Sercin Karahuseyinoglu

Biology of stem cells in human umbilical cord stroma: in situ and in vitro surveys.

Cells in the umbilical cord stroma have gained attention in recent years; however, differentiation to certain lineages in humans has been demonstrated in few studies. Unlike bone marrow MSCs, human umbilical cord stroma cells (HUCSCs) are far from being well characterized. This study attempts to describe proliferation, structural, and differentiation properties of these cells to account for their exceptional nature in many aspects. Cellular dynamics, cellular structure, and the degree of transformations during expansion and differentiation into mesenchymal and neuronal lineages were examined in vitro over a 10‐month period. Comparisons with human bone marrow MSCs regarding differentiation were performed. HUCSCs in culture revealed two distinct cell populations, type 1 and type 2 cells, that possessed differential vimentin and cytokeratin filaments. Corresponding cells were encountered in cord sections displaying region‐specific localization. α‐Smooth muscle actin and desmin filaments, which were evident in cord sections, diminished through passages. No difference was noted regarding type 1 and type 2 cells in differentiation to chondrogenic, adipogenic, and osteogenic lineages, whereas a preferential differentiation was noted in neuronal lineage. Relative success was achieved by production of chondrocytic spheres and osteogenic monolayers, whereas adipocytes were immature compared with bone marrow MSCs. The presence of neuronal markers suggests that they transform into a certain state of maturity under neurogenic induction. Conclusively, HUCSCs retain their original phenotype in culture without spontaneous differentiation, have a limited lifespan, and bear multipotent stem cell characteristics. Given these characteristics, they may be generally considered progenitor cells if manipulated under appropriate conditions and deserve further study to be potentially used in cell‐based therapies.

Concise Review: Human Umbilical Cord Stroma with Regard to the Source of Fetus‐Derived Stem Cells

Human umbilical cord (UC) has been a tissue of increasing interest in recent years. Many groups have shown the stem cell potency of stromal cells isolated from the human UC mesenchymal tissue, namely, Whartons jelly. Since UC is a postnatal organ discarded after birth, the collection of cells does not require an invasive procedure with ethical concerns. Stromal cells, as the dominant cells of this fetus‐derived tissue, possess multipotent properties between embryonic stem cells and adult stem cells. They bear a relatively higher proliferation rate and self‐renewal capacity. Although they share common surface markers with bone marrow‐derived MSCs, they also express certain embryonic stem cell markers, albeit in low levels. Without any spontaneous differentiation, they can be successfully differentiated into mature adipocytes, osteoblasts, chondrocytes, skeletal myocytes, cardiomyocytes, neurons, and endothelial cells. While causing no immunorejection reaction, they effectively function in vivo as dopaminergic neurons, myocytes, and endothelial cells. Given these characteristics, particularly the plasticity and developmental flexibility, UC stromal cells are now considered an alternative source of stem cells and deserve to be examined in long‐term clinical trials. This review first aims to document the published findings so far regarding the nature of human UC stroma with special emphasis on the spatial distribution and functional structure of stromal cells and matrix, which serves as a niche for residing cells, and, secondly, to assess the in vitro and in vivo experiments in which differential stem cell potencies were evaluated.

Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa

PurposeCryopreservation of sperm is a widely used technique to maintain and protect the fertility in various occasions such as infertility and malignancy treatments. This study aims to reveal the effects of freezing and thawing on human spermatozoa.Materials and methodsTo evaluate the effects of freeze–thawing, semen samples were evaluated by light microscopy by means of morphology, motility and viability, by scanning and transmission electron microscopy for detailed ultrastructural changes.ResultsAfter cryopreservation, a significant decrease in spermatozoa viability was observed (p < 0.01). Group a, b and c motility according to World Health Organization criteria decreased considerably (p < 0.05, p < 0.01, p < 0.05, respectively), whereas there was a substantial increase in group d motility. A strong correlation between rise in number of immotile spermatozoa and decrease in viability was also noted (r = −0.848, p < 0.01). Post-thaw light microscopic studies revealed a considerable decrease in rate of normal spermatozoa (p < 0.05). A considerable decline in the rate of normal sperm was also observed by TEM (p < 0.05). Statistically, acrosomal changes and subacrosomal swelling were found to be significantly increased (both p < 0.05), where the latter appears to be a novel finding in literature.ConclusionCryopreservation has deleterious effects on spermatozoa, especially on plasmalemma, acrosomes and tails. Electron microscopy is the ultimate modality to investigate spermatogenic cells.

