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C Wulff, M Weigand, R Kreienberg, and HM Fraser

Normal embryonic development is dependent upon a sufficient oxygen, nutrient and waste exchange through the placenta. In primates including humans, this exchange is attained by successful haemochorial placentation which requires the transformation of maternal intramyometrial spiral arterioles by trophoblast invasion to gain uteroplacental circulation, and establishment and maintenance of a competent fetoplacental vasculature. Thus, trophoblast and endothelial cell differentiation, proliferation and invasion occurring during placentation have to be tightly regulated. This review focuses on the diverse developmental steps during haemochorial placentation in humans and other primates and the possible involvement of angiogenic growth factors (vascular endothelial growth factor (VEGF) and angiopoietins (Ang)) in these processes, highlighting the importance of specific actions of angiogenic ligand-receptor pairs. It is hypothesized that VEGF/VEGF-R1 and Ang-1/Tie receptor 2 (Tie-2) may regulate trophoblast differentiation and invasion; VEGF/VEGF-R2 and Ang-1/Tie-2 may promote fetoplacental vascular development and stabilization; and Ang-2/Tie-2 may be involved in maternal vascular remodelling. The importance of a tight regulation of angiogenic factors and their endogenous antagonists for normal development of the placenta is demonstrated by failure of this system, resulting in abnormal placenta vascularization and trophoblast invasion associated with intrauterine growth retardation or pre-eclampsia.

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D Herr, M Rodewald, H M Fraser, G Hack, R Konrad, R Kreienberg, and C Wulff

This study was performed in order to evaluate the role of angiotensin II in physiological angiogenesis. Human umbilical vein endothelial cells (HUVEC) were stained for angiotensin II type 1 receptor (AGTR1) immunocytochemically and for gene expression of renin–angiotensin system (RAS) components. The regulation of the angiogenesis-associated genes vascular endothelial growth factor (VEGF) and angiopoietins (ANGPT1 and ANGPT2) were studied using quantitative RT-PCR. Furthermore, we examined the effect of angiotensin II on the proliferation of HUVEC using Ki-67 as well as BrdU immunocytochemistry and investigated whether the administration of the AGTR1 blocker candesartan or the VEGF antagonist FLT1-Fc could suppress the observed angiotensin II-dependent proangiogenic effect. AGTR1 was expressed in HUVEC and the administration of angiotensin II significantly increased the gene expression of VEGF and decreased the gene expression of ANGPT1. Since the expression of ANGPT2 was not affected significantly the ratio of ANGPT1/ANGPT2 was decreased. In addition, a significantly increased endothelial cell proliferation was observed after stimulation with angiotensin II, which was suppressed by the simultaneous administration of candesartan or the VEGF antagonist FLT1-Fc. These results indicate the potential capacity of angiotensin II in influencing angiogenesis by the regulation of angiogenesis-associated genes via AGTR1. Since VEGF blockade opposed the effect of angiotensin II on cell proliferation, it is hypothesised that VEGF mediates the angiotensin II-dependent effect in concert with the changes in angiopoietin expression. This is the first report of the RAS on the regulation of angiogenesis-associated genes in physiology.

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V Isachenko, I Lapidus, E Isachenko, A Krivokharchenko, R Kreienberg, M Woriedh, M Bader, and J M Weiss

Cryopreservation as a process can be divided into two methods: conventional freezing and vitrification. The high effectiveness of vitrification in comparison with conventional freezing for human oocytes and embryos is shown, whereas data on human ovarian tissue are limited. The aim of this study was to compare the safety and effectiveness of conventional freezing and vitrification of human ovarian tissue. Ovarian tissue fragments from 15 patients were transported to the laboratory within 22–25 h in a special, isolated transport box that can maintain a stable temperature of between 5 and 8 °C for 36 h. Small pieces of ovarian tissue (0.3–1×1–1.5×0.7–1 mm) were randomly distributed into three groups: group 1, fresh pieces immediately after receiving transport box (control); group 2, pieces after vitrification; and group 3, pieces after conventional freezing. After thawing, all the pieces were cultured in vitro. The viability and proliferative capacity of the tissue by in vitro production of hormones, development of follicles, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression after culture were evaluated. A difference between freezing and vitrification was not found in respect to hormonal activity and follicle quality. The supernatants showed 17-β estradiol concentrations of 365, 285, and 300 pg/ml respectively, and progesterone concentrations of 3.82, 1.99, and 1.95 ng/ml respectively. It was detected that 95, 80, and 83% follicles respectively were morphologically normal. The molecular biological analysis, however, demonstrated that the GAPDH gene expression in ovarian tissue after vitrification was dramatically decreased in contrast to conventional freezing. For cryopreservation of human ovarian tissue, conventional freezing is more promising than vitrification, because of higher developmental potential.

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E Isachenko, V Isachenko, J M Weiss, R Kreienberg, I I Katkov, M Schulz, A G-M I Lulat, M J Risopatrón, and R Sánchez

This study investigates the ability of sucrose to protect spermatozoa against mitochondrial damage, artificial cryoinduction of capacitation, and acrosome reaction. Spermatozoa were isolated using the swim-up procedure performed using three different media: (a) human tubal fluid (HTF, control) medium; (b) HTF with 1% human serum albumin (HSA); and (c) HTF with 1% HSA and 0.25 M sucrose. From each group, 30 μl suspensions of cells were dropped directly into liquid nitrogen and stored for at least 24 h. Cells were thawed by quickly submerging the spheres in HTF with 1% HSA at 37 °C with gentle agitation. Sperm motility, viability, mitochondrial membrane potential integrity, spontaneous capacitation, and acrosome reaction were investigated. Sperm viability, acrosome reaction, and capacitation were detected using the double fluorescence chlortetracycline-Hoechst 33258 staining technique. Mitochondrial function was evaluated using a unique fluorescent cationic dye, 5,5′,6,6′-tetrachloro-1-1′,3,3′-tetraethyl-benzamidazolocarbocyanin iodide, commonly known as JC-1. The number of progressively motile spermatozoa was significantly higher in the sucrose-supplemented medium group (57.1±3.2%, P<0.05) when compared with controls (19.4±1.9%). The combination of HSA and sucrose (65.2±2.6%) has a stronger cryoprotective effect on the integrity of mitochondrial membrane potential (P<0.05) compared with HSA alone (32.6±4.7%). In conclusion, vitrification of human spermatozoa with non-permeable cryoprotectants such as HSA and sucrose can effectively cryopreserve the cells without significant loss of important physiological parameters.