The effect of female tobacco smoking on IVF outcomes.

BACKGROUND: Cigarette smoking is widely believed to be associated with decreased fecundity in naturally conceiving populations; however, the effect of female smoking on pregnancy outcomes in patients undergoing IVF is unclear. METHODS: A retrospective analysis of 389 consecutive patients undergoing first cycle IVF was performed. Outcomes of peak estradiol (E(2)) levels, log mean ovarian volume, number of oocytes retrieved, oocyte maturity in ICSI, fertilization rate, cleavage rate, embryo quality, percentage of high-quality embryos, pregnancy and live birth were assessed in patients reported as never smokers, past smokers and current smokers. Potential confounding variables evaluated included day 3 FSH, number of oocytes retrieved, embryo quality, caffeine and alcohol consumption. The population was also stratified by female age (<35 and >or=35 years). RESULTS: A total of 9.3% of our patients reported current smoking and 12.1% reported a history of smoking. Smoking status did not significantly affect pregnancy outcome, live birth rate or any other indicated outcome. CONCLUSIONS: A total of 21.4% of IVF patients in this study had past or present exposure to cigarette smoking with no measurable effect on IVF outcome.

Mullerian inhibiting substance levels at the time of HCG administration in IVF cycles predict both ovarian reserve and embryo morphology.

BACKGROUND: Pre-antral and early antral follicles secrete Müllerian inhibiting substance (MIS), suggesting that MIS may directly reflect ovarian reserve. Since little is known about how ovarian reserve affects oocyte quality, we attempt here to assess the predictive value of MIS on embryo morphology and IVF outcome. To do so, we measured MIS at the time of HCG administration 36 h prior to oocyte retrieval. METHODS: A total of 257 patients undergoing IVF were prospectively recruited. We measured MIS levels by enzyme-linked immunosorbent assay at the time of HCG, and compared the MIS values to day 3 FSH levels in the prediction of embryo morphology and IVF outcome. RESULTS: The distribution of MIS levels was skewed, with a median of 2.7 ng/ml (range 0 to 28.5 ng/ml). MIS values at the time of HCG administration inversely correlated with basal FSH levels (P = 0.002), and both correlated significantly with patient age, number of mature follicles, number of oocytes retrieved and serum estradiol levels. MIS levels correlated significantly with a greater number of 6-cell embryos and better embryo morphology score, while basal FSH levels did not correlate with these outcome variables. MIS levels > or =2.7 ng/ml portended improved oocyte quality as reflected in a higher implantation rate (P = 0.001) and a trend toward a better clinical pregnancy rate (P = 0.084). CONCLUSIONS: MIS levels seem to predict not only ovarian reserve, but also embryo morphology. Measurement of MIS at the time of HCG administration may, therefore, in the future improve management of patients undergoing treatments with assisted reproductive technology.

Sequential assessment of individually cultured human embryos as an indicator of subsequent good quality blastocyst development.

BACKGROUND: It is of fundamental importance for IVF clinics to determine the most viable embryos for transfer. The challenge for ART clinics is to transfer fewer embryos, thereby minimizing the risk of multiple-infant births, while still maintaining the greatest chance of pregnancy for their patients. In this study, an investigation was made to determine if developmental markers on the day of fertilization (day 1) can predict good subsequent blastocyst development. METHODS AND RESULTS: A total of 1550 individually cultured 2PN embryos from 191 patients undergoing IVF/ICSI treatment at the Yale University Center for Reproductive Medicine and Infertility from February to December 2001 was included. The results showed a significant positive relationship between early-cleaving 2-cell embryos and subsequent good quality > or =4-cell, > or =7-cell and blastocyst development (P < 0.05). PN symmetry (the relative size of the PN to each other), when checked at the time fertilization, is also a significant indictor of good quality > or =4-cell, > or =7-cell stage embryos and blastocysts. Combined, a developing embryo showing PN symmetry with early cleavage and subsequent good > or =4-cell and > or =7-cell cleavage, has a one in two chance of developing into a good-quality blastocyst. CONCLUSION: Early embryo assessment can be used as an indicator of subsequent good blastocyst development.

