New method of FACS analyzing and sorting of intact whole ovarian fragments (COPAS) after long time (24 h) cooling to 5 °C before cryopreservation.

As recently announced by the American Society for Reproductive Medicine (ASRM), human ovarian tissue cryopreservation is an established option for fertility preservation in prepubertal girls and young women undergoing gonadotoxic treatments for cancer as well as some autoimmune diseases. Proper ovarian tissue assessment before and after cryopreservation is essential to increase success rates. Ovarian fragments from 16 patients were divided into small pieces in form of cortex with medulla, and randomly divided into the following two groups. Pieces of Group 1 (n = 16) were frozen immediately after operation, thawed and just after thawing their quality was analyzed. Group 2 pieces (n = 16) after operation were cooled to 5 °C for 24 h, then frozen after 24 h pre-cooling to 5 °C, thawed and just after thawing their quality was analyzed. The effectiveness of the pre-freezing cooling of tissue was evaluated by the development and viability of follicles (Calcein-AM and Propidium Iodide) using complex object parametric analyzer and sorter machine (COPAS). Positive effect of cooling of cells to low supra-zero temperatures on their future development after re-warming has been observed. New flow cytometry- technique is suitable for the evaluation and sorting of cryopreserved whole human whole intact ovarian fragments. Long time (24 h) cooling of ovarian tissue to 5 °C before cryopreservation has a trend of a cell viability increasing.

Searching new targets for anthelminthic strategies: Interference with glycosphingolipid biosynthesis and phosphorylcholine metabolism affects development of Caenorhabditis elegans.

Nematode infections are amongst the most abundant diseases of man and animals. They are characterised by a low mortality but high morbidity, thus reflecting the adaptation of these parasites to their hosts. Resistance as well as severe side-effects and efficacies restricted to distinct larval stages or parasites of the anthelmithics used at present require the urgent development of new and more nematode-specific drugs, targeting enzymes of parasite restricted biosynthetic routes. Caenorhabditis elegans has been found to be a good model system for parasitic nematodes, drug screening and developmental studies. Structural analyses have revealed nematode-specific glycosphingolipid structures of the arthro-series, carrying in part, phosphorylcholine substituents. These biomolecules appear to play important roles in nematode development, fertility and survival within the host and are, therefore, good target-candidates for the development of new anthelminthic strategies. Here we show that RNAi experiments targeting enzymes of glycosphingolipid biosynthesis or choline metabolism result, in part, in a drastic reduction of fertility. We further tested various chemical inhibitors of these pathways and found significant effects on the development of the worms, resulting in developmental arrest, sterility and, in part, lethality. Such inhibitors can, therefore, help to define new classes of anthelminthics.