Functional Structure of Adipocytes Differentiated from Human Umbilical Cord Stroma‐Derived Stem Cells

It has been previously demonstrated that human umbilical cord stroma‐derived stem cells (HUCSCs) are competent to differentiate into adipocytes. However, controversies have arisen as to whether HUCSCs can become mature adipocytes or not, and to what extent these cells can be induced in adipogenic pathway. Here, we extensively analyzed their adipogenic potency with a structural and functional approach by determining lipid formation dynamics in concordance to adipocyte‐specific markers. During a 35‐day period, HUCSCs respond to adipogenic induction, at which point 88% of cells exhibited multilocular lipid granules (LGs) having a mean diameter of 3 μm in round‐shaped, F‐actin‐poor cells. Although the 1st week of induction did not generally display typical lipidogenic phenotypes, the degree of adipogenesis was dissected and confirmed by mRNA expressions of peroxisome proliferator‐activated receptor γ, C/EBP‐β, sterol regulatory element‐binding transcription factor 1, adipophilin, stearoyl‐CoA desaturase, glycerol 3‐phosphate dehydrogenase 1, LIPE, adiponectin, and leptin. All markers tested were found elevated in various amounts (3–70‐fold) around day 7 and reached a plateau after day 14 or 21 (5–335‐fold). Perilipin as a surface protein around the LGs was confined exclusively to the enlarging LGs. Conclusively, we propose that after the termination of proliferation, HUCSCs possess the biochemical and cellular machinery to successfully differentiate into maturing adipocytes under adipogenic conditions, and this feature will ultimately allow these fetus‐derived stem cells to be used for various therapeutic or esthetic purposes.

GnRH agonist leuprolide acetate does not confer any protection against ovarian damage induced by chemotherapy and radiation in vitro

STUDY QUESTION Is there any in vitro evidence for or against ovarian protection by co-administration of a GnRH agonist with chemotherapy in human? SUMMARY ANSWER The co-administration of GnRH agonist leuprolide acetate with cytotoxic chemotherapy agents does not preserve ovarian reserve in vitro. WHAT IS KNOWN ALREADY Randomized controlled trials of the co-administration of gonadotrophin-releasing hormone (GnRH) agonists with adjuvant chemotherapy to preserve ovarian function have shown contradictory results. This fact, together with the lack of a proven molecular mechanism of action for ovarian protection with GnRH agonist (GnRHa) places this approach as a fertility preservation strategy under scrutiny. We therefore aimed in this study to provide in vitro evidence for or against the role of GnRHa in the prevention of chemotherapy-induced damage in human ovary. STUDY DESIGN, SETTINGS, SIZE AND DURATION This translational research study of ex vivo and in vitro models of human ovary and granulosa cells was conducted in a university hospital between 2013 and 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS Ovarian cortical pieces (n = 15, age 14-37) and mitotic non-luteinized (COV434 and HGrC1) and non-mitotic luteinized human granulosa cells (HLGC) expressing GnRH receptor were used for the experiments. The samples were treated with cyclophosphamide, cisplatin, paclitaxel, 5-FU, or TAC combination regimen (docetaxel, adriamycin and cyclophosphamide) with and without GnRHa leuprolide acetate for 24 h. DNA damage, apoptosis, follicle reserve, hormone markers of ovarian function and reserve (estradiol (E2), progesterone (P) and anti-mullerian hormone (AMH)) and the expression of anti-apoptotic genes (bcl-2, bcl-xL, bcl-2L2, Mcl-1, BIRC-2 and XIAP) were compared among control, chemotherapy and chemotherapy + GnRHa groups. MAIN RESULTS AND THE ROLE OF CHANCE The greatest magnitude of cytotoxicity was observed in the samples treated with cyclophosphamide, cisplatin and TAC regimen. Exposure to these drugs resulted in DNA damage, apoptosis and massive follicle loss along with a concurrent decline in the steroidogenic activity of the samples. GnRHa co-administered with chemotherapy agents stimulated its receptors and raised intracellular cAMP levels. But it neither activated anti-apoptotic pathways nor prevented follicle loss, DNA damage and apoptosis induced by these drugs. LIMITATIONS, REASONS FOR CAUTION Our findings do not conclusively rule out the possibility that GnRHa may offer protection, if any, through some other mechanisms in vivo. WIDER IMPLICATIONS OF THE FINDINGS GnRH agonist treatment with chemotherapy does not prevent or ameliorate ovarian damage and follicle loss in vitro. These data can be useful when consulting a young patient who may wish to receive GnRH treatment with chemotherapy to protect her ovaries from chemotherapy-induced damage.

