Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography

In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during the course of the observation period. The female pronucleus was significantly smaller in diameter than the male pronucleus (24.1 and 22.4 microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0 respectively, P < 0.0001). After time-lapse recording, oocytes were cultured for 48 h prior to embryo transfer or cryopreservation. Embryo quality was related to fertilization events and periodicity of the cytoplasmic wave, and it was found that good quality embryos arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.      

Looking for ferns: optimization of digestion pretreatment in fluorescence in situ hybridization (FISH) technique on paraffin-embedded tissues.

Fluorescence in situ hybridization (FISH) is a useful cytogenetic technique for the detection of chromosome aberrations. However, applying this technique routinely on paraffin-embedded tissue is hampered by technical problems. The efficiency of hybridization is influenced by formalin fixation time, and this may vary considerably between specimens. We present a simple method for improving hybridization by microscopically monitoring the time of enzymatic digestion. To establish optimal digestion time, enzymatic digestion was stopped at 3-minute intervals for biopsies and 10-minute intervals for autopsies in 24 paraffin-embedded samples. At every stop, tissue morphology was examined under light microscopy to determine if observed changes could be correlated with subsequent FISH results. The appearance of fernlike formations was found to mark the optimal digestion time that produced the strongest hybridization signals. Using this method of digestion time control, an additional 41 cases were evaluated for FISH with various types of probe. Monitoring under the microscope could be more spaced if the morphology did not change after the first visual control and could be adapted to the type of sample (in general, endoscopic samples, total digestion time of about 10 min; routine biopsies, 15 to 30 min; autopsy samples, 20 to 40 min). In every case, the appearance of the fernlike pattern correlated with proper hybridization signal. Monitoring digestion time for the appearance of fernlike structures is a useful method for improving reproducibility of FISH technique on paraffin-embedded samples. It is particularly useful when dealing with samples under heterogeneous fixation conditions (consultations, autopsies, etc.), because it eliminates the need for repetition.     

Comparison of sevoflurane and propofol in the maintenance of and recovery from anesthesia

OBJECTIVE: To compare the recovery of patients after anesthesia with sevoflurane or propofol during open urological surgery or lumbar column surgery of intermediate duration. PATIENTS AND METHODS: Thirty-six ASA I, II or II patients were enrolled prospectively and randomly assigned to two groups to receive either sevoflurane (n = 19) or proporol (n = 17). Anesthetic induction was accomplished with thiopental, fentanil and vecuronium. During anesthetic maintenance a mixture of 60% nitrous oxide in oxygen plus the drug under study was adjusted to keep blood pressure and/or heart rate within +/- 20% of baseline. After surgery we recorded time until eye opening, spontaneous breathing, extubation, orientation, and identification of parts of the body. Side effects were likewise recorded. In the postanesthetic recovery ward patient condition was assessed using the Aldrete scale, the Newman-Trieger test and a visual analog scale for postoperative pain. Consumption of analgesic during the first 24 h after surgery was monitored. RESULTS: No significant differences were found in demographic data; duration of anesthesia; anesthetic doses; or time until spontaneous breathing, extubation, orientation or identification of parts of the body. Only time until eye opening was shorter in the sevoflurane group than in the propofol group (6.9 +/- 3.3 vs 11.5 +/- 4.8 min; p < 0.05). No differences were recorded on scales reflecting intermediate-term recovery. Analgesic consumption and the incidence of side effects were similar in both groups. CONCLUSIONS: Sevoflurane and propofol are comparable for anesthetic maintenance in urological and neurological procedures of intermediate duration.     

The role of pancreatic venous sampling in the localization of occult insulinomas

The palpation and enucleation of occult insulinomas (less than 15 mm) can be a difficult surgical problem even with good arteriographic localization. In the authors' limited experience, confirmation of arteriographic findings by pancreatic venous sampling provided little additional localizing information. However, if arteriography is negative or equivocal, venous sampling can indicate the segment of pancreas to be "blindly" resected if the adenoma is not palpable. Venous sampling may be misleading in polyendocrine syndromes because of the frequency of multiple adenomas and variable hormone production.     

