Early and widespread engagement of the cerebellum during hippocampal epileptiform activity Format: Brief Communication.

Despite research illustrating the cerebellum may be a critical circuit element in the epilepsies, remarkably little is known about cerebellar engagement during seizures. We therefore implemented a novel method for repeated imaging of the cerebellum in awake, chronically epileptic animals. We found widespread changes in cerebellar calcium signals during behavioral seizures and during hippocampal seizures that remained electrographic only, arguing against cerebellar modulation simply reflecting motor components. Moreover, even brief interictal spikes produced widespread alterations in cerebellar activity. Changes were noted in the anterior and posterior cerebellum, along the midline, and both ipsilaterally and contralaterally to the seizure focus. Remarkably, changes in the cerebellum also occurred prior to any noticeable change in the hippocampal electrographic recordings, suggesting a special relationship between the cerebellum and hippocampal epileptiform activity. Together these results underscore the importance of the cerebellum in epilepsy, warranting a more consistent consideration of the cerebellum when evaluating epilepsy patients.

Good practice recommendations on add-ons in reproductive medicine†.

STUDY QUESTION: Which add-ons are safe and effective to be used in ART treatment? SUMMARY ANSWER: Forty-two recommendations were formulated on the use of add-ons in the diagnosis of fertility problems, the IVF laboratory and clinical management of IVF treatment. WHAT IS KNOWN ALREADY: The innovative nature of ART combined with the extremely high motivation of the patients has opened the door to the wide application of what has become known as 'add-ons' in reproductive medicine. These supplementary options are available to patients in addition to standard fertility procedures, typically incurring an additional cost. A diverse array of supplementary options is made available, encompassing tests, drugs, equipment, complementary or alternative therapies, laboratory procedures, and surgical interventions. These options share the common aim of stating to enhance pregnancy or live birth rates, mitigate the risk of miscarriage, or expedite the time to achieving pregnancy. STUDY DESIGN, SIZE, DURATION: ESHRE aimed to develop clinically relevant and evidence-based recommendations focusing on the safety and efficacy of add-ons currently used in fertility procedures in order to improve the quality of care for patients with infertility. PARTICIPANTS/MATERIALS, SETTING, METHODS: ESHRE appointed a European multidisciplinary working group consisting of practising clinicians, embryologists, and researchers who have demonstrated leadership and expertise in the care and research of infertility. Patient representatives were included in the working group. To ensure that the guidelines are evidence-based, the literature identified from a systematic search was reviewed and critically appraised. In the absence of any clear scientific evidence, recommendations were based on the professional experience and consensus of the working group. The guidelines are thus based on the best available evidence and expert agreement. Prior to publication, the guidelines were reviewed by 46 independent international reviewers. A total of 272 comments were received and incorporated where relevant. MAIN RESULTS AND THE ROLE OF CHANCE: The multidisciplinary working group formulated 42 recommendations in three sections; diagnosis and diagnostic tests, laboratory tests and interventions, and clinical management. LIMITATIONS, REASONS FOR CAUTION: Of the 42 recommendations, none could be based on high-quality evidence and only four could be based on moderate-quality evidence, implicating that 95% of the recommendations are supported only by low-quality randomized controlled trials, observational data, professional experience, or consensus of the development group. WIDER IMPLICATIONS OF THE FINDINGS: These guidelines offer valuable direction for healthcare professionals who are responsible for the care of patients undergoing ART treatment for infertility. Their purpose is to promote safe and effective ART treatment, enabling patients to make informed decisions based on realistic expectations. The guidelines aim to ensure that patients are fully informed about the various treatment options available to them and the likelihood of any additional treatment or test to improve the chance of achieving a live birth. STUDY FUNDING/COMPETING INTEREST(S): All costs relating to the development process were covered from ESHRE funds. There was no external funding of the development process or manuscript production. K.L. reports speakers fees from Merck and was part of a research study by Vitrolife (unpaid). T.E. reports consulting fees from Gynemed, speakers fees from Gynemed and is part of the scientific advisory board of Hamilton Thorne. N.P.P. reports grants from Merck Serono, Ferring Pharmaceutical, Theramex, Gedeon Richter, Organon, Roche, IBSA and Besins Healthcare, speakers fees from Merck Serono, Ferring Pharmaceutical, Theramex, Gedeon Richter, Organon, Roche, IBSA and Besins Healthcare. S.R.H. declares being managing director of Fertility Europe, a not-for-profit organization receiving financial support from ESHRE. I.S. is a scientific advisor for and has stock options from Alife Health, is co-founder of IVFvision LTD (unpaid) and received speakers' fee from the 2023 ART Young Leader Prestige workshop in China. A.P. reports grants from Gedeon Richter, Ferring Pharmaceuticals and Merck A/S, consulting fees from Preglem, Novo Nordisk, Ferring Pharmaceuticals, Gedeon Richter, Cryos and Merck A/S, speakers fees from Gedeon Richter, Ferring Pharmaceuticals, Merck A/S, Theramex and Organon, travel fees from Gedeon Richter. The other authors disclosed no conflicts of interest. DISCLAIMER: This Good Practice Recommendations (GPRs) document represents the views of ESHRE, which are the result of consensus between the relevant ESHRE stakeholders and are based on the scientific evidence available at the time of preparation.ESHRE GPRs should be used for information and educational purposes. They should not be interpreted as setting a standard of care or bedeemedinclusive of all proper methods of care, or be exclusive of other methods of care reasonably directed to obtaining the same results.Theydo not replace the need for application of clinical judgement to each individual presentation, or variations based on locality and facility type.Furthermore, ESHRE GPRs do not constitute or imply the endorsement, or favouring, of any of the included technologies by ESHRE.