Development and application of a self-referencing glucose microsensor for the measurement of glucose consumption by pancreatic beta-cells.

Glucose gradients generated by an artificial source and beta-cells were measured using an enzyme-based glucose microsensor, 8-microm tip diameter, as a self-referencing electrode. The technique is based on a difference measurement between two locations in a gradient and thus allows us to obtain real-time flux values with minimal impact of sensor drift or noise. Flux values were derived by incorporation of the measured differential current into Fick's first equation. In an artificial glucose gradient, a flux detection limit of 8.2 +/- 0.4 pmol.cm(-2).s(-1) (mean +/- SEM, n = 7) with a sensor sensitivity of 7.0 +/- 0.4 pA/ mM (mean +/- SEM, n = 16) was demonstrated. Under biological conditions, the glucose sensor showed no oxygen dependence with 5 mM glucose in the bulk medium. The addition of catalase to the bulk medium was shown to ameliorate surface-dependent flux distortion close to specimens, suggesting an underlying local accumulation of hydrogen peroxide. Glucose flux from beta-cell clusters, measured in the presence of 5 mM glucose, was 61.7 +/- 9.5 fmol.nL(-1).s(-1) (mean +/- SEM, n = 9) and could be pharmacologically modulated. Glucose consumption in response to FCCP (1 microM) transiently increased, subsequently decreasing to below basal by 93 +/- 16 and 56 +/- 6%, respectively (mean +/- SEM, n = 5). Consumption was decreased after the application of 10 microM rotenone by 74 +/- 5% (mean +/- SEM, n = 4). These results demonstrate that an enzyme-based amperometric microsensor can be applied in the self-referencing mode. Further, in obtaining glucose flux measurements from small clusters of cells, these are the first recordings of the real-time dynamic of glucose movements in a biological microenvironment.

In situ analysis of pH gradients in mosquito larvae using non-invasive, self-referencing, pH-sensitive microelectrodes.

The alkaline environment, pH approximately 11, in the anterior midgut lumen of mosquito larvae is essential for normal nutrition and development. The mechanism of alkalization is, however, unknown. Although evidence from immunohistochemistry, electron microscopy and electrophysiology suggests that a V-ATPase is present in the basal membranes of the epithelial cells, its physiological role in the alkalization process has not been demonstrated. To investigate a possible role of the V-ATPase in lumen alkalization, pH gradients emanating from the hemolymph side of the midgut in semi-intact mosquito larvae were measured using non-invasive, self-referencing, ion-selective microelectrodes (SERIS). Large H+ concentration gradients, with highest concentrations close to the basal membrane (outward [H+] gradients), were found in the anterior midgut, whereas much smaller gradients, with concentrations lowest close to this membrane (inward [H+] gradients), were found in the gastric caeca and posterior midgut. Similar region-specific pH gradients, with consistent anterior-to-posterior profiles, were observed in individuals of two Aedes species, Aedes aegypti from semi-tropical Florida and Aedes canadensis from north-temperate Massachusetts. The gradients remained in a steady state for up to 6 h, the maximum duration of the recordings. Bafilomycin A1 (10(-5), 10(-7 )mol x l(-1)) on the hemolymph side greatly diminished the [H+] gradients in the anterior midgut but had no effect on the gradients in the gastric caecum and posterior midgut. These physiological data are consistent with the previous findings noted above. Together, they support the hypothesis that a basal, electrogenic H+ V-ATPase energizes luminal alkalization in the anterior midgut of larval mosquitoes.

Noninvasive measurement of potassium efflux as an early indicator of cell death in mouse embryos.

