Developmentally delayed cleavage-stage embryos maintain comparable implantation rates in frozen embryo transfers

Purpose

In fresh IVF cycles, embryos reaching the eight-cell stage on day 3 of development are thought to have a higher chance of implantation than those reaching this stage on day 4. To determine whether this difference persists after cryopreservation, we compared pregnancy and implantation rates between frozen embryo transfer (FET) cycles using delayed cleavage-stage embryos (cryopreserved day 4) and normal cleavage-stage embryos (cryopreserved day 3).

Methods

Participants underwent FET between 2008 and 2012 using embryos cryopreserved on either day 3 (n = 76) or day 4 (n = 48), depending on the length of time needed to achieve the eight-cell stage. All embryos, regardless of day of cryopreservation, were thawed and transferred on the 4th day of vaginal progesterone following endometrial preparation with oral estradiol. Chi-square and Mann-Whitney U tests were used to compare patient demographics and cycle outcomes.

Results

More women in the day 4 group had diminished ovarian reserve (44 vs 16 %, p = 0.003). Pregnancy outcomes in preceding fresh cycles were not different between the two groups. Pregnancy, implantation, and live birth rates following FET did not differ between the day 3 and day 4 groups.

Conclusions

This is the first study to address outcomes using day 3 versus day 4 cryopreserved embryos. Despite a higher prevalence of diminished ovarian reserve (DOR) in the day 4 group, delayed cleavage-stage embryos utilized in FET cycles performed as well as embryos growing at the normal rate, suggesting delayed embryo development does not affect embryo implantation as long as endometrial synchrony is maintained.     

Prediction Model for Two-Year Risk of Opioid Overdose Among Patients Prescribed Chronic Opioid Therapy

Background

Naloxone is a life-saving opioid antagonist. Chronic pain guidelines recommend that physicians co-prescribe naloxone to patients at high risk for opioid overdose. However, clinical tools to efficiently identify patients who could benefit from naloxone are lacking.

Objective

To develop and validate an overdose predictive model which could be used in primary care settings to assess the need for naloxone.

Design

Retrospective cohort.

Setting

Derivation site was an integrated health system in Colorado; validation site was a safety-net health system in Colorado.

Participants

We developed a predictive model in a cohort of 42,828 patients taking chronic opioid therapy and externally validated the model in 10,708 patients.

Main Measures

Potential predictors and outcomes (nonfatal pharmaceutical and heroin overdoses) were extracted from electronic health records. Fatal overdose outcomes were identified from state vital records. To match the approximate shelf-life of naloxone, we used Cox proportional hazards regression to model the 2-year risk of overdose. Calibration and discrimination were assessed.

Key Results

A five-variable predictive model showed good calibration and discrimination (bootstrap-corrected c-statistic?=?0.73, 95% confidence interval [CI] 0.69–0.78) in the derivation site, with sensitivity of 66.1% and specificity of 66.6%. In the validation site, the model showed good discrimination (c-statistic?=?0.75, 95% CI 0.70–0.80) and less than ideal calibration, with sensitivity and specificity of 82.2% and 49.5%, respectively.

Conclusions

Among patients on chronic opioid therapy, the predictive model identified 66–82% of all subsequent opioid overdoses. This model is an efficient screening tool to identify patients who could benefit from naloxone to prevent overdose deaths. Population differences across the two sites limited calibration in the validation site.

     

Increased Coronary Artery Disease Severity in Black Women Undergoing Coronary Bypass Surgery

Race and sex disparities are believed to play an important role in heart disease. The purpose of this study was to examine the association between race, sex, and number of diseased vessels at the time of coronary artery bypass grafting (CABG), and subsequent postoperative outcomes.The 13,774 patients undergoing first-time, isolated CABG between 1992 and 2011 were included. Trend in the number of diseased vessels between black and white patients, stratified by sex, were analyzed using a Cochran–Armitage trend test. Models were adjusted for age, procedural status (elective vs. nonelective), and payor type (private vs. nonprivate insurance).Black female CABG patients presented with an increasingly greater number of diseased vessels than white female CABG patients (adjusted P_trend = 0.0021). A similar trend was not observed between black and white male CABG patients (adjusted P_trend = 0.18). Black female CABG patients were also more likely to have longer intensive care unit and hospital lengths of stay than other race–sex groups.Our findings suggest that black female CABG patients have more advanced coronary artery disease than white female CABG patients. Further research is needed to determine the benefit of targeted preventive care and preoperative workup for this high-risk group.     