Morphokinetics of vitrified and warmed blastocysts predicts implantation potential.

PURPOSE: It was studied whether morphokinetics of blastocoele re-expansion and hatching in vitrified-warmed blastocysts is predictive of implantation, clinical pregnancy, and live birth. METHODS: In 144 patients aiming for single warmed blastocyst transfer, blastocysts were cultured in a new time-lapse system (Miri® TL) immediately after warming. Video sequences with an image interval of 5 min were annotated and the corresponding morphokinetic variables were correlated with pregnancy outcome. In detail, tRE (start of re-expansion), tCRE (completion of re-expansion), tAH (hatching from the manipulated zona pellucida), and presence of collapses were recorded. RESULTS: In the pregnant group, tRE and tCRE were significantly lower (0.69 ± 0.45 h and 2.16 ± 0.94 h) as compared to the non-pregnant group (1.23 ± 1.08 h and 2.70 ± 1.20 h). Both variables and the duration of re-expansion (tCRE-tRE) allowed for distinction between "non-pregnant," "loss of pregnancy," and live birth/ongoing pregnancy. Presence and number of collapses showed no correlation with outcome. CONCLUSIONS: Time-lapse imaging of vitrified-warmed blastocysts offers additional selection criteria allowing for prediction of implantation potential. As a consequence, cumulative pregnancy rate could be increased and time-to-pregnancy reduced.

Pharmacokinetic Evaluation of a Drug Transporter Cocktail Consisting of Digoxin, Furosemide, Metformin, and Rosuvastatin.

This article reports the clinical investigation of a probe drug cocktail containing substrates of key drug transporters. Single oral doses of 0.25 mg digoxin (P-gp), 5 mg furosemide (OAT1 and OAT3), 500 mg metformin (OCT2, MATE1, and MATE2-K), and 10 mg rosuvastatin (OATP1B1, OATP1B3, and BCRP) were administered separately or as a cocktail in a randomized six-period crossover trial in 24 healthy male volunteers. As a cocktail, relative bioavailabilities of digoxin and metformin and furosemide AUC0-tz were similar to separate dosing. However, when administered as a cocktail the Cmax of furosemide was 19.1% lower and the Cmax and AUC0-tz of rosuvastatin were 38.6% and 43.4% higher, respectively. In addition, the effects of increased doses of metformin or furosemide on the cocktail were investigated in 11 and 12 subjects, respectively. The cocktail explored in this trial has the potential to be used for the in vivo screening of transporter-mediated drug-drug interactions. © 2016 American Society for Clinical Pharmacology and Therapeutics.

Artificial oocyte activation: evidence for clinical readiness.

