Ultrastructure and intracellular calcium response during activation in vitrified and slow-frozen human oocytes.

BACKGROUND: The sensitivity of human oocytes to cryodamage may compromise their developmental competence following cryopreservation. Herein, we compared the ultrastructure and the response to the calcium (Ca²âº) ionophore A23187 of fresh, slow-frozen and vitrified metaphase II (MII) human oocytes. METHODS: Supernumerary fresh MII oocytes, donated under written informed consent, were cryopreserved through either a slow cooling procedure based on propane-1,2-diol and 0.3 M sucrose or a closed vitrification system based on dimethylsulphoxide (DMSO) and ethylene glycol (EG). Ultrastructure of fresh and cryopreserved oocytes was assessed by transmission electron microscopy and compared through morphometrical analysis; intracellular calcium ([Ca²âº](i)) dynamics was studied by evaluating the response to the Ca²âº ionophore A23187. RESULTS: Morphometric analysis demonstrated a markedly higher proportion of oocytes with large vacuoles, inward displacement of organelles from the pericortical toward the deep cytoplasm, and mitochondrial damage in slow-frozen compared with both fresh and vitrified oocytes. A23187 increased the [Ca²âº](i) in all oocyte groups and the peak average increase in slow-frozen oocytes was significantly higher than in both fresh and vitrified oocytes. Moreover, the ability of slow-frozen oocytes to recover [Ca²âº](i) to basal levels was significantly reduced compared with both fresh and vitrified oocytes. CONCLUSIONS: Closed vitrification based on DMSO and EG preserves the ultrastructural features and the ability to respond to the Ca²âº ionophore A23187 significantly better than does slow freezing with 0.3 M sucrose. Damage to organelles involved in the [Ca²âº](i) modulation might reduce the developmental competence of cryopreserved oocytes.

Is there a role for endocannabinoids in sperm-oviduct interaction?

The endocannabinoid system (ECS) has been found in reproductive cells and tissues in several mammals. Spermatozoa are able to respond to anandamide, and the oviduct is able to synthesize and modulate the concentration of this endocannabinoid along the isthmic and ampullary regions. The main aim of this study was to understand whether the ECS has a role during sperm storage and release within the oviduct in cattle. Data showed that 1) the endocannabinoid receptors 1 and 2 (CB1 and CB2) are present in bovine spermatozoa both in the initial ejaculate and in spermatozoa bound to the oviduct in vitro; 2) CB1 receptor is still detectable in spermatozoa released from the oviduct through penicillamine but not in those released through heparin; 3) arachidonylethanolamide (AEA) does not affect sperm viability, whereas it depresses sperm progressive motility and kinetic values; 4) sperm-oviduct binding and release in vitro are not influenced by AEA; 5) AEA depresses sperm-zona pellucida (ZP) binding; 6) binding of heparin-capacitated spermatozoa to the ZP is not affected by AEA; 7) N-acylphosphatidylethanolamine-selective phospholipase D, the main enzyme involved in anandamide synthesis, is expressed in oviductal epithelial cells. In conclusion, secretion of AEA from epithelial cells might contribute to the oviduct sperm-reservoir function, prolonging the sperm fertile life through the depression of motility and capacitation. Capacitation signals, such as heparin, that promote sperm release, might remodel the sperm surface and cause a loss of the sperm sensitivity to AEA.

Evaluation of patients' engagement in radiation therapy safety.

PURPOSE: Treatment safety has become a priority in health policies after several incidents occurred around the world in radiation oncology departments. The aim of this study was to analyse the patients' contribution in that field and to understand which actions empower the patient in that regard. METHODS: Several methods were used in a general hospital and in a comprehensive cancer centre to analyse the activities of the radiation therapists and the patients and the interactions between them: treatment session observations, semidirective interviews with radiation therapists and patients, self and alloconfrontation with radiation therapists and explanatory interviews with patients. RESULTS: Cooperation of the patients in treatment safety acts as an additional step that contributes to safer treatments. Radiation therapy sessions are a creative opportunity for the patient to observe, learn and analyse what is happening. Changes between treatment sessions are a source of anxiety for the patients. This study highlights the factors that favour the patients' participation. A trusting relationship and support from the health professionals can be leveraged in that manner. CONCLUSION: There is a common will shared between the patients and the health professionals towards better treatment safety. The cooperation is still not well-known and underused. This empowerment of the patient cannot be mandatory but should be promoted and developed.

