Prevalence and risk factors of zygotic splitting after 937 848 single embryo transfer cycles.

STUDY QUESTION: What is the prevalence of multiple pregnancy with zygotic splitting after single embryo transfer (SET)? SUMMARY ANSWER: The prevalence of multiple pregnancy with zygotic splitting after SET was 1.36%. WHAT IS KNOWN ALREADY: In 2008, the Japan Society of Obstetrics and Gynaecology (JSOG) recommended the adoption of SET to reduce multiple births. Since then, to improve the clinical pregnancy rate, elective SET using blastocyst transfer and frozen-warmed ET has increased. Blastocyst culture and zona pellucida manipulation, including ICSI and AH, have been widely reported as risk factors for monozygotic twinning. However, all these studies may have included cases with dizygotic pregnancies produced by a transferred embryo and a spontaneous conception. STUDY DESIGN, SIZE, DURATION: A retrospective observational study was performed, based on 937 848 SET cycles in registered ART data from the JSOG between 2007 and 2014. The study was approved by the Registration and Research Subcommittee of the JSOG and Juntendo University Ethics Committee. PARTICIPANTS/MATERIALS, SETTING, METHODS: To identify possible factors affecting the prevalence of zygotic splitting, we identified pregnancies, in which the number of foetuses exceeded the number of gestational sacs (GSs), to restrict our analysis to 'true' zygotic splitting. Multiple logistic regression analysis was performed using singleton pregnancy after SET, as control. P < 0.05 was considered statistically significant. MAIN RESULTS AND THE ROLE OF CHANCE: Fresh and frozen-warmed SET produced 276 934 clinical pregnancies (29.5%/SET), including 4310 twins (1.56% of pregnancies) and 109 triplets (0.04% of pregnancies). Based on sex analysis of dichorionic twins after SET, the prevalence of multiple pregnancy with zygotic splitting was 1.36%. Statistical analysis revealed that compared to singleton pregnancies zygotic splitting pregnancies were associated with frozen-warmed ET cycles (odds ratio [OR] = 1.34; 95% CI: 1.16-1.55), blastocyst culture (OR = 1.79; 95% CI: 1.54-2.09) or AH (OR = 1.21; 95% CI: 1.08-1.35). In fresh ET cycles, the prevalence rate of zygotic splitting pregnancy after single blastocyst transfer was significantly higher than that after SET cycles with cleavage embryos (OR = 2.20; 95% CI: 1.83-2.66). However, no significant difference in ovarian stimulation and fertilization methods was recognized. LIMITATIONS, REASONS FOR CAUTION: In the current Japanese ART registry system, data regarding frozen-warmed ET do not include information about ovarian stimulation and fertilization methods. Registration for AH only began in 2010. There is no way of validating if data submitted by clinics is correct. WIDER IMPLICATIONS OF THE FINDINGS: Clinicians should consider whether to counsel couples about the small increase in the risk of zygotic splitting associated with some embryo manipulations. STUDY FUNDING/COMPETING INTEREST(S): No external funds were used for the study. The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: None.

Comparing normal walking and compensated walking: their stability and perturbation resistance. A simulation study.

People usually develop different kinds of compensated gait in response to local function deficits, such as muscle weakness, spasticity in specific muscle groups, or joint stiffness, in order to overcome the falling risk factors. Compensated walking has been analysed empirically in the impaired gait analysis area. However, the compensation could be identified spatially and temporally. The stability and perturbation resistance of compensated walking have not been analysed quantitatively. In this research, a biomimetic human walking simulator was employed to model one individual paraplegic subject with plantarflexor spasticity. The pes equinus was expressed by biasing the outputs of plantarflexor neurons corresponding to the spastic muscles. Then, the compensatory mechanism was explored by adjusting the outputs of the other muscles. It was shown that this approach can be used for quantitative analysis of the spastic gait and compensated walking. Thus, this research can improve the understanding of the behaviour of compensated walking, bringing insights not only for building useful walking assist systems with high safety but also for designing effective rehabilitation interventions.

Crystallization and vitrification of electrons in a glass-forming charge liquid.