Programmed cell death (apoptosis) occurs in nearly all cell types examined, including mammalian oocytes and embryos, where it may underlie some forms of infertility in humans. Although the molecular machinery participating in apoptosis have been intensely investigated, the accompanying physiological changes have not received similar attention. In this study, a novel electrophysiology technique has been employed to monitor real-time perturbations in the physiology of mouse embryos undergoing apoptosis evoked by hydrogen peroxide, diamide, and staurosporine. Despite differences in their mode of action, these agents evoked a similar early change in cellular physiology; namely, a pronounced, transient, potassium efflux through tetraethylammonium-sensitive potassium channels accompanied by cell shrinkage. Mouse zygotes exposed to 200 microM H(2)O(2) exhibited potassium efflux that elevated the potassium concentration of the media surrounding embryos by 1.4 +/- 0.1 microM. Pretreatment with tetraethylammonium inhibited this increase (0.2 +/- 0.1 microM). Our results indicate that potassium efflux through potassium channels and concurrent cell shrinkage are early indicators of cell death in embryos and that noninvasive measurements of potassium pathophysiology may identify embryos undergoing cell death prior to the manifestation of other morphological or molecular hallmarks of cell death.

Increased birefringence in the meiotic spindle provides a new marker for the onset of activation in living oocytes.

The newly developed Pol-Scope allows imaging of spindle retardance, which is an optical property of organized macromolecular structures that can be observed in living cells without fixation or staining. Experiments were undertaken to examine changes in meiotic spindles during the initial stages of activation of living mouse oocytes using the Pol-Scope. Parthenogenetic activation of oocytes treated with calcium ionophore evoked a dynamic increase in meiotic spindle retardance, particularly of the midregion, before spindle rotation and second polar body extrusion. The pronounced increase in spindle retardance, which could, for the first time to our knowledge, be quantified in living oocytes, was maintained during polar body extrusion. Spindle retardance of newly in vivo fertilized oocytes was significantly higher than that of ovulated, metaphase II oocytes. Pol-Scope imaging of fertilized oocytes did not affect subsequent development. These results establish that increased spindle retardance precedes polar body extrusion and pronuclear formation. The increased birefringence in the spindle provides an early indicator of oocyte activation. Thus, noninvasive, quantitative imaging of the onset of activation in living oocytes might improve the efficiency of assisted fertilization and other embryo technologies.

A non-invasive method for measuring preimplantation embryo physiology.

The physiology of the early embryo may be indicative of embryo vitality and therefore methods for non-invasively monitoring physiological parameters from embryos could improve preimplantation diagnoses. The self-referencing electrophysiological technique is capable of non-invasive measurement of the physiology of individual cells by monitoring the movement of ions and molecules between the cell and the surrounding media. Here we use this technique to monitor gradients of calcium, potassium, oxygen and hydrogen peroxide around individual mouse preimplantation embryos. The calcium-sensitive electrode in self-referencing mode identified a region of elevated calcium concentration (approximately 0.25 pmol) surrounding each embryo. The calcium gradient surrounding embryos was relatively steep, such that the region of elevated calcium extended into the medium only 4 microns from the embryo. By contrast, using an oxygen-sensitive electrode an extensive gradient of reduced dissolved oxygen concentration was measured surrounding the embryo and extended tens of micrometres into the medium. A gradient of neither potassium nor hydrogen peroxide was observed around unperturbed embryos. We also demonstrate that monitoring the physiology of embryos using the self-referencing technique does not compromise their subsequent development. Blastocyts studied with the self-referencing technique implanted and developed to term at the same frequency as did unexamined, control embryos. Therefore, the self-referencing electrode provides a valuable non-invasive technique for studying the physiology and pathophysiology of individual embryos without hindering their subsequent development.

Involvement of mitochondria in oxidative stress-induced cell death in mouse zygotes.

