Fast and furious: successful survival and resumption of meiosis in immature human oocytes vitrified and warmed using a short protocol.

RESEARCH QUESTION: Can immature oocytes vitrified and warmed using a short protocol survive and resume meiosis? DESIGN: This study examined modifications of oocyte vitrification and warming protocols that reduce the length of exposure to vitrification and warming solutions. In total, 561 germinal vesicles and 218 metaphase I oocytes that were immature at oocyte retrieval were vitrified at room temperature for 2 min. Warming was performed at 37°C for 2 min. Resumption of meiotic activity was evaluated after 24 and 48 h of culture. Two different commercially available vitrification and warming kits were used for comparison. RESULTS: Ninety-five percent of germinal vesicles survived, with no difference observed between the kits. The survival of metaphase I oocytes was, on average, 95.4% and did not differ significantly between the kits. Of the 533 germinal vesicles that survived, 491 converted to metaphase I oocytes (92.1%). After culture for 48 h, 54.4% converted to metaphase II oocytes. In addition, of the 208 metaphase I oocytes that survived warming, 84.1% converted to metaphase II oocytes after 24 h of culture. These maturation rates were similar to those of non-vitrified oocytes. CONCLUSIONS: Vitrification and warming of oocytes at different nuclear maturation stages can be performed with 2 min of exposure to hypertonic solution and 2 min of exposure to hypotonic solution, respectively. This approach reduces exposure of the oocytes to room temperature during dehydration and rehydration. Warming in 0.5M sucrose helps to maintain and support the potential of oocytes to resume nuclear meiotic activity, and conversion from germinal vesicles to metaphase I and metaphase II oocytes.

Aseptic vitrification of blastocysts from infertile patients, egg donors and after IVM.

During embryo vitrification, it is advisable that cooling and storage should occur in a carrier device in which there is complete separation of the embryos from liquid nitrogen to ensure asepsis. The consequence of a reduction in the cooling rate resulting from the heat-insulating barrier aseptic devices has to be counteracted by gradually increasing intracellular concentrations of cryoprotectants without inducing a toxic effect. Blastocysts originating from couples with male and/or female factor infertility (group 1) or from oocyte donors (group 2) or from in-vitro matured oocytes (group 3) were gradually exposed to increasing concentrations of dimethylsulphoxide/ethylene glycol (5/5%, 10/10% and 20/20%) before aseptic vitrification using a specially designed carrier (VitriSafe), a modification of the open hemi-straw plug device. A total of 120 aseptic vitrification/warming cycles were performed in group 1, 91 in group 2 and 22 in group 3. Survival rates before embryo transfer, ongoing pregnancy and implantation rates were as follows: for group 1, 73, 43 and 26%; for group 2, 88, 53 and 34%; and for group 3, 69, 50 and 38%, respectively. In spite of reduced cooling rates due to aseptic vitrification conditions, a three-step exposure to cryoprotectant solutions protects the embryos effectively from cryo-injuries and guaranties high survival rates.

Cryoloop vitrification in assisted reproduction: analysis of survival rates in > 1000 human oocytes after ultra-rapid cooling with polymer augmented cryoprotectants.

While human oocytes have been successfully cryopreserved using traditional slow-rate freezing protocols, inconsistent results post-thaw have limited the routine clinical application of oocyte cryopreservation. Despite interest in the potential benefits of vitrification as an alternative laboratory approach to long-term oocyte preservation in assisted reproduction, there is little agreement on how best to configure such cryopreservation protocols to optimize oocyte viability. To comparepost-thaw oocyte survival rates,we performed cryoloop vitrification of human oocytes utilizing two different cryoprotectant mixtures that included polymer macromolecules. Human oocytes (n = 1120) were obtained from consenting patients undergoing in vitro fertilization, but only failed-matured (uninseminated) or failed-fertilized (inseminated but without 2pn development) were included in this investigation. Protocol A consisted of 20% ethylene glycol and 20% dimethyl sulphoxide + 0.4 M sucrose and 20% synthetic serum substitute. Protocol B consisted of 20% ethylene glycol and 20% dimethyl sulphoxide + 0.65 M sucrose, 1 mg/ml polyethylene glycol, 10 mg/ml Ficoll and 20% synthetic serum substitute. Following cryostorage for 10-14 d at -196 degrees C, the survival rate for oocytes vitrified with protocol A was 80.9%, whereas the post-thaw viability among protocol B oocytes was 80.6% (p > 0.005). Our results indicate that an ethylene glycol + dimethyl sulphoxide mixture (with or without polymer macromolecules) can be an effective cryoprotectant strategy for human oocyte vitrification; either approach can be employed without any observed compromise in post-warming survival and/or morphology.