Artificial oocyte activation using Ca(2+)ionophores or similar compounds is a widely applied technique in IVF laboratories. This is all the more interesting as most of the agents aiming for intracellular Ca(2+) increase do not result in physiological Ca(2+) oscillations but much rather cause a single Ca(2+) transient. Two observations from mammals may explain why a rather non-physiological single Ca(2+) peak caused by ionophores is sufficient to rescue cycles showing severe male factor infertility, deficient oocyte maturation, developmental problems in humans, or both. On the one hand, it has been shown that it is mainly the initial Ca(2+) rise that drives further downstream events, in particular calcium/calmodulin-dependent protein kinase II (CaMKII) action, and on the other, it is possible that this enzyme remains active even in the absence of Ca(2+). It therefore seems that mammalian oocytes can respond to a wide range of intracellular Ca(2+) signals and have a surprisingly high degree of tolerance for changes in cytosolic Ca(2+). As epigenetic consequences or differences in gene expression have not been studied to date, artificial oocyte activation has to be considered as experimental and should only be applied with a proper indication.

Healthy twin live-birth after ionophore treatment in a case of theophylline-resistant Kartagener syndrome.

PURPOSE: To evaluate whether it is a feasible option to target the oocyte (with Ca(2+)-ionophore) in case that sperm motility cannot be restored in Kartagener syndrome. METHODS: A case of a male Kartagener syndrome with exclusively immotile spermatozoa that did not react to the dimethylxanthine theophylline. Thus, half of the associated oocytes were treated for 15 min with the ready-to-use- ionophore CultActive immediately after ICSI whereas the other 50 % were injected with routine ICSI without artificial oocyte activation. Rates of fertilization, blastulation, pregnancy and live birth were evaluated. RESULTS: Fertilization check revealed that none of the conventionally injected but 4/6 (66.7 %) of the artificially activated oocytes showed two pronuclei. Three embryos were of good and one of fair quality. Corresponding blastocyst formation rate was 3 out of 4 (75 %). A double embryo transfer led to a healthy twin birth in the 34th week of gestation (two boys with a birth weight of 1724 g and 2199 g). CONCLUSIONS: This case indicates that Ca(2+)-ionophore treatment in cycles from theophylline-resistant Kartagener syndrome patients is a feasible option. The future will show if routine application of A23187 in Kartagener or primary cilia dyskinesis patients will be of benefit.

Live birth after artificial oocyte activation using a ready-to-use ionophore: a prospective multicentre study.

Artificial oocyte activation has been proposed as a suitable means to overcome the problem of failed or impaired fertilization after intracytoplasmic sperm injection (ICSI). In a multicentre setting artificial oocyte activation was applied to 101 patients who were diagnosed with fertilization abnormalities (e.g. less than 50% fertilized oocytes) in a previous conventional ICSI cycle. Female gametes were activated for 15 min immediately after ICSI using a ready-to-use Ca(2+)-ionophore solution (A23187). Fertilization, pregnancy and live birth rates were compared with the preceding cycle without activation. The fertilization rate of 48% in the study cycles was significantly higher compared with the 25% in the control cycles (P < 0.001). Further splitting of the historical control group into failed (0%), low (1-30%) and moderate fertilization rate (31-50%) showed that all groups significantly benefitted (P < 0.001) in the ionophore cycle. Fewer patients had their embryo transfer cancelled compared with their previous treatments (1/101 versus 15/101). In total, 99% of the patients had an improved outcome with A23187 application resulting in a 28% live birth rate (35 babies). These data suggest that artificial oocyte activation using a ready-to-use compound is an efficient method.

Influence of intracervical and intravaginal seminal plasma on the endometrium in assisted reproduction: a double-blind, placebo-controlled, randomized study.