Charge ordering (CO) is a phenomenon in which electrons in solids crystallize into a periodic pattern of charge-rich and charge-poor sites owing to strong electron correlations. This usually results in long-range order. In geometrically frustrated systems, however, a glassy electronic state without long-range CO has been observed. We found that a charge-ordered organic material with an isosceles triangular lattice shows charge dynamics associated with crystallization and vitrification of electrons, which can be understood in the context of an energy landscape arising from the degeneracy of various CO patterns. The dynamics suggest that the same nucleation and growth processes that characterize conventional glass-forming liquids guide the crystallization of electrons. These similarities may provide insight into our understanding of the liquid-glass transition.

Comparison of force-time parameters and EMG in static explosive gripping by various exertion conditions: muscle fatigue state and submaximal exertion.

AIM: The purpose of this study was to compare the exertion and electromyography (EMG) properties during the developmental phase (DFmax) in static explosive grip (SEG), rapid submaximal exertion grip, namely fake SEG exertion (FAKE), and SEG in a muscle fatigue state. METHODS: Thirty healthy males and females performed the SEG and FAKE exertions (50% and 75% of peak value as a target value). Then, they performed sustained repeated rhythmic grip for 6 min (30 times x min(-1)), and SEG after 1-min, 4-min, and 7-min (SEG after the exertion). EMG was measured concurrently to compare with the muscle activation property during each grip exertion. Eight force-time parameters evaluating the DFmax in addition to the peak value were selected. RESULTS: The peak value significantly decreased, and the mean power spectrum density shifted to the low-wave in SEG after the exertion as compared with before. Therefore, SEG after the exertion was judged to be a muscle fatigue state. In addition, because the frequency properties in each exertion differed, the muscle activation properties during their DFmaxs were considered to differ. From the comparison between SEG before and after the exertion and FAKE, it is suggested that the time of reaching the peak value and the relative muscle strength when reaching an inflection point are not useful as parameters to evaluate the explosive muscle function during SEG. CONCLUSIONS: The maximal increasing volume during the DFmax and integrated area until 0.25 and 0.5 s could discriminate a difference of the DFmaxs according to each exertion and they are useful parameters.

The characteristics of simple muscle power by gripping: gender differences and reliability of parameters using various loads.

AIM: There are few studies on muscle power during local muscle contractions with a small range of motion such as in gripping. The purposes of this study were to clarify the properties of the developmental phase based on time series of muscle power output, the reliability of the parameters, their relationships and the load intensity derived peak power by gender differences, and to examine the possibility of evaluating muscle power using gripping. METHODS: Fifteen young males and 15 females participated in this study. Based on a crossover experimental design, each subject carried out 2 explosive grips at 20%, 30%, 40% and 50% loads of maximal using a voluntary grip contraction (MVC). The grip contraction velocities, sampled at 100 Hz, were measured accurately using a power instrument with an accelerometer. Muscle power curves were drawn from the product of the velocity and the set-up load. RESULTS: The cross-correlation coefficients between the trials for the average curve of the time-series moving distance, the velocity, and the power in any load were very high (over 0.95) in both genders. The reliability of each parameter was mostly good in both genders (intraclass correlation coefficient, ICC>0.75). The peak power curve differed between genders, and the curve around the peak value in females was irregular. CONCLUSIONS: A gender difference was found in the maximal power and the properties of the power curve. The maximal muscle power appeared at 30-50% MVC in males, and at 20-40% MVC in females. The peak power during the whole contraction, and the time to peak may reflect the conditions throughout the whole of the contraction. The new device used in this study to evaluate local regional muscle power (grip) is a very reliable and useful tool.

Bonding and electronic states of boron in silicon nanowires characterized by an infrared synchrotron radiation beam.

The infrared synchrotron radiation (IR-SR) beamline of SPring-8 as an IR light source was applied to characterize boron (B) atoms in silicon nanowires (SiNWs). The use of an IR-SR beam with much higher brilliance than conventional IR light sources and a wide range of wavenumbers from visible to far IR regions made it possible to detect a local vibrational mode of B in SiNWs. The use of this technique has also made it possible to detect other IR peaks related to transitions of a bound hole from the ground state of a B acceptor atom to excited states, clarifying the electronic state of B acceptors in SiNWs.