ALENDRONATE ENHANCES ANTIBIOTIC-IMPREGNATED BONE GRAFTS IN THE TREATMENT OF OSTEOMYELITIS

Bisphosphonates are systemic drugs. There is limited knowledge about their effects when applied locally and in osteomyelitis treatment. A prospective longitudinal randomised controlled study was designed in rat tibia to test the efficacy of local or systemically administered bisphosphonates for controlling the osteolytic reactions and possible effects on local infection control. Tibial osteomyelitis was induced in 72 Wistar albino rats with Staphylococcus aureus ATCC 25923 strain. Débridement was performed on all rats in all groups. No other treatment was given to the control group. Treatment groups received “plain bone grafts”, “vancomycin-loaded bone grafts”, “vancomycin-loaded bone grafts+systemic alendronate”, “alendronate-impregnated bone grafts” and “vancomycin+alendronate-impregnated grafts”. Study results were evaluated by swab cultures, radiology, quantitative computed tomography, dual-energy X-ray absorptiometry (DEXA) and histopathology. S. aureus was eradicated in groups II and IV by the sixth week. Diaphyseal widening, bone deformation, diaphyseal widening and osteolysis scores were significantly lower (p < 0.05), and bone mineral content, density measurements and DEXA scores were significantly higher (p = 0.001) with alendronate administration. Histology revealed marked osteoblastic activity. Local alendronate interfered with local infection control. Even though local alendronate at the given dose has stronger effects, the possible effects on the local inflammatory process needs to be clarified.RésuméLes Bisphosphonates sont des médicaments utilisés par voie systémique avec un effet systémique. Nous n’avons aucune connaissance sur leurs effets lorsqu’ils sont utilisés localement, notamment dans le traitement de l’ostéomyélite. Une étude prospective longitudinale randomisée a été réalisée sur le tibia de rat afin de tester l’efficacité de l’administration systémique ou locale des bisphosphonates sur les réactions ostéolytiques et sur de possibles effets sur une infection locale. L’ostéomyélite tibiale a été induite chez 72 rats albinos Wistar avec une inoculation d’un staphylocoque Aureus ATCC 25923. Une mise à plat a été réalisée chez tous les rats, dans tous les groupes. Il n’y a pas eu d’autres traitements dans le groupe contrôle. Les autres groupes ont reçu une greffe osseuse, soit une greffe osseuse imprégnée de vancomycine soit une greffe osseuse imprégnée de vancomycine et avec injection systémique d’alendronate, soit une greffe osseuse imprégnée d’alendronate et enfin une greffe osseuse imprégnée d’alendronate et de vancomycine. Les résultats ont été évalués par cultures bactériologiques, radio, QCT, DEXA et histopathologie. Le staphylocoque doré a été éradiqué dans les groupes II et IV après six semaines. Le gonflement diaphysaire, la déformation osseuse et l’élargissement diaphysaire avec lésions ostéolytiques ont été de façon significative diminués (p < 0.05), de même que la densité minérale osseuse et le score DEXA ont été significativement plus élevés (p = 0.001) ceci grâce à l’administration de l’alendronate. L’examen histologique a mis en évidence une activité ostéoblastique et l’on peut affirmer que l’administration locale d’alendronate a une influence sur une infection locale. Cependant, l’alendronate administré localement peut avoir des effets importants au niveau du processus inflammatoire qu’il faudra étudier secondairement.