Effect of carrier system on the yield of human oocytes and embryos as assessed by survival and developmental potential after vitrification.

The present study was undertaken to examine the effect of a cooling regimen during vitrification on survival and morphological appearance of human oocytes after warming and the developmental potential of day 3 embryos. Aged human oocytes that had failed to fertilize and human embryos derived from abnormally fertilized zygotes that showed one pronucleus or three or more pronuclei were used as models in this study. In the first part of the study, 928 aged human oocytes that had failed to fertilize were vitrified. The viability of oocytes after vitrification using the hemi-straw system was slightly higher than it was using the cryoloop after warming (85.4% (410 of 480) versus 80.6% (361 of 448)), but the difference was not significant. In the second part of the study, 266 embryos were vitrified. The survival of day 3 embryos after vitrification was improved by using a mixture of ethylene glycol and dimethyl sulphoxide in combination with the hemi-straw system method rather than the cryoloop method (89.7% (122 of 136) versus 83.8% (109 of 130)), but the difference was again not significant. The potential for development up to compaction on day 4 in embryos vitrified using the hemi-straw system method was significantly higher (37.7%) than it was in embryos vitrified using the cryoloop (29.4%; chi(2), P = 0.002). The hemi-straw system and cryoloop methods of vitrification are both successful, easy to perform, and demonstrate the ability of both carriers to vitrify different stages of development (oocytes and day 3 embryos). In addition, the success of both methods appears to be related to the rate of cooling and the lower concentration of cryoprotectant used.

[Telemedicine: perspectives and multidisciplinary approach]. / Télémédecine: perspectives et approche pluridisciplinaire.

BACKGROUND: Telemedicine is use of the new computer-based communication technologies for medical purposes. It augments the exchange of scientific information, while its applications in the fields of patient care and medical education cover remote diagnosis and therapy as well as remote education and training. METHOD: This article reviews the development of telemedicine and its application to specialties such as anaesthesiology, dermatology, medicine, surgery and pathology at the University Hospital of Basle, Switzerland. RESULTS: Since 1980 the Department of Medicine has held multidisciplinary teleconferences for expert consultation and medical education. Since 1992 the Institute of Pathology has been linked to remote hospitals for real-time biopsy, and, since 1997, remote dermato-histopathological diagnosis has been performed in conjunction with a number of centres and practitioners. International academic teleconferences have been held in the field of surgery since 1986 and there is an interactive education programme via telemedicine in the field of anaesthesiology. The technology in use must be adapted to needs: since few practitioners are currently connected to the Internet, teleconferencing will still be the rule in the Department of Medicine. Remote diagnosis in dermatology and pathology requires high-resolution images transmitted by self-developed software via 64 Kb/s ISDN connection, while surgery works with ISDN teleconferencing at 384 Kb/s to ensure live transmission of surgical procedures with high-quality images. CONCLUSION: Our practice, based on several hundred cases, suggests that telemedicine is useful in simplifying and expanding access to remote interdisciplinary expertise, as well as improving medical education in a number of specialties. Telemedicine's multidisciplinary approach is to be recommended.

Perforin-independent CD8(+) T-cell-mediated cytotoxicity of alveolar epithelial cells is preferentially mediated by tumor necrosis factor-alpha: relative insensitivity to Fas ligand.