Empowering patients for radiation therapy safety: Results of the EMPATHY study.

PURPOSE: With the increase of treatment complexity, enhancing safety is a key concern in radiation oncology. Beyond the involvement of the healthcare professional, patient involvement and empowerment could play a major role in that setting. We explored how patients perceived and fulfilled that role during their radiation treatment. MATERIALS AND METHODS: A voluntary and anonymous questionnaire was administered to all patients treated in our department between November 2013 and May 2014. The following data were collected: sociodemographic profile; information received and initiatives to search for additional information; behavior when an unusual treatment event was perceived; active involvement in the safety of the treatment; nature and perception of their own involvement. A statistical analysis was performed to assess behavioral predictors. RESULTS: A total of 155 patients answered the survey. Most of them were treated for prostate (n=58, 37.4%), lung (n=27, 17.4%), head and neck (n=26, 16.8%) and breast (n=25, 16.1%). Only eight patients (5%) had previously received radiation therapy. Ninety-five percent of the patients estimated they had received enough information about their treatment, but 48% would have wanted more. When patients noticed an unusual event during their treatment session, most of them (61%) reported it to the radiation therapist. CONCLUSION: Patient participation to radiation therapy safety should be encouraged to ensure a cooperative risk management. Healthcare professionals need to inform the patients on the basic technical processes involved in their treatment. Patient empowerment should be added to the verifications made by the radiation therapists and physicians but should not replace them.

Bovine oviductal monolayers cultured under three-dimension conditions secrete factors able to release spermatozoa adhering to the tubal reservoir in vitro.

Different in vitro models have been developed to understand the interaction of gametes and embryos with the maternal reproductive tract. We recently showed that bovine oviductal monolayers three-dimensionally cultured in Gray's medium on collagen-coated microporous polycarbonate inserts under liquid-air interface conditions are well polarized, develop cilia, remain viable for at least 3 weeks postconfluence, and mantain the viability of bound spermatozoa significantly better than bidimensionally cultured monolayers. Herein, we used these culture conditions to understand whether: (1) spermatozoa adhering to three-dimensionally cultured oviductal monolayers can be released by heparin or penicillamine as previously shown with bidimensionally cultured oviductal monolayers and explants; and (2) media conditioned by three-dimensionally cultured oviductal monolayers were able to release spermatozoa adhering to oviductal explants. Findings demonstrated that (1) spermatozoa adhering to three-dimensionally cultured oviductal monolayers are readily released by heparin and penicillamine, (2) media conditioned by three-dimensionally cultured oviductal monolayers are able to release spermatozoa bound to oviductal explants, (3) do not depress sperm motility and viability, (4) they improve sperm kinetics, and (5) promote binding to the zona pellucida. In conclusion, in vitro data suggest that the release of spermatozoa adhering to the oviductal reservoir in vivo can be triggered by factors secreted by the oviduct itself that induce sperm capacitation.

Long-term viability and differentiation of bovine oviductal monolayers: bidimensional versus three-dimensional culture.

Different in vitro models have been developed to study the interaction of gametes and embryos with the maternal tract. In cattle, the interaction of the oviduct with gametes and embryos have been classically studied using oviductal explants or monolayers (OMs). Explants are well differentiated but have to be used within 24 h after collection, whereas OMs can be used for a longer time after cell confluence but dedifferentiate during culture, losing cell polarity and ciliation. Herein, OMs were cultured either in M199 plus 10% fetal calf serum or in a semidefined culture medium (Gray's medium), in an immersed condition on collagen-coated coated microporous polyester or polycarbonate inserts under air-liquid interface conditions. The influence of culture conditions on long-term viability and differentiation of OMs was evaluated through scanning electron microscopy, localization of centrin and tubulin at the confocal laser scanning microscope, and assessment of maintenance of viability of sperm bound to OMs. Findings demonstrated that OMs cultured in an immersed condition with Gray's medium retain a better morphology, do not exhibit signs of crisis at least until 3 wks postconfluence, and maintain the viability of bound sperm significantly better than parallel OMs cultured in M199 plus 10% fetal calf serum. OM culture with Gray's medium in air-liquid interface conditions on porous inserts promotes cell polarity, ciliation, and maintenance of bound sperm viability at least until 3 wks postconfluence. In conclusion, oviduct culture in Gray's medium in an immersed or air-liquid condition allows long-term culture and, in the latter case, also ciliation of bovine OMs, and may represent in vitro systems that mimick more closely the biological processes modulated by the oviduct in vivo.