DNA methylation profiling identifies novel markers of progression in hepatitis B-related chronic liver disease

BackgroundChronic hepatitis B infection is characterized by hepatic immune and inflammatory response with considerable variation in the rates of progression to cirrhosis. Genetic variants and environmental cues influence predisposition to the development of chronic liver disease; however, it remains unknown if aberrant DNA methylation is associated with fibrosis progression in chronic hepatitis B.ResultsTo identify epigenetic marks associated with inflammatory and fibrotic processes of the hepatitis B-induced chronic liver disease, we carried out hepatic genome-wide methylation profiling using Illumina Infinium BeadArrays comparing mild and severe fibrotic disease in a discovery cohort of 29 patients. We obtained 310 differentially methylated regions and selected four loci comprising three genes from the top differentially methylated regions: hypermethylation of HOXA2 and HDAC4 along with hypomethylation of PPP1R18 were significantly linked to severe fibrosis. We replicated the prominent methylation marks in an independent cohort of 102 patients by bisulfite modification and pyrosequencing. The timing and causal relationship of epigenetic modifications with disease severity was further investigated using a cohort of patients with serial biopsies.ConclusionsOur findings suggest a linkage of widespread epigenetic dysregulation with disease progression in chronic hepatitis B infection. CpG methylation at novel genes sheds light on new molecular pathways, which can be potentially exploited as a biomarker or targeted to attenuate inflammation and fibrosis.

The role of ovarian reserve markers in prediction of clinical pregnancy

Abstract To evaluate the role of ovarian reserve markers in the prediction of clinical pregnancy and embryo transfer accomplishment among poor responder IVF applicants. 304 female poor responder IVF applicants were included in this prospective cohort study conducted at the IVF-unit. Antral follicle count, FSH, LH, E2, AMH and IVF outcomes were compared in pregnant and non-pregnant groups as well as in ET vs. non-ET groups. The number of retrieved oocytes was significantly correlated positively with AMH and AFC, and negatively with FSH and age. Quartiles of FSH and AFC were similar to the rate of pregnancy. Quartiles of AMH (<25%/25–75% and <25%/>75%) were statistically significant. Mean serum levels for AMH were significantly lower in the non-ET group. Our findings seem to indicate that day 3 AMH values can predict ET accomplishment with a sensitivity of 96% and a specificity of 35%. Quartiles of AMH <25% (< 0.21 ng/mL) can predict the IVF results among poor responder IVF applicants. Impact statement Various cut-off values have been determined for day 3 serum AMH values. These values help to determine the groups that are expected to give normal, high or low response to stimulation and decide the treatment options. In contrast to other groups of patients, poor responders cannot reach the embryo transfer stage for several reasons. These are; absence of a mature oocyte after oocyte pick-up, fertilisation failure without male factor or poor embryo quality. In the present study; a cut-off value of 0.33 ng/mL for the prediction of ET accomplishment in poor responder patients was determined with a sensitivity of 96%. Additionally, clinical pregnancy could not be achieved under the value of 0.21 ng/mL day 3 AMH values. It is important to clarify the embryo transfer success of poor responder patients prior to expected treatment success. Pre-treatment counselling for these patients would lessen the disappointment that may develop after treatment. The cost-effectiveness of treatments below these AMH values can be determined by further studies.