Action of enflurane on cholinergic transmission in identified Aplysia neurones.

Effects of enflurane on the cholinergic transmission in Aplysia neurones were studied by current and voltage clamp methods. Acetylcholine (ACh) evoked three types of postsynaptic responses on different identified neurones: (1) a depolarizing response due to an increase in Na and K conductances (D-response), (2) a fast hyperpolarizing response due to an increase in C1 conductance (C1-response), and (3) a slow hyperpolarizing response due to an increase in K conductance (K-response). Enflurane altered neither the action potential nor the membrane resistance of the neurones but depressed the three ACh-induced responses, non-competitively, in a dose-dependent manner. The K-response was less suppressed than the other two. Blockade of the closed state of ion channel was suggested by a reduction in the first ACh response evoked 1 min after administration of enflurane. The anaesthetic facilitated the decay of the neurally evoked e.p.s.c. and i.p.s.c. in suggesting a reduction in the mean open time of the postsynaptic ion channel. It is concluded that enflurane depresses excitatory and inhibitory cholinergic transmission by reducing the postsynaptic currents.

Kinetic and pharmacological properties of the GABA-induced chloride current in Aplysia neurones: a 'concentration clamp' study.

1. gamma-Aminobutyric acid (GABA) was applied by the 'concentration clamp' technique to isolated neurones of Aplysia. GABA induced a chloride current (ICl) due to activation of a single class of chloride-channel. 2. The concentration-response curve for the peak ICl gave an apparent dissociation constant of 6.4 X 10(-5) M and a Hill coefficient of 0.88. The current-voltage relationship was linear in the voltage range examined (-40 to +10 mV). 3. The activation phase of the ICl could be fitted to a single exponential function and desensitization followed the sum of two exponential functions. The time constants of activation and desensitization decreased with increasing concentrations of GABA but were voltage-independent. The recovery process from desensitization also followed the sum of two exponential functions. 4. As for the rate-limiting step of the channel activation, the hyperbolic relationship between the activation rate and GABA concentration showed that the rapid binding assumption holds, suggesting that the isomerization step is rate-limiting. The apparent channel closing rate constant was estimated to be 10 s-1 from the ordinate intercept of the linear part of the above relationship at lower concentrations. 5. Muscimol and beta-alanine induced a ICl, which cross-desensitized with that evoked by GABA. The GABA-ICl was not enhanced by diazepam (10(-6) M) or alpha-chloralose (10(-3) M), in fact depressant effects were evident. 6. Pentobarbitone decreased the GABA-ICl non-competitively without altering activation or desensitization kinetics. The concentration-inhibition curve gave a KD value of 8.9 x 10(-5) M and a Hill coefficient of 1.0. 7. These results suggest that GABA activates a single class of Cl channel in Aplysia neurones, which have one binding site for the agonist. The GABA receptor-Cl channel complex in Aplysia is pharmacologically and perhaps structurally different from that in vertebrates.

Blockade by local anaesthetics of the single Ca(2+)-activated K+ channel in rat hippocampal neurones.

1. Effects of local anaesthetics on single Ca(2+)-activated K+ channels were investigated using the inside-out configuration of the patch-clamp technique in single pyramidal neurones, which were freshly dissociated from rat hippocampus by use of proteolytic enzymes. 2. No significant effect was observed when 2 mM benzocaine was applied on either side of the membrane patch, or when 2 mM lignocaine or QX-314 was applied to the external surface of the membrane. 3. Lignocaine 1 mM, applied to the internal surface, slightly reduced the amplitude of the single K+ channel current. When applied to the internal surface QX-314 reduced the amplitude of the K+ channel current, accompanied by an increase in noise in the open channel current, suggesting a fast flickering block. The blocking effect of QX-314 on the outward current increased with depolarization, suggesting a binding site for the drug at an electrical distance of about 0.5 across the membrane field. 4. The open time histogram showed one exponential component and the closed time histogram showed at least two components. The mean open time of the outward current was increased when the amplitude was reduced by the drugs. 5. The ionized form of the local anaesthetics had a similar action on the Ca(2+)-activated K+ channels to that on Na+ channels, that is, they enter into the channel from the cytoplasmic side to induce open channel block. The blocking kinetics, however, might be so fast that they were beyond the frequency response of our recording apparatus, thus the recorded current amplitude was decreased. In contrast the K+ channel was not accessible via hydrophobic pathways for the neutral form, which is also known to block the sodium channel.