OBJECTIVE: To investigate the effect of intracervical and intravaginal application of seminal plasma on the endometrium, as assessed by endometrial/subendometrial vascularization and endometrial volume between the day of oocyte retrieval and the day of embryo transfer in an in-vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycle. METHODS: This was a double-blind, placebo-controlled, randomized study including patients undergoing a first or second IVF/ICSI cycle. Homologous seminal plasma or placebo (sodium chloride) was injected into the cervix and posterior vaginal fornix just after follicle aspiration. Three-dimensional power Doppler examination was performed 30 min before oocyte retrieval and 30 min before embryo transfer. Main outcome measures were changes in vascularization flow index (VFI), flow index (FI) and vascularization index (VI) of the endometrium/subendometrium using VOCAL™ (Virtual Organ Computer-aided AnaLysis) and endometrial volume. RESULTS: One hundred patients agreed to participate in the study. Twenty-three patients were excluded, mainly as a result of canceled embryo transfer. Data were analyzed from 40 patients receiving seminal plasma and 37 receiving placebo. No significant differences between the two groups were seen in VFI, FI or VI of the endometrium or subendometrium or in endometrial volume on the day of oocyte pick-up and on the day of embryo transfer. CONCLUSION: Neither endometrial/subendometrial vascularization parameters nor endometrial volume seem to be affected by the application of seminal plasma in patients undergoing their first or second IVF/ICSI cycle.

Treatment with Ca2+ ionophore improves embryo development and outcome in cases with previous developmental problems: a prospective multicenter study.

STUDY QUESTION: Does calcium ionophore treatment (A23187, calcimycin) improve embryo development and outcome in patients with a history of developmental problems/arrest? SUMMARY ANSWER: Application of A23187 leads to increased rates of cleavage to 2-cell stage, blastocyst formation and clinical pregnancy/live birth. WHAT IS KNOWN ALREADY: Studies on lower animals indicate that changes in intracellular free calcium trigger and regulate the events of cell division. In humans, calcium fluctuations were detected with a peak shortly before cell division. Interestingly, these calcium oscillations disappeared in arrested embryos. Mitotic division blocked with a Ca(2+) chelator could be restored by means of ionophores in an animal model. STUDY DESIGN, SIZE, DURATION: This prospective, multicenter (five Austrian centers), uncontrolled intervention study (duration 1 year) includes 57 patients who provided informed consent. PARTICIPANTS/MATERIALS, SETTING, METHODS: Inclusion criteria were complete embryo developmental arrest in a previous cycle (no transfer), complete developmental delay (no morula/blastocyst on Day 5), or reduced blastocyst formation on Day 5 (≤15%). Severe male factor patients and patients with <30% fertilization rate after ICSI were excluded because these would be routine indications for ionophore usage. The total of the 57 immediately preceding cycles in the same patients constituted the control cycles/control group. In the treatment cycles, all metaphase II-oocytes were exposed to a commercially available ready-to-use ionophore for 15 min immediately after ICSI. After a three-step washing procedure, in vitro culture was performed as in the control cycles, up to blastocyst stage when achievable. MAIN RESULTS AND THE ROLE OF CHANCE: Fertilization rate did not differ (75.4 versus 73.2%); however, further cleavage to 2-cell stage was significantly higher (P < 0.001) in the ionophore group (98.5%) when compared with the control cycles (91.9%). In addition, significantly more (P < 0.05) blastocysts formed on Day 5 in the study compared with the control group (47.6 versus 5.5%, respectively) and this was associated with a significant increase (P < 0.01) in the rates of implantation (44.4 versus 12.5%), clinical pregnancy (45.1 versus 12.8%) and live birth (45.1 versus 12.8%). All babies born at the time of writing (22/28) were healthy. LIMITATIONS, REASONS FOR CAUTION: The frequency of patients showing embryo developmental problems was expected to be low; therefore, a multicenter approach was chosen in order to increase sample size. In one-third of the cycles, the clinician or patient requested a change of stimulation protocol; however, this did not influence the developmental rate of embryos. WIDER IMPLICATIONS OF THE FINDINGS: This is the first evidence that developmental incompetence of embryos is an additional indication for ionophore treatment. The present approach is exclusively for overcoming cleavage arrest. STUDY FUNDING/COMPETING INTERESTS: No funding received. T.E. reports fees from Gynemed, outside the submitted work. All co-authors have no interest to declare.

Efficacy of a sperm-selection chamber in terms of morphology, aneuploidy and DNA packaging.