KDM2B, an H3K36-specific demethylase, regulates apoptotic response of GBM cells to TRAIL

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively kill tumor cells. TRAIL resistance in cancers is associated with aberrant expression of the key components of the apoptotic program. However, how these components are regulated at the epigenetic level is not understood. In this study, we investigated novel epigenetic mechanisms regulating TRAIL response in glioblastoma multiforme (GBM) cells by a short-hairpin RNA loss-of-function screen. We interrogated 48 genes in DNA and histone modification pathways and identified KDM2B, an H3K36-specific demethylase, as a novel regulator of TRAIL response. Accordingly, silencing of KDM2B significantly enhanced TRAIL sensitivity, the activation of caspase-8, -3 and -7 and PARP cleavage. KDM2B knockdown also accelerated the apoptosis, as revealed by live-cell imaging experiments. To decipher the downstream molecular pathways regulated by KDM2B, levels of apoptosis-related genes were examined by RNA-sequencing upon KDM2B loss, which revealed derepression of proapoptotic genes Harakiri (HRK), caspase-7 and death receptor 4 (DR4) and repression of antiapoptotic genes. The apoptosis phenotype was partly dependent on HRK upregulation, as HRK knockdown significantly abrogated the sensitization. KDM2B-silenced tumors exhibited slower growth in vivo. Taken together, our findings suggest a novel mechanism, where the key apoptosis components are under epigenetic control of KDM2B in GBM cells.

Comparison of estradiol and progesterone priming/antagonist/letrozole and microdose flare-up protocols for poor responders undergoing intracytoplasmic sperm injection

Abstract Background: To compare the effect of the GnRH antagonist/letrozole/gonadotropin protocol with the microdose GnRH agonist flare-up protocol in poor ovarian responders for intracytoplasmic sperm injection. Materials and methods: One hundred twenty-one consecutive patients suspected of having or with a history of poor ovarian response between January 2009 and June 2010, who were undergoing ICSI were enrolled. The microdose flareup (MF) protocol was used in 79 patients and the estradiol + progesterone/letrozole + gonadotropin and GnRH antagonist (EP/ALG) protocol was used in 42 patients. Results: Age of the patients, duration of infertility, basal FSH, the total gonadotropin consumption, duration of stimulation, E2 level on the day of hCG administration, the number of embryo transferred, the fertilization rate, implantation rate, clinical pregnancy rate and the live birth rate were not statistically different (p > 0.05). Only the number of oocytes retrieved was significantly higher in the EP/LGA group (1.7 ± 0.7 versus 2.6 ± 0.6). Conclusion: The EP/LGA protocol has no significant improvement against the microdose flare-up protocol in poor responder patients. Chinese abstract 背景：比较卵巢低反应性卵胞浆内单精子注射中GnRH拮抗剂/来曲唑/促性腺激素方案与微量GnRH激动剂flare-up方案的效果。 材料与方法：研究对象为2009年1月到2010年7月之间、怀疑卵巢低反应性或曾有卵巢低反应性史的121例行ICSI的患者。79例患者应用微量flare-up（microdose flare-up，MF）方案，42例患者应用雌二醇+黄体酮/来曲唑+促性腺激素和GnRH拮抗剂（estradiol + progesterone/letrozole + gonadotropin and GnRH antagonist，EP/ALG）方案。 结果：两组患者年龄、不孕时间、基础FSH水平、促性腺激素总用量、刺激时间、hCG日雌二醇水平、移植卵母细胞数量、受精率、移植率、临床妊娠率、活产率无统计学差异（p＞0.05）。EP/LGA方案只有获卵数明显高于MF方案（1.7 ± 0.7 vs 2.6 ± 0.6）。 结论：EP/LGA方案与微量刺激方案相比并不显著改善卵巢低反应性患者的反应性。