CD8(+) T cells appear to play an important pathophysiologic role in many inflammatory lung diseases. The primary effector function of this T-cell subset is cytolysis of virus-infected cells, and it is widely believed that there are two primary molecular mechanisms by which this occurs: the perforin/granzyme-mediated pathway of cytolysis, and the Fas ligand (FasL)-Fas (CD95/APO-1) pathway of induction of target-cell apoptosis. This conclusion is based primarily on data obtained with hematopoetic cell lines as target cells. There is also a growing body of evidence that Fas is involved in the transduction of apoptotic signals in a variety of inflammatory disease states, particularly involving the liver and the lung. In the study reported here we took advantage of a novel in vitro assay to directly assess the effector mechanisms employed in CD8(+) T-cell-mediated cytolysis of alveolar epithelial cells. We present evidence that FasL-induced, Fas-mediated apoptosis does not directly contribute to T-cell-mediated cytolysis of alveolar epithelial-derived cells, even though Fas is expressed and functional on these cells. We also demonstrated that the perforin-independent cytolytic activity of CD8(+) T cells against alveolar epithelial-derived cells is explained entirely by tumor necrosis factor-alpha (TNF-alpha), which is expressed on CD8(+) T cells. Furthermore, we show that bystander cytolysis of alveolar epithelial-derived cells by antiviral CD8(+) T cells is entirely perforin-independent. This activity is mediated exclusively by TNF-alpha. Both alveolar epithelial-derived cells and primary murine type II cells show susceptibility to apoptosis triggered by soluble TNF-alpha, without the need for transcriptional or translational inhibition. We also confirmed the resistance of alveolar type II cells to FasL in vivo by performing adoptive transfer of perforin-deficient antiviral CD8(+) T cells into transgenic mice expressing a target antigen in type II epithelial cells. Significant lung injury developed in the transgenic CD8(+) T-cell recipients, whether or not Fas was expressed in these animals. Furthermore, preincubation of the T cells with antibody to TNF-alpha completely abolished the injury. These results suggest that alveolar epithelial cells are relatively sensitive to T cell-triggered, TNF-alpha-mediated apoptosis, and resistant to apoptosis triggered by FasL. These observations may have important ramifications for understanding of the pathophysiology of interstitial and inflammatory lung diseases.

Evaluation of a cut-off value for sperm motility after different hours of incubation to select the suitable reproductive technology (IVF or ICSI).

BACKGROUND: The aim of this study was to enhance the predictability of conventional semen parameters for in-vitro fertilization outcome. The utility of late sperm motility in presence of a cumulus-oocyte complex after different hours of incubation was investigated to predict the outcome of IVF in borderline and normal ejaculates (at least 5 x 10(6) motile sperm). METHODS: The study was done on 52 infertile couples undergoing conventional in-vitro fertilization and embryo transfer. Sperm were prepared by the Percoll cushion centrifugation with swim-down. Cocultures were established by inseminating the cumulus-oocyte complexes with 100000 motile spermatozoa and incubating them for 48 hours. Another 100000 spermatozoa were incubated in culture medium for 48 hours. Sperm motility (WHO a+b) was determined at 0, 4, 24 and 48 hours of incubation. RESULTS: The fertilization rate was 65.5% (42.9-88.1). The conventional semen parameters did not correlate with the fertilization rate. Sperm motility measured after different hours of incubation was found to be significantly positively correlated with the fertilizing ability of sperm in vitro in Spearman's rank correlation test: motility after 0 h (p<0.02), after 4 h (p=0.0025). after 24 h (n.s.) and after 48 h (p=0.0071). Cut-off values for late sperm motility were determined and differences in fertilization rates were calculated for these cut-off values after different hours of incubation. A cut-off value of 20% progressive motile spermatozoa after 48 hours gave the best statistical power (fertilization rate 71.7 vs. 50.2%, p<0.001). Significant differences in the fertilization rates were also observed for a cut-off value of 35% after 24 hours of incubation (70.1 vs. 46.2%, p=0.001) and for a cut-off point of 60% after 4 hours (72.4 vs. 51.5%, p=0.001). CONCLUSIONS: The predictive power of sperm motility after 48 h for fertilization outcome provides support in the decision-making process within the assisted reproduction setting. If less than 20% of sperm are motile after 48 h micromanipulatory techniques should be considered.

Arithmetical abilities in Alzheimer disease.

The purpose of this research was to analyze the calculation abilities in Alzheimer Disease (AD). Twenty right-handed patients meeting the DSM-IV (American Psychiatric Association, 1994) criteria for AD were studied. Age ranged from 64- to 88-year-old. A neuropsychological test battery including language, memory, constructional abilities, attention, mathematics, and abstraction tests was administered. In addition, the Mini-Mental State Exam (MMSE) (Folstein, Folstein, McHugh, 1975) was also administered. Mathematical subtests correlated higher than the MMSE with the scores in the different neuropsychological tests. Highest correlations of the mathematical subtests were observed with language repetition, non-verbal memory, and attention tasks. It is proposed that mathematical ability tests represent in AD an excellent predictor of general intellectual performance. It is further proposed that disturbances in arithmetical ability should be included as a diagnostic criteria for AD.