Effect of temperature on the inhibition of the GABA-gated response by intracellular calcium.

The effects of the voltage-dependent Ca2+ inward current (ICa) on gamma-aminobutyric acid (GABA)-induced Cl- current (ICl) in isolated frog sensory neurons were examined at different temperatures and with different concentrations of external Ca2+ using the 'concentration-clamp' technique. The total amount of inhibition of GABA-induced ICl by a preceding ICa increased in a hyperbolic manner with increasing Ca2+ influx. The time course of recovery of the GABA response after inhibition by Ca2+ influx followed a single exponential and was facilitated by warming but slowed dramatically by a slight cooling from 20 to 15 degrees C in spite of a decrease in Ca2+ influx. It is discussed that the energy-dependent, temperature-sensitive ionic pump and exchange systems at the surface membrane and intracellular organelles regulate the cytoplasmic free Ca2+, thus explaining the quantitative effects of Ca2+ influx and temperature on the inhibition of the GABA-gated Cl- channel.

Blockade of the voltage-dependent sodium current in isolated rat hippocampal neurons by tetrodotoxin and lidocaine.

The effects of tetrodotoxin and lidocaine on the voltage-dependent sodium current (INa) were studied in the CA1 pyramidal neurons isolated acutely from rat hippocampus using a 'concentration-clamp' technique which combines the intracellular perfusion with a rapid external solution change within a few ms. Tetrodotoxin (TTX) exerted its inhibitory action in time- and dose-dependent manner on the peak amplitude of INa without any apparent effects on both the current activation and inactivation processes of the current. The time course for reaching a steady-state of the inhibitory action shortened with increasing TTX concentration, but the time course of recovery from the inhibition after washing out the toxin was quite the same at any concentrations used. Lidocaine also inhibited dose-dependently the INa, though with slightly accelerating both the activation and inactivation processes. The time courses for reaching the steady-state inhibition and the recovery from the inhibition were much shorter than those in the case of TTX. The results indicate that the voltage-dependent sodium channel of mammalian brain neuron is TTX-sensitive as well as that of peripheral neuron and that the mode of TTX inhibition on the INa is quite different from that of lidocaine.

Scorpion toxin prolongs an inactivation phase of the voltage-dependent sodium current in rat isolated single hippocampal neurons.

The effects of scorpion toxin on the voltage-dependent sodium current (INa) of CA1 pyramidal neurons isolated from rat hippocampus were studied under the single-electrode voltage-clamp condition using a 'concentration-clamp' technique. The toxin increased the peak amplitude of INa and prolonged its inactivation phase in a time- and dose-dependent manner. Inactivation phase of INa proceeded with two exponential components in the absence (control) and presence of the toxin. In the toxin-treated neurons, both the time constant of slow component and its fractional contribution to the total current increased dose-dependently while the fractional contribution of the fast one decreased in a dose-dependent fashion without changing its time constant. Actions of scorpion toxin on the sodium channels of hippocampal pyramidal neurons were essentially similar to those of peripheral preparations. Therefore, it can be concluded that the sodium channels of mammalian brain neurons have structures and functions similar to peripheral channels.

Ketamine depression of excitatory and inhibitory cholinergic responses in Aplysia neurons.

The effects of ketamine on the cholinergic excitatory and inhibitory (Cl component) responses in Aplysia neurons were examined using the iontophoretic application of ACh and the two-microelectrode voltage clamp technique. Ketamine reduced both responses non-competitively to the same extent in a dose-dependent and reversible manner at concentrations between 10(-6) and 10(-3) M, without changing the reversal potentials. These findings suggest that ketamine depresses the cholinergic responses by affecting the gating mechanism at postsynaptic membranes.

The glutamate-induced chloride current in Aplysia neurones lacks pharmacological properties seen for excitatory responses to glutamate.