Accumulation of reactive oxygen species during aging leads to programmed cell death (PCD) in many cell types but has not been explored in mammalian fertilized eggs, in which mitochondria are "immature," in contrast to "mature" mitochondria in somatic cells. We characterized PCD in mouse zygotes induced by either intensive (1 mM for 1.5 h) or mild (200 microM for 15 min) hydrogen peroxide (H(2)O(2)) treatment. Shortly after intensive treatment, zygotes displayed PCD, typified by cell shrinkage, cytochrome c release from mitochondria, and caspase activation, then terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining in condensed pronuclei. On the other hand, after mild treatment, zygotes arrested developmentally and showed neither cytochrome c release nor caspase activation over 48 h; until 72 h, 46% zygotes exhibited TUNEL staining, and 88% of zygotes lost plasma membrane integrity. Interestingly, mild oxidative treatment induced a decline in mitochondrial membrane potential and disruption of the mitochondrial matrix. Taken together, these results suggest that oxidative stress caused by H(2)O(2) induces PCD in mouse zygotes and that mitochondria are involved in the early phase of oxidative stress-induced PCD. Furthermore, mitochondrial malfunction also may contribute to cell cycle arrest, followed by cell death, triggered by mild oxidative stress.

Oxidative phosphorylation-dependent and -independent oxygen consumption by individual preimplantation mouse embryos.

The self-referencing electrode technique was employed to noninvasively measure gradients of dissolved oxygen in the medium immediately surrounding developing mouse embryos and, thereby, characterized changes in oxygen consumption and utilization during development. A gradient of depleted oxygen surrounded each embryo and could be detected >50 microm from the embryo. Blastocysts depleted the surrounding medium of 0.6+/-0.1 microM of oxygen, whereas early cleavage stage embryos depleted the medium of only 0.3+/-0.1 microM of oxygen, suggesting a twofold increase in oxygen consumption at the blastocyst stage. Mitochondrial oxidative phosphorylation (OXPHOS) accounted for 60-70% of the oxygen consumed by blastocysts, while it accounted for only 30% of the total oxygen consumed by cleavage-stage embryos. The amount of oxygen consumed by non-OXPHOS mechanisms remained relatively constant throughout preimplantation development. By contrast, the amount of oxygen consumed by OXPHOS in blastocysts is greater than that consumed by OXPHOS in cleavage-stage embryos. The amount of oxygen consumed by one-cell embryos was modulated by the absence of pyruvate from the culture medium. Treatment of one-cell embryos and blastocysts with diamide, an agent known to induce cell death in embryos, resulted in a decline in oxygen consumption, such that the medium surrounding dying embryos was not as depleted of oxygen as that surrounding untreated control embryos. Together these results validate the self-referencing electrode technique for analyzing oxygen consumption and utilization by preimplantation embryos and demonstrate that changes in oxygen consumption accompany important physiological events, such as development, response to medium metabolites, or cell death.

A reliable, noninvasive technique for spindle imaging and enucleation of mammalian oocytes.

Factors affecting the efficiency of animal cloning remain to be elucidated. Enucleation of recipient oocytes is a critical step in cloning procedures and typically is performed by aspirating a portion of the cytoplasm underlying the first polar body. Enucleation is evaluated using epifluorescence after Hoechst staining for DNA, which may disrupt functions of the cytoplast, especially mitochondria. Mitochondrial DNA in Dolly and other cloned sheep has been shown to derive exclusively from recipient oocytes. Not only might evaluation of the aspirated karyoplast portion inadequately reflect the state of the cytoplast, it is also time consuming. Here we report a reliable, noninvasive technique for spindle imaging and enucleation of oocytes using a new microscope, the Pol-Scope. The efficiency of enucleation was 100%, and only 5.5% of the oocytes' mitochondria entered the karyoplast upon Pol-Scope-directed removal of the spindle. Moreover, Pol-Scope imaging of spindles and micromanipulation did not compromise the developmental competence of reconstituted oocytes and cytoplasts.

Thiol oxidation-induced embryonic cell death in mice is prevented by the antioxidant dithiothreitol.