Le syndrome de Smith-Magenis

Smith-Magenis syndrome is caused by a 17p11.2 deletion. It associates mental retardation, facial dysmorphism and brachydactyly; aberrant behavior and major sleep problems are present in 70% of the cases. It is probably under-diagnosed because the facial abnormalities are mild and the behavioral problems with hyperactivity and self-injuries are dominant, leading to the diagnosis of psychiatric pathology. However these behavioral problems are sufficiently characterized to allow the diagnosis of the syndrome and look for a 17p11.2 microdeletion. Otorhinolaryngologic, ophtalmologic, cardiac and renal abnormalities can be associated and their evaluation is necessary. Smith-Magenis syndrome is considered as a contiguous gene syndrome. Genes have been mapped and isolated to the critical region, but their participation in the pathogenesis of the syndrome remains unclear.     

Angiogenesis is an early event in the development of chemically induced skin tumors

In this study we have analyzed the vascular response induced in the two- stage carcinogenesis model in SENCAR mice. The role of angiogenesis has not been explored in this model, which is the paradigm of multistage carcinogenesis and a model for neoplastic lesions derived from exophytic premalignant lesions (e.g. colon carcinoma, bladder papilloma). We investigated if angiogenesis is involved in the formation of papillomas and in the progression from papilloma to carcinoma. To this end we analyzed the vasculature of normal and hyperplastic skin, focal epidermal hyperplasias that are precursors of papillomas, papillomas at different stages and squamous cell carcinomas. We also analyzed the vascularization of papillomas induced in two strains of mice that differ in their susceptibility to malignant progression. We show here that angiogenesis is turned on in the earliest stages of papilloma formation. In late stages, regardless of state of progression, the predominant response is an increase in the size of blood vessels. Thus, in the SENCAR mouse model, representative of exophytic tumors, the angiogenesis switch is a very early event, probably mechanistically related to the development of the primarily exophytic lesions. Therefore, the density of blood vessels cannot be used as a predictor of malignant progression in this model.      

Pulmonary microvessel density is a marker of angiogenesis in children after cavopulmonary anastomosis

OBJECTIVE: Pulmonary arteriovenous malformations cause progressive cyanosis in children after cavopulmonary anastomosis and may be due to abnormal angiogenesis. We determined the microvessel density, a marker of angiogenesis, in the lungs of children after cavopulmonary anastomosis. METHODS: Lung biopsy specimens were obtained from 8 children after cavopulmonary anastomosis and from 4 control patients. Three of the 8 children undergoing cavopulmonary anastomosis had clinical and angiographic evidence of pulmonary arteriovenous malformations, whereas the other 5 were free of symptoms. Routine histologic and immunohistologic stains were performed with a primary antibody to von Willebrand factor. Microvessel staining for von Willebrand factor was determined for 10 fields (200x) per patient. RESULTS: Patients with and without pulmonary arteriovenous malformations after cavopulmonary anastomosis demonstrated significantly increased microvessel density compared with control subjects (32.7 +/- 2.8 vs 9.3 +/- 4.6, P =.02, and 31.5 +/- 15.7 vs 9.3 +/- 4.6, P =.01, respectively). There was no difference in microvessel density in children with and without clinically apparent pulmonary arteriovenous malformations after cavopulmonary anastomosis (P =.9). The children with pulmonary arteriovenous malformations had numerous greatly dilated vessels that were absent in the asymptomatic children after cavopulmonary anastomosis. CONCLUSIONS: After cavopulmonary anastomosis, pulmonary microvessel density is increased even in the absence of clinically apparent pulmonary arteriovenous malformations, supporting the presence of a constant angiogenic stimulus. Children with clinically apparent pulmonary arteriovenous malformations possess large numbers of greatly dilated pulmonary microvessels, which are absent in asymptomatic children after cavopulmonary anastomosis. These results suggest that the transition to clinically apparent pulmonary arteriovenous malformations may be due to mechanisms that lead to vessel dilation and remodeling.