Since most current techniques analysing spermatozoa will inevitably exclude these gametes from further use, attempts have been made to enrich semen samples with physiological spermatozoa with good prognosis using special sperm-processing methods. A particular sperm-selection chamber, called the Zech-selector, was found to be effective in completely eliminating spermatozoa with DNA strand breaks. The aim of this study was to further analyse the subgroup of spermatozoa accumulated using the Zech-selector. In detail, the potential of the chamber to select for proper sperm morphology, DNA status and chromatin condensation was tested. Two samples, native and processed semen, of 53 patients were analysed for sperm morphology (×1000, ×6300), DNA packaging (fragmentation, chromatin condensation) and chromosomal status (X, Y, 18). Migration time (the time needed for proper sperm accumulation) was significantly correlated to fast progressive motility (P=0.002). The present sperm-processing method was highly successful with respect to all parameters analysed (P<0.001). In particular, spermatozoa showing numeric (17.4% of patients without aneuploidy) or structural chromosomal abnormalities (90% of patients without strand-breaks) were separated most effectively. To summarize, further evidence is provided that separating spermatozoa without exposure to centrifugation stress results in a population of highly physiological spermatozoa.

In vitro glucuronidation of the angiotensin II receptor antagonist telmisartan in the cat: a comparison with other species.

Glucuronidation of telmisartan comprises nearly its entire metabolic clearance in several mammalian species including human. However, data were lacking for the cat, a species noted for its inability to glucuronidate some drugs. Therefore, the glucuronidation of telmisartan was investigated using feline liver microsomes and compared to liver microsomes of rats, dogs, and human, intestinal human microsomes and cell lines expressing human UDP-glucuronosyltransferases (UGT). Incubation of telmisartan with cat liver microsomes readily yielded telmisartan glucuronide, and pooled (N = 3 for each gender) cat liver microsomes even showed the highest glucuronidation rate (cat > dog >> human > rat). Michaelis Menten kinetics were observed with Km of 7.5 and 10 µm and Vmax of 3.9 and 3.3 nmol/min/mg for male and female cats, respectively. Confirming the in vitro data, telmisartan glucuronide was detected as the major circulating metabolite in cat plasma. To elucidate which UGT enzymes are involved, telmisartan was incubated with cell lines expressing human UGTs. The highest glucuronidation activity was observed for UGT1A8, UGT1A7, and UGT1A9. In conclusion, telmisartan was effectively glucuronidated in cats. Defects of the UGT1A6 gene in cats do not affect the glucuronidation of telmisartan as it is not a substrate of human UGT1A6.

Planar embryos have poor prognosis in terms of blastocyst formation and implantation.

Normally, day-2 embryos show a crosswise arrangement of four cells with three blastomeres lying side by side. Cleavage anomalies include embryos that are characterized by a particular planar constellation of four blastomeres with presumed incomplete cleavage. Since little is known on the developmental fate of such conceptuses, within a 10-month period all consecutive patients were screened for day-2 planar embryos. A total of 64/2070 embryos with suboptimal blastomere configuration were detected (3.1%). In conventional IVF, planar embryos were significantly less frequent (0.7%) as compared with intracytoplasmic sperm injection (2.8%; P<0.05) and cases of testicular sperm extraction (5.4%; P<0.01). Interestingly, embryos with a cleavage anomaly showed better morphology both on day 2 (P<0.005) and day 3 (P<0.001). In contrast, blastocyst formation (P<0.001) and blastocyst quality (P=NS) was higher in tetrahedral embryos. There was a significant increase in implantation rate if tetrahedral embryos could be transferred compared with when planar embryos had to be transferred (P<0.01). It may be postulated that, in planar embryos, the mitotic spindle might have been affected, e.g. sperm centrosome composition or function, which in turn might have led to the observed cleavage anomaly. Normally, day-2 embryos show a crosswise arrangement of four cells with three blastomeres lying side by side. Cleavage anomalies include more planar embryos that are characterized by a particular flat constellation of four blastomeres with presumed premature cleavage (like a tetrafoliate clover). Since little is known on the developmental fate of such embryos within a 10-month study period, all consecutive patients were screened for the presence of day-2 planar embryos (study group). A total of 64 (out of 2070) embryos with abnormal blastomere configuration were detected (3.1%). Interestingly, in conventional IVF (0.7%), the presence of planar embryos was significantly less frequent as compared with intracytoplasmic sperm injection (2.8%; P<0.05) and cases of testicular biopsy (5.4%; P<0.01). Embryos from the study group showed better morphology both on day 2 (P<0.005) and day 3 (P<0.001). In contrast, blastocyst formation (survival to day 5 of preimplantation development) was higher in the normally cleaved control group (P<0.001) and so was blastocyst quality; however, the latter parameter did not reach level of significance. This was also reflected in a significantly higher implantation rate in the control group (P<0.01). Based on present data, it may be postulated that, in planar embryos, the mitotic spindle (which involves the sperm centrosome) might have been affected, which in turn might have led to an incomplete cleavage.