Calcineurin mediates homeostatic synaptic plasticity by regulating retinoic acid synthesis

Homeostatic synaptic plasticity is a form of non-Hebbian plasticity that maintains stability of the network and fidelity for information processing in response to prolonged perturbation of network and synaptic activity. Prolonged blockade of synaptic activity decreases resting Ca²⁺ levels in neurons, thereby inducing retinoic acid (RA) synthesis and RA-dependent homeostatic synaptic plasticity; however, the signal transduction pathway that links reduced Ca²⁺-levels to RA synthesis remains unknown. Here we identify the Ca²⁺-dependent protein phosphatase calcineurin (CaN) as a key regulator for RA synthesis and homeostatic synaptic plasticity. Prolonged inhibition of CaN activity promotes RA synthesis in neurons, and leads to increased excitatory and decreased inhibitory synaptic transmission. These effects of CaN inhibitors on synaptic transmission are blocked by pharmacological inhibitors of RA synthesis or acute genetic deletion of the RA receptor RARα. Thus, CaN, acting upstream of RA, plays a critical role in gating RA signaling pathway in response to synaptic activity. Moreover, activity blockade-induced homeostatic synaptic plasticity is absent in CaN knockout neurons, demonstrating the essential role of CaN in RA-dependent homeostatic synaptic plasticity. Interestingly, in GluA1 S831A and S845A knockin mice, CaN inhibitor- and RA-induced regulation of synaptic transmission is intact, suggesting that phosphorylation of GluA1 C-terminal serine residues S831 and S845 is not required for CaN inhibitor- or RA-induced homeostatic synaptic plasticity. Thus, our study uncovers an unforeseen role of CaN in postsynaptic signaling, and defines CaN as the Ca²⁺-sensing signaling molecule that mediates RA-dependent homeostatic synaptic plasticity.Synaptic plasticity, a fundamental feature of the nervous system, is defined as modification of synaptic strength based on experience and activity history. Homeostatic synaptic plasticity is a type of compensatory mechanism activated during chronic elevation or reduction of network activity to modulate synaptic strength in the opposite direction (for example, reduced network activity leads to increased synaptic strength) (1, 2). Retinoic acid (RA) is a key signaling molecule in a form of homeostatic synaptic plasticity induced by reduced excitatory synaptic activity (3–5). Prolonged inhibition of excitatory synaptic transmission leads to a compensatory increase in synaptic excitation and a decrease in synaptic inhibition (4, 5). Both of these processes require RA synthesis. It also has been shown that dendritic Ca²⁺ levels directly govern the synthesis of RA; basal Ca²⁺ levels maintained by normal synaptic transmission are sufficient to suppress RA synthesis. Upon synaptic activity inhibition, reduced Ca²⁺ levels de-repress RA synthesis and activate RA-dependent homeostatic synaptic mechanisms (6). Thus, a Ca²⁺-dependent signaling molecule that is sensitive to changes in basal Ca²⁺ levels must be involved in synaptic RA signaling.Ca²⁺-dependent protein kinases and phosphatases are critical components of signaling pathways involved in synaptic plasticity (7, 8). For example, regulation of the phosphorylation of key serine residues in the C-terminal sequences of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptor (AMPAR) subunit GluA1 by kinases (i.e., PKA, PKC, and CaMKII) and phosphatases [i.e., calcineurin (CaN) and PP2A] is thought to play major roles in governing AMPAR trafficking in and out of the synaptic membrane and to mediate the expression of well-established forms of synaptic plasticity, such as long-term potentiation (LTP) and long-term depression (LTD) (9–12). A recent study examining the stoichiometry of AMPAR phosphorylation revealed surprisingly low levels of phosphorylated GluA1 in neurons (13), however, suggesting that the phosphorylation of AMPARs might not be involved in homeostatic plasticity, and that other mechanisms may be in play.In the present study, we identified CaN as the Ca²⁺-dependent signaling molecule that regulates RA synthesis in neurons in an activity-dependent manner. Inhibition of CaN activity triggers RA synthesis, suggesting that basal CaN activity, supported by normal synaptic transmission, is sufficient to suppress RA synthesis in an active neural network. In CaN-deficient neurons, synaptic activity blockade-induced RA-dependent forms of homeostatic synaptic plasticity are absent. Similar to direct RA application, CaN inhibition enhances excitatory synaptic transmission and reduces inhibitory synaptic transmission. Blocking RA synthesis or genetic deletion of the RA receptor RARα prevents CaN inhibitor-induced regulation of synaptic strength, indicating that CaN acts upstream of RA. Importantly, neurons bearing GluA1 S831A or S845A knockin (KI) mutations, which eliminate phosphorylation at these two serine residues, respond normally to CaN inhibitors and RA treatment. Taken together, our results indicate that CaN participates in RA-dependent homeostatic synaptic plasticity through regulation of RA synthesis independent of the modulation of GluA1 phosphorylation.     

