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Rahimi G.,University of Cologne | Isachenko V.,University of Ulm | Kreienberg R.,University of Ulm | Sauer H.,Justus Liebig University | And 4 more authors.
European Journal of Obstetrics Gynecology and Reproductive Biology | Year: 2010

Objective: In ovarian tissue grafts there is a massive loss of follicles during the ischaemic period until re-vascularisation is established. The aim of our study was to investigate the influence of different cryopreservation techniques on the ability for the re-vascularisation of ovarian tissue transplanted to SCID mice. Study design: Ovarian fragments from five patients were cut into pieces (∼0.5 mm × 1.0 mm × 1.0 mm) and randomly distributed into three groups: fresh non-treated tissue (group A); tissue conventionally frozen in standard 0.5 ml insemination straws with 1.5 M ethylene glycol + 0.1 M sucrose, with thawing in a 40 °C water bath and step-wise removal of cryoprotectants at room temperature in 0.5 M, 0.25 M and 0.15 M sucrose with gentle agitation (group B); tissue vitrified in 2.62 M dimethylsulphoxide + 2.6 M acetamide + 1.31 M propylene glycol + 0.0075 M polyethylene glycol, with warming by direct plunging of solid specimens with ovarian pieces into 20 ml of 50% vitrification solution pre-warmed to 40 °C and dilution of cryoprotectants in a decreasing concentration of vitrification solution (25%, 12.5%) at room temperature (group C). We used a xenograft model in which ovarian tissue pieces of all three groups were subcutaneously transplanted in SCID mice. The animals were sacrificed on the third day after ovarian tissue transplantation and then weekly during 1 month to obtain the ovarian tissue grafts. These samples were examined by immunohistochemical staining with the endothelial cell-specific marker platelet endothelial cell adhesion molecule-1 (PECAM-1) to determine angiogenesis. Histological observation of tissue after explantation was performed and quality and quantity of follicles were assessed. Results: No PECAM-1 staining was observed in all treatment groups prior to grafting. After warming and in vivo culture of ovarian tissue, the beginning of angiogenesis in pieces from all treatment groups on the third day was detected by PECAM-1 staining. After 4 weeks of in vivo culture the overall area of PECAM-1-positive blood vessels significantly increased (P < 0.05), independent of the type of cryopreservation (groups B and C vs. group A). It was found that transplantation technique had negative influence on the integrity of follicles independent of the type of treatment during in vivo culture. The duration of in vivo culture has a negative, but not statistically significant, influence on follicle quality in long-cultured transplants inside each treatment group (P > 0.5). Conclusion: The process of re-vascularisation of transplanted ovarian tissue is independent of the type of treatment and does not influence follicle quality. © 2009 Elsevier Ireland Ltd. All rights reserved.


Jabeen A.,University of Essex | Miranda-Sayago J.M.,University of Essex | Obara B.,Durham University | Spencer P.S.,University of Essex | And 5 more authors.
Biology of Reproduction | Year: 2013

Human placental syncytiotrophoblasts lack expression of most types of human leukocyte antigen (HLA) class I and class II molecules; this is thought to contribute to a successful pregnancy. However, the HLA class Ib antigens HLA-G,-E, and-F and the HLA class Ia antigen HLA-C are selectively expressed on extravillous trophoblast cells, and they are thought to play a major role in controlling feto-maternal tolerance. We have hypothesized that selective expression, coupled with the preferential physical association of pairs of HLA molecules, contribute to the function of HLA at the feto-maternal interface and the maternal recognition of the fetus. We have developed a unique analytical model that allows detection and quantification of the heterotypic physical associations of HLA class I molecules expressed on the membrane of human trophoblast choriocarcinoma cells, ACH-3P and JEG-3. Automated image analysis was used to estimate the degree of overlap of HLA molecules labeled with different fluorochromes. This approach yields an accurate measurement of the degree of colocalization. In both JEG-3 and ACH-3P cells, HLA-C,-E, and-G were detected on the cell membrane, while the expression of HLA-F was restricted to the cytoplasm. Progesterone treatment alone induced a significant increase in the expression level of the HLA-G/HLA-E association, suggesting that this heterotypic association is modulated by this hormone. Our data shows that the cell-surface HLA class I molecules HLA-G,-E, and-C colocalize with each other and have the potential to form preferential heterotypic associations. © 2013 by the Society for the Study of Reproduction, Inc.


