRs play abundant immunologic and physiologic roles in reproductive system. Therefore, the aim of this study is to investigate TLR1.2, 3, 5, 7, 8 genes expression in FOLLICULAR CELLS obtained from ovarian poor response women in compare to normal women.Materials and Methods: All procedures were approved by the Royan Ethics committee and informed consent was obtained prior to the collection of samples.Forty patients (20 infertile ovarian poor responder patients and 20 normal women with male factor infertility) underwent controlled ovarian stimulation with monitoring E2 levels and pelvic ultrasounds. Gonadotropin doses were then adjusted accordingly and monitoring was continued until patients received 10, 000 IU hCG intramuscular.Oocyte retrieval was performed approximately 36 h after hCG. The FOLLICULAR fluid was obtained from the largest follicle (>18 mm) visualized on ultrasound before using any flushing medium. This follicle was aspirated with a 17-gauge Cook needle attached to 100 mm Hg pump-operated aspirator. It was the first puncture of the oocyte retrieval. The FOLLICULAR fluid was transferred to a sterile Petri dish, and the oocytes were then removed. The FOLLICULAR fluid was placed into a 15- mL conical tube and centrifuged at 300g for 5 min. The supernatant was removed for further proteomic study.Total RNA was extracted separately from cellular pallet in each group using TRI reagent and treated with DNaseI.First strand cDNA synthesis was performed using oligo dT primers and Superscript II reverse transcriptase system. RT-PCR and Quantitative PCR was performed using the prepared cDNA and primer for TLR1-10. Relative TLR expression quantities were compared between two groups. The threshold cycle values were normalized against the threshold value of humanβ-actin. Differences in normalized expression values between samples were tested for significance using ANOVA statistical test. The results were expressed as mean ±SEM. The level of statistical significance was set at p<0.05.Results: TLR1-10 genes were expressed in FOLLICULAR cell of both, case and control groups. The mean relative expression of TLR1, 2, 4, 6 genes were significantly higher in poor ovarian responder, TLR5 and 8 expressions were higher in poor ovarian responder but not significant. TLR 3, 7, 9 and TLR10 was lower in patients with poor ovarian responder in compare to normal women.Conclusion: Our findings suggested that TLRs are involved in pathophysiology of ovarian poor response. It’s been proposed that inflammatory markers such as IL6, IL8 and TNF-a are vary in poor responders. Since these markers are produced by TLRs signaling, therefore it’s possible that these changes are a result of TLRs activation in poor responders.

Background: Polycystic ovarian syndrome (PCOS) is one of the causes of infertility for which treatment methods do not have a high rate of pregnancy. In this study, the stem CELLS in the FOLLICULAR fluid (FF) of patients were grown in the normal FF, and their differentiation into oocytes was evaluated. Materials and Methods: The FF of PCOS patients was centrifuged, and their CELLS were cultured with and without 20% normal FF for 2 weeks. The CELLS were evaluated for their morphology by inverted microscope and for markers of stem CELLS (NANOG and OCT4) and oocytes (zona pellucida (ZP) 2 and ZP3) by RT-PCR and immunocytochemistry. The amount of steroids was measured by enzyme-linked immunosorbent assay (ELISA). Results: The CELLS were all round on day 0. After that, they had a heterogeneous morphology (fibroblast-like CELLS, epithelial-like CELLS, and round oocyte-like CELLS). In the first week, NANOG and OCT4 genes in the study group were less expressed than those in the control group (P < 0. 0001) (~0. 5-fold), while ZP2 and Z3 genes were more expressed (P < 0. 0001) (~2-fold). In the second week, stem cell genes were more expressed in the control group (~2 fold), and oocyte genes were more expressed in the study group (P < 0. 0001) (~2. 5–3. 11 fold). These results were also confirmed by immunocytochemistry. The amount of steroids was much higher in the study group (three times and five times in two weeks) (P < 0. 0001). Conclusions: Stem CELLS can be obtained from the FF of PCOS, and normal FF has a positive effect on the growth and maturation of oocyte-like CELLS in vitro.

Type 1 diabetes (T1D) is an autoimmune disease resulting from the damage of pancreatic 𝛽-CELLS mediated by autoreactive CD4+ and CD8+ T CELLS. In recent years, FOLLICULAR T helper (Tfh) CELLS have been recognized as a subpopulation of CD4+ T CELLS providing help for B CELLS differentiation and antibody production. In this study, we examined the frequency of circulating CD4+CXCR5+ and CD4+CXCR5+ICOS+ (representing Tfh) CELLS as well as serum levels of anti-glutamic acid decarboxylase 65 (GAD65) and islet cell autoantibodies (ICA) in children with type I diabetes. We analyzed the percentage of Tfh CELLS within peripheral blood mononuclear CELLS in 20 children with T1D (≤ 300 days from disease onset; Mean age 6. 8± 4. 6 years) and 18 healthy individuals (Mean age 8. 8± 2. 2 years) using flow cytometry. Anti-glutamic acid decarboxylase (GAD) and islet-cell cytoplasmic autoantibodies (ICA) levels were determined by ELISA and indirect immunofluorescence respectively. We found that the frequency of CD4+CXCR5+ and CD4+CXCR5+ICOS+ (Tfh) CELLS were significantly increased in the peripheral blood of patients compared with healthy controls (p<0. 001). Furthermore, elevated levels of anti-GAD and ICA antibodies were detected in children with T1D (p=0. 001 and p=0. 02 respectively). There was no correlation between Tfh CELLS frequency and the autoantibody levels. The results of our study indicate an increased frequency of Tfh CELLS in children With T1D that could suggest a possible role of these CELLS in the disease pathogenesis.