[Can or must the patient participate to risk management in radiotherapy?]. / Le patient peut-il et doit-il participer à la gestion des risques en radiothérapie ?

PURPOSE: The present study aimed at analyzing if patient participation constitutes a promising way of improvement of patient safety, or not. The hypothesis is that patient participation is a means to develop a safety culture based on the cooperation between patients and healthcare providers, to improve patients' satisfaction and to reduce the costs associated to adverse events. PATIENTS AND METHODS: A half-day session was organized on this theme during a training of radiotherapy professionals on risk management. Professionals were first distributed in three subgroups according to their specialty (radiation oncologists, radiation physicists and medical technicians), and had to work on four main questions relating to participation, among which the collection of real situations in which patients effectively contributed (positively or negatively) to patient safety. Results were then collectively discussed. RESULTS: Patient participation allows not only to detect and recover some mistakes or errors made by the professionals (error of identity), but also to decrease patients' risk behaviors (purposely taking the place of another patient in order to be treated faster). However, it must be seen as a possibility offered to patients, and not as an obligation. CONCLUSION: Patient participation to patient safety is a field of study, which requires to be developed in order to define the conditions enhancing such participation and to implement a set of actions to improve healthcare safety by a cooperative management of this one.

Ability of sulfated glycoconjugates and disulfide-reductants to release bovine epididymal sperm bound to the oviductal epithelium in vitro.

In Bos taurus, at ejaculation, epididymal sperm acquire a number of proteins secreted in the seminal plasma that increase their ability to interact with the female reproductive tract. Sperm-oviduct interaction comprises a transient sperm adhesion to the isthmus, the lower portion of the oviduct, followed by sperm release around ovulation. Oviductal fluid molecules, such as sulfated glycoconjugates and disulfide-reductants, are able to release bovine ejaculated sperm bound to the oviductal epithelium in vitro through the reduction of sperm surface protein disulfides to sulfhydryls. To understand whether the sperm molecules sensitive to releasing signals are already exposed on the surface of epididymal sperm, we studied the ability of cauda epididymal sperm to adhere to the oviductal epithelium and to be released by sulfated glycoconjugates and the disulfide-reductant penicillamine. Surface protein sulfhydryls in cauda epididymal sperm were analyzed in the initial suspension, in sperm bound to the in vitro-cultured oviductal epithelium, and in released sperm. Results showed that epididymal sperm are able to bind the oviductal epithelium in vitro, although at a lower extent than frozen-thawed ejaculated sperm; the interaction is mediated by oviductal cell microvilli that closely bind to the plasma membrane of the sperm head rostral region, as previously shown for ejaculated sperm. The sulfated glycoconjugates heparin, fucoidan, and dextran sulfate, as well as the disulfide-reductant penicillamine, are all powerful inducers of sperm release. The level of sulfhydryls in sperm surface proteins was (1) high in the initial sperm suspension; (2) low in bound sperm; (3) markedly increased in sperm released by heparin or by penicillamine. In conclusion, epididymal sperm are already able to bind the oviductal epithelium and to respond to the inducers of release through the reduction of sperm surface protein disulfides to sulfhydryls.

[Risk assessment in radiation therapy]. / La gestion des risques en radiothérapie.

Radiation therapy was one of the first medical specialities to implement quality assurance (QA) programs in clinical practice. These QA programs have mainly focused on equipment performance neglecting human factors. Risk management is a relatively new approach in medical disciplines. Methods of evaluation and risk management must be identified, applied and adapted to the specificities of this domain. Based on the experience of industry, risk management will allow us to substantially improve safety and develop our own models of management. Recent experiences use these approaches to implement risk management in radiation therapy departments.