The pharmacological properties of the L-glutamate (Glu)-induced chloride current (ICl) in enzymatically isolated Aplysia neurones were examined using the 'concentration clamp' technique. The Glu-ICl did not cross-desensitize with the ICl evoked by gamma-aminobutyric acid or acetylcholine. Quisqualate, kainate (one out of eight) and N-methyl-D-aspartate (one out of nine) induced a small, non-desensitizing ICl in Glu-responding neurones. The quisqualate- and kainate-ICl did not cross-desensitize with the Glu-ICl. L-Aspartate did not induce a ICl in 11 neurones tested, which showed a Glu-ICl. Glutamate diethyl ester, Joro Spider toxin and ketamine did not suppress the Glu-ICl. Concanavalin A had no effect on the time course of desensitization. These results suggest that the Glu receptor-Cl channel complex in Aplysia neurones has pharmacological properties which differ from those of the excitatory Glu receptor-channel complexes in the crustacean muscle fibres and in the central neurones of vertebrates.

Kinetic analysis of glutamate-induced chloride current in Aplysia neurones: a 'concentration clamp' study.

L-Glutamate (Glu) applied by the 'concentration clamp' technique to isolated neurones of Aplysia induced a chloride current (ICl) by activating a single population of the channel. The concentration-response curve for the peak ICl gave a dissociation constant of 1.3 x 10(-4) M and a Hill coefficient of 1.8. The current-voltage relationship was linear in the voltage range examined (-60 to +10 mV). The activation phase of the ICl followed a single-exponential time course and desensitization was complete with a double-exponential time course. The time constants for activation and desensitization decreased with increasing concentrations of Glu but were voltage-independent. The process of recovery from desensitization was also double-exponential. The single-channel conductance estimated by ensemble noise analysis was 50 +/- 4.7 pS (n = 4). These results suggest that the Glu receptor-Cl channel complex in Aplysia neurones consists of a single population with two binding sites for the agonist.

Blockade by trifluoperazine of a Ca(2+)-activated K+ channel in rat hippocampal pyramidal neurons.

The effects of trifluoperazine, a phenothiazine derivative, on the large-conductance Ca(2+)-activated K+ channel (BKCa) in dissociated rat hippocampal pyramidal neurons were examined using the inside-out configuration of the patch-clamp technique. The BKCa was activated by 12.6 microM Ca2+ on the internal surface of the membrane patch. The single channel conductance of the BKCa was 244 +/- 17.5 pS (n = 10) in symmetrical solutions of 150 mM K+. Trifluoperazine, applied on the internal surface of the membrane, decreased the open probability of the channel without changing the single channel conductance. The reduction in the open probability was well described by a block of the open state of the channel in a simple sequential model. The apparent dissociation constant (KD) for the reduction was calculated to be 1.4 microM and the Hill coefficient 0.69 at +20 mV. The inhibition was voltage dependent, being more pronounced at depolarized voltages. The voltage dependence enabled us to estimate that the binding site for the agent in the channel lies about half way across the membrane electrical field. It is concluded that trifluoperazine blocks the open state of the BKCa, which is known to provide an outward current for repolarization and afterhyperpolarization of the neuronal action potential. This may result in a decrease in spike intervals during burst firing of neurons.

Local anesthetics reduce the inhibitory neurotransmitter-induced current in dissociated hippocampal neurons of the rat.

The effects of local anesthetics on amino acid-induced currents were examined using the whole-cell configuration of the patch clamp technique in dissociated hippocampal pyramidal neurons of the rat. Lidocaine (3 mM) decreased the glycine-induced Cl- current (Gly-ICl) more potently (to 46% of the control value) than the gamma-aminobutyric acid-induced Cl- current (GABA-ICl; to 75%), whereas the agent had little effect on the excitatory glutamate response. The reduction in the Gly-ICl was dose-dependent, with a dissociation constant (KD) of 3 mM and a Hill coefficient of 0.96. A non-competitive inhibition was suggested by a double reciprocal plot of the effects of lidocaine on the concentration-response curve of the Gly-ICl. Benzocaine, a neutral local anesthetic at physiological pH, decreased the Gly-ICl more potently than lidocaine, while QX314, a permanently charged quaternary derivative of lidocaine, produced a much smaller inhibition, thereby indicating that the neutral form of local anesthetics is more effective in reducing the Gly-ICl. The depression of the Gly-ICl and GABA-ICl in central neurons may contribute to local anesthetic-induced convulsions.