Effects of local growth factors on the secretory function of bovine corpus luteum during the oestrous cycle and pregnancy in vitro.

The impact of insulin-like growth factor-I (IGF-I) basic fibroblast growth factor (bFGF), endothelin-1 (ET-1), tumour necrosis factor-alpha (TNF-alpha), transforming growth factor-alpha (TGF-alpha) and platelet-derived growth factor (PDGF) on the release of progesterone (P4) and oxytocin (OT) from individual bovine corpora lutea at different stages of the oestrous cycle and pregnancy was evaluated with a microdialysis system (MDS) in vitro. IGF-I (1 microgram mL-1) induced significantly the acute effects on P4 release at the late luteal stage (Days 15-18) and early pregnancy (Days 60-120), whereas bFGF (100 ng mL-1) was extremely effective in stimulating P4 release particularly during the mid-luteal stage (Days 8-12). Both peptides stimulated (P < 0.05) the release of OT throughout the three luteal stages and during early and late pregnancy (Days 30-60 and Days 150-210). ET-1 (100 ng mL-1) clearly inhibited P4 release during the early (Days 5-7) and mid-luteal phase and stimulated OT release only during the mid-luteal stage (P < 0.001). TNF-alpha (100 ng mL-1) stimulated the release of P4 exclusively at the early luteal phase (P < 0.05), whereas OT secretion was increased by TNF-alpha during all stages of the oestrous cycle (P < 0.001). TGF-alpha and PDGF (100 ng mL-1) were effective in stimulating P4 release particularly during late pregnancy (P < 0.05). In contrast, stimulation of OT secretion by TGF-alpha was maximal during the late-luteal stage (P < 0.001), whereas PDGF significantly increased OT secretion during the oestrous cycle (except the early luteal stage) and pregnancy (P < 0.001). The data demonstrate distinct and stage-specific effects of growth factors on P4 and OT secretion in vitro. IGF-I, bFGF and TGF-alpha may play an important role in corpus luteum (CL) function during the oestrous cycle and pregnancy since they are locally expressed and synthesized, there are receptors for these growth factors, and they have been demonstrated to exert biological effects on the CL.

Is methylphenidate like cocaine? Studies on their pharmacokinetics and distribution in the human brain.

BACKGROUND: The purposes of this study were to investigate the pharmacokinetics of methylphenidate hydrochloride (Ritalin) in the human brain, to compare them with those of cocaine, and to evaluate whether cocaine and methylphenidate compete for the same binding sites. METHODS: We used positron emission tomography to measure the temporal and spatial distribution of carbon 11 (11C)-labeled methylphenidate. These results were compared with those obtained previously for [11C]cocaine. Eight healthy male subjects, 20 to 51 years of age, were scanned with [11C]methylphenidate. Three were tested twice to assess test-retest variability, four were tested at baseline and after administration of methylphenidate, and one was tested with [11C]methylphenidate and [11C]cocaine. Two baboons were scanned to evaluate whether there was competition between cocaine and methylphenidate for the same binding sites in the brain. RESULTS: The uptake of [11C]methylphenidate in the brain was high (mean +/- SD, 7.5% +/- 1.5%), and the maximal concentration occurred in striatum. Pretreatment with methylphenidate decreased binding only in striatum (40%). Although the regional distribution of [11C]methylphenidate, was identical to that of [11C]cocaine and they competed with each other for the same binding sites, these two drugs differed markedly in their pharmacokinetics. Clearance of [11C]methylphenidate from striatum (90 minutes) was significantly slower than that of [11C]cocaine (20 minutes). For both drugs, their fast uptake in striatum paralleled the experience of the "high." For methylphenidate, the high decreased very rapidly despite significant binding of the drug in the brain. In contrast, for cocaine, the decline in the high paralleled its fast rate of clearance from the brain. CONCLUSION: We speculate that because the experience of the high is associated with the fast uptake of cocaine and methylphenidate in the brain, the slow clearance of methylphenidate from the brain may serve as a limiting factor in promoting its frequent self-administration.

Actions of somatotrophin on oxytocin and progesterone release from the microdialysed bovine corpus luteum in vitro.