Prevention of type I diabetes transfer by glutamic acid decarboxylase 65 peptide 206-220-specific T cells

Glutamic acid decarboxylase (GAD) 65 is one of the major pancreatic antigens targeted by self-reactive T cells in type I diabetes mellitus. T cells specific for GAD65 are among the first to enter inflamed islets and may be important for the initiation of autoimmune diabetes. However, we previously reported that nonobese diabetic (NOD) mice transgenic for a T cell antigen receptor (TCR) specific for one of the immunodominant epitopes of GAD65, peptide 286-300 (G286), are protected from insulitis and diabetes. To examine whether other GAD65-reactive T cells share this phenotype, we have generated TCR transgenic NOD mice for a second immunodominant epitope of GAD65, peptide 206-220 (G206). As in G286 mice, G206 mice do not develop islet inflammation or diabetes. When adoptively transferred along with diabetogenic T cells, activated G206 T cells significantly delayed the onset of diabetes in NOD.scid recipients. Both G206 and G286 T cells produce immunoregulatory cytokines IFN-gamma and IL-10 at low levels when activated by cognate antigens. These data suggest that GAD65-specific T cells may play a protective role in diabetes pathogenesis by regulating pathogenic T cell responses. A better understanding of the functions of autoreactive T cells in type I diabetes will be necessary for choosing desirable targets for immunotherapy.     

Distinct von Hippel-Lindau gene and hypoxia-regulated alterations in gene and protein expression patterns of renal cell carcinoma and their effects on metabolism

During the last decade the knowledge about the molecular mechanisms of the cellular adaption to hypoxia and the function of the “von Hippel Lindau” (VHL) protein in renal cell carcinoma (RCC) has increased, but there exists little information about the overlap and differences in gene/protein expression of both processes. Therefore the aim of this study was to dissect VHL- and hypoxia-regulated alterations in the metabolism of human RCC using ome-based strategies. The effect of the VHL- and hypoxia-regulated altered gene/protein expression pattern on the cellular metabolism was analyzed by determination of glucose uptake, lactate secretion, extracellular pH, lactate dehydrogenase activity, amino acid content and ATP levels. By employing VHL⁻/VHL⁺ RCC cells cultured under normoxic and hypoxic conditions, VHL-dependent, HIF-dependent as well as VHL-/HIF-independent alterations in the gene and protein expression patterns were identified and further validated in other RCC cell lines. The genes/proteins differentially expressed under these distinct conditions were mainly involved in the cellular metabolism, which was accompanied by an altered metabolism as well as changes in the abundance of amino acids in VHL-deficient cells. In conclusion, the study reveals similarities, but also differences in the genes and proteins controlled by VHL functionality and hypoxia thereby demonstrating differences in the metabolic switch of RCC under these conditions.