Todorov P.,Institute of Biology and Immunology of Reproduction | Hristova E.,Institute of Biology and Immunology of Reproduction | Konakchieva R.,Institute of Biology and Immunology of Reproduction | Dimitrov J.,Sofia University
Cell Biology International | Year: 2010

Fetal stem cells possess some intriguing characteristics, which delineate them as promising cellular therapeutics. They are less immunogenic, at lower stage of differentiation and have higher potential for repopulation and migration. Furthermore, the fetal stem cells secrete a set of cytokines and growth factors, which stimulate the regeneration of the recipient tissue. The present study indicated that the adhesive fraction of human fetal liver cells possessed the morphological characteristics of mesenchymal stem cells, as well as potential to differentiate into adipocyte and osteoblast lineages. The immunophenotypic analysis showed that the cells expressed CD13, CD73, CD90 and CD105 (typical for mesenchymal stem cells) and lacked the haematopoietic lineage markers CD34 and CD45. Addressing the issue of the low-temperature storage of the human fetal liver cells, four different methods for cryopreservation were assessed: conventional slow freezing, program freezing and two vitrification protocols. The obtained results demonstrated that the cells were cryotolerant and maintained their properties and differentiation potential after thawing. Program freezing showed to be the most efficient method for cryopreservation of the investigated cells. © The Author(s) Journal compilation © 2010 Portland Press Limited.


Lech A.,Military Institute of Medicine | Lech A.,University of Warsaw | Daneva T.,Institute of Biology and Immunology of Reproduction | Pashova S.,Institute of Biology and Immunology of Reproduction | And 5 more authors.
Frontiers in Bioscience | Year: 2013

Ovarian cancer is characterized by the highest mortality rate among gynecologic malignancies. Therefore, there is a growing need for innovative therapies and techniques for monitoring and prevention of this disease. The exact cause of most ovarian tumors usually remains unknown. Ovarian cancer is believed to be caused by a range of different variables. This review is an attempt to summarize some genetic factors involved in the disruption of certain signaling pathways responsible for ovarian tumor transformation and development. Those factors considerably contribute to accurate diagnostics, treatment and prognosis in ovarian cancer.


Abadjieva D.,Institute of Biology and Immunology of Reproduction | Grigorova S.,Institute of Animal Science | Petkova M.,Institute of Animal Science
Asian Journal of Animal and Veterinary Advances | Year: 2016

Objective: The objective of the present study was to investigate the effect of jodis concentrate (Ukraine) on testicular morphometry and histological changes in rabbit bucks. Methodology: An experiment was conducted with a total of 30 white New Zealand weaned male rabbits with average body weight of 1.5 kg. The animals were distributed in three groups viz., a control and two experimental groups, with 10 animals in each. The growing rabbits from both experimental groups received jodis concentrate in a dose of 2 mL LG1 of drinking water. Results: The rabbits from the 1st experimental group were born from mothers not supplemented with jodis concentrate during the pregnancy and the sucking periods. The animals from the 2nd experimental group were born from mothers who were given the tested product during the pregnancy and sucking periods. The trial lasted for 48 days. Testes were weighed and prepared for histological examination. Conclusion: The iodine from jodis concentrate in a dose of 2 mL LG1 of drinking water has a potential to improve the testicular morphometry and histology of experimental rabbit bucks born from mothers who were not given the tested additive. On the other hand, a clear negative impact was observed on the morphological and histological changes in the testes of rabbit bucks born from mothers supplemented with jodis concentrate during pregnancy. Therefore, serious attention should be paid to the total level of iodine in the feed for rabbits intended for breeding. © 2016 D. Abadjieva et al.


