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Measurement of ovarian artery blood flow and follicle aspiration On the hCG administration day and the follicle aspiration day, both sides of intraovarian artery flow for each patient were measured by transvaginal color ultrasonographic pulsed wave Doppler. The values of pulsatility indices (PI) were printed on a wave-form image. The PI of each patient was defined as the mean value of the two measured sides. Oocyte retrieval was performed 35 h after the hCG administration by follicle aspiration under transvaginal ultrasonographic guidance. All the follicles with a mean diameter >12 mm were aspirated, using an 18-gauge needle connected to a connecting tube and 20-ml syringe for suction. Granulosa cell treatment The oocyte-cumulus complex was removed Protease Inhibitor Library price from Selleckchem BGJ398 the follicular fluid, washed twice in culture medium (human tubal fluid) and placed in fresh culture medium. The cumulus granulosa cells (ApoC) were mechanically separated from the oocyte using 26-gauge needles under the microscope and placed on glass slides. The mural cell mass was collected from the follicular fluid and washed twice in the culture medium. The ApoC and mural granulosa cells (ApoM) were dispersed separately by hyaluronidase (0.1% w/v) and fixed with 4% neutral formalin on glass slides. The granulosa cells were fixed Unoprostone was counted. Apoptotic cells were defined as those with fragmented and uniformly dense nuclei or fragmented cytoplasm containing a uniformly dense fragmented nucleus. Follicular fluids collection and steroids assay Each follicular fluid sample was centrifuged at 300 x g for 10 min and the cleared fluid was stored at ?20?C until the assay for E2, P and T. The quantification of the hormonal concentration was performed using commercially available immunoassays: E2 by time-resolved fluoroimmunoassay (DELFIA Estradiol; Pharmacia, Tokyo, Japan), P by radioimmunoassay (RIA) (DPC progesterone; DPC, Inc., Tokyo, Japan) and free T by RIA (DPC progesterone kit and DPC free testosterone kit; DPC, Inc.). Reliability criterions for all the assays were established. The hormones were assayed in duplicate within the same assay (8). Assessment of oocyte Oocyte quality Oocyte quality was scored by the incidence of apoptotic granulosa cells with condensed and fragmented nuclei. Oocyte maturity Oocyte maturity was judged by the condition of the corona radiata and the ApoC, as described previously.