A plea for a more physiological ICSI.

Intracytoplasmic sperm injection (ICSI) can be considered the most 'revolutionary' in vitro insemination technique because it has efficiently allowed the treatment of male factor infertility. Although ICSI has been successfully and safely applied worldwide for almost 20 years, currently, we have no real knowledge regarding the hypothetical long-term side effects on ICSI adults, given the increased likelihood of spermatozoa with defective nuclear content fertilising the oocytes. The aim of this review article is to investigate the most recent advances of performing ICSI in the safest possible manner, thus, minimising the theoretical hazards of this procedure. To allow for substantiated recommendation which male gametes to choose for physiological ICSI an updated search was performed in Medline and Embase, from 1996 to June 2011. Recent technical advances allow operators to more or less simulate physiological conditions in the laboratory, reducing potential damage to the gametes. It seems possible to prevent fertilisation by DNA-damaged and chromosomal-unbalanced spermatozoa by selecting ICSI sperm by motility and/or maturation markers such as hyaluronic acid or other zona pellucida receptors. Furthermore, novel non-invasive imaging techniques can be valid tools for helping in the morphological selection of ICSI spermatozoa.

Easy sperm processing technique allowing exclusive accumulation and later usage of DNA-strandbreak-free spermatozoa.

Sperm DNA fragmentation is increased in poor-quality semen samples and correlates with failed fertilization, impaired preimplantation development and reduced pregnancy outcome. Common sperm preparation techniques may reduce the percentage of strandbreak-positive spermatozoa, but, to date, there is no reliable approach to exclusively accumulate strandbreak-free spermatozoa. To analyse the efficiency of special sperm selection chambers (Zech-selectors made of glass or polyethylene) in terms of strandbreak reduction, 39 subfertile men were recruited and three probes (native, density gradient and Zech-selector) were used to check for strand breaks using the sperm chromatin dispersion test. The mean percentage of affected spermatozoa in the ejaculate was 15.8 ± 7.8% (range 5.0­42.1%). Density gradient did not significantly improve the quality of spermatozoa selected(14.2 ± 7.0%). However, glass chambers completely removed 90% spermatozoa showing strand breaks and polyethylene chambers removed 76%. Both types of Zech-selectors were equivalent in their efficiency, significantly reduced DNA damage (P < 0.001) and,with respect to this, performed better than density gradient centrifugation (P < 0.001). As far as is known, this is the first report ona sperm preparation technique concentrating spermatozoa unaffected in terms of DNA damage. The special chambers most probably select for sperm motility and/or maturity.

Group culture of human zygotes is superior to individual culture in terms of blastulation, implantation and life birth.

This prospective study tested a new type of culture dish for the effects of individual culture and autotrophic factors. Within a 6-month period, 72 patients with nine or more fertilized eggs were enrolled in this prospective evaluation. Their 936 zygotes were split into three subgroups (individual culture, individual culture with contact to neighbours, group culture). All concepti were cultured in 30 µl drops (medium change on day 3) until blastocyst stage. On day 5, a single-blastocyst transfer was performed and the remaining blastocysts of good quality were vitrified. Fertilization rates were 69% for IVF and 81% for intracytoplasmic sperm injection. Blastulation was 48%. Single-blastocyst transfer resulted in a clinical pregnancy rate of 54%. Group culture was superior in terms of compaction (P<0.01) and blastulation (P<0.001) as compared with individual culture. A better blastocyst quality was observed in group culture (P<0.05). As a trend, more life births were achieved with blastocysts derived from group culture. As far as is known, this is the first evidence that grouping embryos improves preimplantation development in human and it is recommended that culture volume should be reduced or embryo density increased.

Excretion mass balance evaluation, metabolite profile analysis and metabolite identification in plasma and excreta after oral administration of [14C]-meloxicam to the male cat: preliminary study.