PubMed | Institute of Biology and Immunology of Reproduction, Trakia University and Goce Delcev University of Štip
Type: Journal Article | Journal: In vitro cellular & developmental biology. Animal | Year: 2016

Rabbits are considered as appropriate animal models to study some obesity-associated abnormalities because of the similarity of their blood lipid profile and metabolism to humans. The current study was focused on comparison of adipose differentiation ability in rabbit adipose-derived stem cells (ADSC) in vitro. Subcutaneous and visceral stromal vascular fractions (SVF) were isolated from three 28-d-old New Zealand rabbits by collagenase digestion. Supernatants from both isolates were collected 24h after the initial plating. On the fourth passage, all isolated cell types undergo triplicate adipogenic induction. The adipose induction potential was calculated as percentage of increasing optical density (OD) values. The data revealed that with increasing the number of induction cycles, the induction tendency in visceral ADSC decreased in contrast to the subcutaneous ones. Although the supernatants did not reach induction levels of their relevant precursors, they follow the same pattern in both subcutaneous and visceral ADSC. All cell types successfully passed osteogenic and chondrogenic differentiation. In conclusion, the best adipose induction ability was observed in directly plated subcutaneous cell population. The increase of induction numbers depressed adipose induction ability in cell populations derived from visceral fat depots.


PubMed | Institute of Biology and Immunology of Reproduction, MedEvent Dr. Merzenich GmbH, University College London, University of Cologne and Bulgarian Academy of Science
Type: Journal Article | Journal: Reproductive biology and endocrinology : RB&E | Year: 2016

Phosphatidylserine is the phospholipid component which plays a key role in cell cycle signaling, specifically in regards to necrosis and apoptosis. When a cell affected by some negative factors, phosphatidylserine is no longer restricted to the intracellular side of membrane and can be translocated to the extracellular surface of the cell. Cryopreservation can induce translocation of phosphatidylserine in response to hypoxia, increasing intracellular CaOvarian fragments from twelve patients were divided into small pieces of two types, medulla-free cortex (Group 1, n=42, 1.5-3.01.5-3.00.5-0.8mm) and cortex with medulla (Group 2, n=42, 1.5-3.01.5-3.01.5-2.0mm), pre-cooled after operative removal to 5C for 24h and then conventionally frozen with 6% dimethyl sulfoxide, 6% ethylene glycol and 0.15M sucrose in standard 5-ml cryo-vials. After thawing at +100C and step-wise removal of cryoprotectants in 0.5M sucrose, ovarian pieces were xenografted to SCID mice for 45days. The efficacy of tissues cryopreservation, taking into account the presence or absence of medulla, was evaluated by the development of follicles (histology with hematoxylin-eosin) and through the intensity of translocation of phosphatidylserine (FACS with FITC-Annexin V and Propidium Iodide).For Groups 1 and 2, the mean densities of follicles per 1mmThe presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue.


PubMed | University Hospital Jena, University of Barcelona, University of Duisburg - Essen, c Microtrac GmbH and 11 more.
Type: Journal Article | Journal: Critical reviews in clinical laboratory sciences | Year: 2016

Extracellular vesicles (EVs) are released from almost all cells and tissues. They are able to transport substances (e.g. proteins, RNA or DNA) at higher concentrations than in their environment and may adhere in a receptor-controlled manner to specific cells or tissues in order to release their content into the respective target structure. Blood contains high concentrations of EVs mainly derived from platelets, and, at a smaller amount, from erythrocytes. The female and male reproductive tracts produce EVs which may be associated with fertility or infertility and are released into body fluids and mucosas of the urogenital organs. In this review, the currently relevant detection methods are presented and critically compared. During pregnancy, placenta-derived EVs are dynamically detectable in peripheral blood with changing profiles depending upon progress of pregnancy and different pregnancy-associated pathologies, such as preeclampsia. EVs offer novel non-invasive diagnostic tools which may reflect the situation of the placenta and the foetus. EVs in urine have the potential of reflecting urogenital diseases including cancers of the neighbouring organs. Several methods for detection, quantification and phenotyping of EVs have been established, which include electron microscopy, flow cytometry, ELISA-like methods, Western blotting and analyses based on Brownian motion. This review article summarises the current knowledge about EVs in blood and cord blood, in the different compartments of the male and female reproductive tracts, in trophoblast cells from normal and pre-eclamptic pregnancies, in placenta ex vivo perfusate, in the amniotic fluid, and in breast milk, as well as their potential effects on natural killer cells as possible targets.