Modulation of miniature inhibitory postsynaptic currents by isoflurane in rat dissociated neurons with glycinergic synaptic boutons.

The effects of a volatile anesthetic, isoflurane, on glycinergic miniature inhibitory postsynaptic currents (IPSCs) were investigated in mechanically dissociated rat trigeminal nucleus neurons with intact glycinergic interneuronal presynaptic nerve terminals. The nystatin-perforated patch recording configuration was used to record the miniature IPSCs under voltage-clamp conditions. Isoflurane shifted in a parallel fashion the glycine (Gly) concentration-response curve of enzymatically dissociated neurons to the left without changing the maximum response. Isoflurane reversibly increased the frequency of the miniature IPSCs and prolonged the decay time constant without affecting the mean amplitude. The increase in the frequency of miniature IPSCs in the presence of isoflurane was also observed in Ca(2+)-free external solution. Thapsigargin prohibited the facilitatory effect of isoflurane on the miniature IPSC frequency. It is concluded that isoflurane increases the Ca(2+) concentration in the glycinergic presynaptic nerve terminal by enhancing the release and/or suppressing the uptake of Ca(2+) into stores.

Effects of isoflurane and hexafluorodiethyl ether on human recombinant GABA(A) receptors expressed in Sf9 cells.

Effects of volatile anesthetics and a volatile convulsant on human recombinant gamma-aminobutyric acid (GABA) type A receptor responses were studied using the whole cell configuration of the patch clamp technique. Sf9 cells were transfected with bacuroviruses carrying cDNAs of alpha1beta2, alpha1beta2gamma2s, alpha3beta2 and alpha3beta2gamma2s subunit combinations of the human GABA(A) receptor. Clinical concentrations of isoflurane (a volatile anesthetic) enhanced the GABA-induced current of the alpha1beta2gamma2s and alpha3beta2gamma2s GABA(A) subunit combinations. On the other hand, isoflurane suppressed the current of the alpha1beta2 and alpha3beta2 subunit combinations, indicating that the anesthetic effects depended upon the presence of gamma2s subunit. A high concentration (2 mM) of isoflurane generated a surge current following the washout of GABA and the anesthetic. Hexafluorodiethyl ether (a volatile convulsant) decreased the GABA-response of the both alpha3beta2gamma2s and alpha3beta2 constructs without generating a surge current. The results suggest that volatile agents affect the receptor-ionophore complex via direct interaction with proteins but not through a perturbation of the membrane lipid environment. A hypothetical sequential model for the anesthetic action is presented.

GABA affects the glutamate receptor-chloride channel complex in mechanically isolated and internally perfused Aplysia neurons.

The effects of gamma-aminobutyric acid (GABA) on the glutamate receptor chloride ion (Cl-) channel complex were examined in mechanically isolated and internally perfused Aplysia neurons using a concentration clamp technique. GABA at concentrations of 3 x 10(-6) M or more, concentration dependently delayed the recovery of the glutamate response from desensitization. This effect was independent of the GABA response and Cl- redistribution. Muscimol (10(-4) M) mimicked the effect of GABA. However, this was not the case for baclofen (10(-3) M). In some isolated neurons, GABA at concentrations of more than 10(-4) M clearly induced an additional Cl- current, the current kinetics of which were different from those induced by lower concentrations of GABA. Even in the continued presence of 10(-4) M GABA, which desensitized the fast GABA response, higher concentrations of GABA (3 x 10(-4) M to 10(-2) M) elicited the additional current in a concentration-dependent manner. The presence of 10(-4) M glutamate completely abolished this current, indicating cross-desensitization between the glutamate and slow GABA responses. High concentrations of GABA (3 x 10(-2) M) did not activate the glutamate receptor coupled to the large cation channel. The results suggest that, in Aplysia neurons, the glutamate receptor-Cl- channel complex has some similarities to the GABA receptor-Cl- channel complex.