In the present investigation, the effect of recombinant (BST) and pituitary-derived (bGH) bovine somatotrophin on progesterone and oxytocin release was examined. Individual copora lutea (CL) were obtained from cows at different stages of the oestrous cycle (days 5-7, 8-12 and 15-18) and also from early pregnancy (days 60-120) and were implanted with an in vitro microdialysis system (MDS). Perfusion with BST for 60 min (0.05, 0.5 and 5 mumol/l) induced a dose-dependent stimulation of progesterone release. Release of oxytocin from CL was significantly stimulated by BST at all dose levels. BST (0.5 mumol/l) stimulated progesterone release most during the early and mid-luteal phases and oxytocin release especially during the early luteal stage (days 5-7) of the oestrous cycle. CL from early pregnancy (days 60-120) treated with BST showed a significant response in progesterone and oxytocin release. bGH showed comparable effects. Our results suggest that somatotrophin acts directly on the secretory function of bovine CL in the MDS, specifically during the early luteal stage (days 5-7) of the oestrous cycle and early pregnancy (days 60-120). Somatotrophin may therefore have physiologically relevant effects associated with the development and maintenance of luteal function.

Effects of prenatal cocaine exposure upon postnatal development of neostriatal dopaminergic function.

Pregnant rats were injected twice daily with 20 mg/kg cocaine (or saline) from gestational day 10 to parturition. Brains from offspring were examined with quantitative receptor autoradiography [D1 receptor (D1R), D2 receptor (D2R) and dopamine transporter (DAT)] and quantitative in situ hybridization [D1R mRNA, D2R mRNA, preproenkephalin (PPE) mRNA] for markers of neostriatal dopaminergic function. Prenatal cocaine exposure did not alter postnatal development of striatal D1R sites, but D1R mRNA levels were reduced by a third at days 14 and 35. D2R sites were increased over control in lateral striatum by day 6, and remained elevated through postnatal day 35. Total D2R mRNA was increased over control in both medial and lateral striatum at 7 and 14 days but was equal to control at 35 days. Prenatal cocaine exposure increased DAT density at postnatal days 1 through 5, but reduced it at days 14 and 35; PPE mRNA expression was reduced at days 7, 14 and 35. Many of these results are similar to those found in experimental animals and humans following cocaine withdrawal.

[The function of the coracoacromial ligament]. / Funktion des Ligamentum coracoacromiale.

The function of the coracoacromial ligament was investigated in 8 dissecting-room scapulae. Strain gauges were attached around the coracoid process and the acromion, and tension (50 or 100 N) applied through the remaining muscle stumps. The results showed that, after division of the ligament, significantly more distortion could be measured in the acromion than in the coracoid process, which suggests that the 'stay' effect of the coracoacromial ligament is stronger for the former. Since the degree of distortion is largely dependent on the direction of pull, the ligament is interpreted as a dynamic brace between the two processes of the scapula.

Second cancer following cancer of the male genital system in Connecticut, 1935-82.

The risk of a second primary cancer developing was evaluated in nearly 20,000 men with cancers of the prostate or testis in Connecticut, 1935-82. Among 18,135 men with prostate cancer, a significant 15% deficit of all second cancers was observed [1,053 vs. 1,241; relative risk (RR) = 0.85; 95% CI = 0.80-0.90], most notably for respiratory (RR = 0.7) and digestive cancers (RR = 0.8). The absence of a colon cancer risk lends little support to the idea of common risk factors such as dietary fat consumption. Only the risk for salivary gland cancer was significantly increased, possibly due to chance. Leukemia was significantly elevated among men observed for 10 and more years (RR = 2.2). In contrast to most other index tumors, the prostate stands out as being associated with an overall low risk of second cancer development. The reasons for these deficiencies have not been explained. Among 1,446 men with testis cancer, a significant twofold risk of second cancers was seen (104 vs. 50.1). A fivefold risk of leukemia (8 vs. 1.5) was not related to treatment or age. Contralateral testis cancer (6 vs. 0.5) was elevated in men treated with and without radiation. Risks for kidney cancer (5 vs. 1.5), bladder cancer (9 vs. 3.4), pancreatic cancer (6 vs. 1.5), non-Hodgkin's lymphoma (6 vs. 1.5), and prostate cancer (12 vs. 5.9) were significantly increased. No trends over time were noted for any cancer. Overall risk of second cancer development tended to be higher in younger men with testis cancer. The relationship of leukemia to testis and prostate cancers should be investigated in future research.