PubMed | Institute of Biology and Immunology of Reproduction
Type: Case Reports | Journal: Ear, nose, & throat journal | Year: 2016

To the best of the authors knowledge, no case of a patient with stapediovestibular ankylosis who was also coinfected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) has been previously described in the literature. This report describes the case of a 36-year-old woman who was diagnosed with all three conditions. The clinical diagnosis of stapes fixation was based on otoscopic, audiometric, tympanometric, and surgical findings. Stapedectomy was performed, and perilymph and serum samples were obtained and tested for anti-HIV and anti-HCV antibodies. While the titers of anti-HCV antibodies in the serum and perilymph were of similar magnitude, there were almost 16 times more anti-HIV antibodies in the serum than in the perilymph. This case offered a unique opportunity to study the titers of anti-HIV/HCV antibodies in both the blood serum and perilymph. Data relating to these titers may provide new insights into the mechanisms of stapediovestibular ankylosis and inner ear immunology.


PubMed | Institute of Biology and Immunology of Reproduction, Praxisklinik Schoenhauserstrasse, Institute of Microbiology and University of Cologne
Type: Journal Article | Journal: PloS one | Year: 2015

To translocation (externalization) of phosphatidylserine lead at least the five negative effects observed during cells cryopreservation: hypoxia, increasing of intracellular Ca2+, osmotic disruption of cellular membranes, generation of reactive oxygen species (ROS) and lipid peroxidation. The aim of this study was to test the intensiveness of the phosphatidylserine translocation immediately after thawing and after 45 d xenografting of human ovarian tissue, which was either frozen just after operative removal from patient or cooled before cryopreservation to 5C for 24 h and then frozen.Ovarian fragments from twelve patients were divided into small pieces in form of cortex with medulla, and randomly divided into the following four groups. Pieces of Group 1 (n=30) were frozen immediately after operation, thawed and just after thawing their quality was analyzed. Group 2 pieces (n=30) after operation were cooled to 5C for 24 h, then frozen after 24 h pre-cooling to 5C, thawed and just after thawing their quality was analyzed. Group 3 pieces (n=30) were frozen immediately after operation without pre-cooling, thawed, transplanted to SCID mice and then, after 45 d of culture their quality was analyzed. Group 4 pieces (n=30) were frozen after 24 h pre-cooling to 5C, thawed, transplanted to SCID mice and then, after 45 d their quality was analyzed. The effectiveness of the pre-freezing cooling of tissuewas evaluated by the development of follicles (histology) and by intensiveness of translocation of phosphatidylserine (FACS with FITC-Annexin V and Propidium Iodide).For groups 1, 2, 3 and 4 the mean densities of follicles per 1 mm3 was 19.0, 20.2, 12.9, and 12.2, respectively (P1-2, 3-4 >0.1). For these groups, 99%, 98%, 88% and 90% preantral follicles, respectively were morphologically normal (P1-2, 3-4 >0.1). The FACS analysis showed significantly decreased intensiveness of translocation of phosphatidylserine after pre-cooling of frozen tissue (46.3% and 33.6% in Groups 2 and 4, respectively), in contrast with tissue frozen without pre-cooling (77.1% and 60.2 % in Groups 1 and 3, respectively, P1, 3-2, 4 <0.05).Long time (24 h) cooling of ovarian tissue to 5C before cryopreservation decreased translocation of phosphatidylserine that evidences about increases the viability of the cells in the tissue after thawing.

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