The objective of this study was to investigate the metabolic pathways and routes of excretion of oral meloxicam in the cat. [(14)C]-meloxicam was administered orally to three fasted male cats. Urine, faeces, vomit and cage washes were collected over the following 144 h period. Blood was collected predosing and at 3 and 12 h postdosing. Metabolites were identified by HPLC/MS/MS. When possible a metabolic structure was proposed for each metabolite detected. Only unchanged meloxicam was identified in plasma. Five major metabolites were detected in urine and four in faeces, which were identified by HPLC/MS/MS as products of oxidative metabolism. No conjugated metabolites were detected. Elimination occurred early (61% during the first 48 h). A total of 21% of the recovered dose was eliminated in urine (2% as unchanged meloxicam, 19% as metabolites) and 79% in the faeces (49% as unchanged meloxicam, 30% as metabolites). The results indicate that after oral administration the major route of excretion of meloxicam in the cat is faecal and that the main pathway of biotransformation of meloxicam in the cat is oxidation.

Morphology of vitrified/warmed day-5 embryos predicts rates of implantation, pregnancy and live birth.

Although some post-thaw morphological predictors of pregnancy have been investigated in slow freezing of blastocysts, no such data have been published for vitrified and warmed blastocysts. Therefore, a prospective four-part score was applied to vitrified/warmed day-5 embryos to evaluate whether certain morphological parameters could serve as predictors of implantation, pregnancy and live birth. All morulae/blastocysts that were considered to be viable after warming were scored according to a previously unpublished grading system based on re-expansion, hatching (out of an artificial gap in the zona pellucida), extensive cytoplasmic granulation and presence of necrotic foci. Overall, 74% (202/273) of the vitrified concepti were found to be viable after warming. Early blastocysts showed better survival versus extended/hatching blastocysts (P < 0.01). Of the morphological parameters analysed, immediate re-expansion (P < 0.05) and hatching (P < 0.001) were positive predictors of the rates of implantation, pregnancy and live birth. The opposite holds for extensive cytoplasmic granulation (P < 0.05), which was negatively related. Accurate scoring of warmed blastocysts (within the first 2 h) allows for prediction of pregnancy outcome, and thus will help to further reduce the number of transferred embryos.

Long-term potentiation of the responses to parallel fiber stimulation in mouse cerebellar cortex in vivo.

Long-term potentiation (LTP) of parallel fiber-Purkinje cell (PF-PC) synapses in the cerebellum has been suggested to underlie aspects of motor learning. Previous in vitro studies have primarily used low frequency PF stimulation conditioning paradigms to generate either presynaptic PF-PC LTP (4-8 Hz) or postsynaptic PF-PC LTP (1 Hz). Little is known about the conditions that evoke PF-PC LTP in vivo. High frequency stimulation in vivo increases PC responsiveness to peripheral stimuli; however, neither the site of action nor the signaling pathways involved have been examined. Using flavoprotein autofluorescence optical imaging in the FVB mouse in vivo, this report describes that a conditioning stimulation consisting of a high frequency burst of PF stimulation (100 Hz, 15 pulse trains every 3 s for 5 min) evokes a long-term increase in the response to PF stimulation. Following the conditioning stimulation, the response to PF stimulation increases over 20 min to approximately 130% above baseline and this potentiation persists for at least 2 h. Field potential recordings of the responses to PF stimulation show that the postsynaptic component is potentiated but the presynaptic, parallel fiber volley is not. Paired-pulse facilitation does not change after the conditioning stimulation, suggesting the potentiation occurs postsynaptically. Blocking non-NMDA (N-methyl-d-aspartic acid) ionotropic glutamate receptors with DNQX (6,7-dinitroquinoxaline-2,3-dione disodium salt, 50 muM, bath application) during the conditioning stimulation has no effect on the long-term increase in fluorescence. However, blocking subtype I metabotropic glutamate receptors (mGLuR(1)) with LY367385 (200 muM) during the conditioning stimulation abolishes the long-term increase in fluorescence. Blocking GABAergic neurotransmission is not required to evoke this long-term potentiation. Blocking GABA(A) receptors reduces but does not eliminate the long-term potentiation. Therefore, this study demonstrates that high frequency PF stimulation generates long-term potentiation of PF-PC synapses in vivo. This novel form of LTP is generated primarily postsynaptically and is mediated by mGluR(1) receptors.