The oxidation of cellular sulfhydryl (SH) groups has been implicated in the induction of apoptosis in various types of cells and in the disturbance of the meiotic spindle of murine oocytes during aging. The objective of this study was to determine whether the SH-specific oxidant diamide could inhibit embryo development and induce cell death, and whether the antioxidant dithiothreitol (DTT) could counteract such effects. Exposure of mouse zygotes to diamide for 3 h at 25 or 50 microM (but not 12.5 microM) resulted in cell cycle arrest and cell death with evidence of apoptosis. At higher concentrations (100 or 200 microM), diamide induced necrosis as evidenced by propidium iodide-positive pronuclei within 24 h of treatment. Simultaneous addition of DTT at equimolar concentration prevented these effects. However, when DTT was added later, it was no longer protective. DTT also effectively protected against the thiol-oxidative damage caused by diamide in blastocysts. These results suggest that altering thiol-redox status in zygotes and blastocysts may result in cell cycle arrest, apoptosis, and/or cell death.

Self-referencing, non-invasive, ion selective electrode for single cell detection of trans-plasma membrane calcium flux.

Biological systems have very different internal ion compositions in comparison with their surrounding media. The difference is maintained by transport mechanisms across the plasma membrane and by internal stores. On the plasma membrane, we can classify these mechanisms into three types, pumps, porters, and channels. Channels have been extensively studied, particularly since the advent of the patch clamp technique, which opened new windows into ion channel selectivity and dynamics. Pumps, particularly the plasma membrane Ca(2+)-ATPase, and porters are more illusive. The technique described in this paper, the self-referencing, ion-selective (or Seris) probe, has the ability to monitor the behavior of membrane transport mechanisms, such as the pumps and porters, in near to real-time by non-invasively measuring local extracellular ion gradients with high sensitivity and square micron spatial resolution. The principles behind the self-referencing technique are described with an overview of systems utilizing ion, electrochemical and voltage sensors. Each of these sensors employs the simple expedient of increasing the system resolution by self-referencing and, thereby, removing the drift component inherent to all electrodes. The approach is described in detail, as is the manner in which differential voltage measurements can be converted into a flux value. For the calcium selective probes, we can resolve flux values in the low to sub pmol.cm(-2)s(-1) range. Complications in the use of the liquid ion exchange cocktail are discussed. Applications of the calcium selective probe are given, drawing on examples from the plant sciences, developmental biology, muscle physiology, and the neurosciences.

The shaking-B2 mutation disrupts electrical synapses in a flight circuit in adult Drosophila.

The shaking-B2 mutation was used to analyze synapses between haltere afferents and a flight motoneuron in adult Drosophila. We show that the electrical synapses among many neurons in the flight circuit are disrupted in shaking-B2 flies, suggesting that shaking-B expression is required for electrical synapses throughout the nervous system. In wild-type flies haltere afferents are dye-coupled to the first basalar motoneuron, and stimulation of these afferents evokes electromyograms from the first basalar muscle with short latencies. In shaking-B2 flies dye coupling between haltere afferents and the motoneuron is abolished, and afferent stimulation evokes electromyograms at abnormally long latencies. Intracellular recordings from the motoneuron confirm that the site of the defect in shaking-B2 flies is at the synapses between haltere afferents and the flight motoneuron. The nicotinic cholinergic antagonist mecamylamine blocks the haltere-to-flight motoneuron synapses in shaking-B2 flies but does not block those synapses in wild-type flies. Together, these results show that the haltere-to-flight motoneuron synapses comprise an electrical component that requires shaking-B and a chemical component that is likely to be cholinergic.

Different neural pathways coordinate Drosophila flight initiations evoked by visual and olfactory stimuli.

To determine the role played by the giant fiber interneurons (GFs) in coordinating the jumping stages of visually elicited and olfactory-induced fight initiation we have recorded extracellularly from the cervical connective nerve during flight initiation. A spike is recorded from the cervical connective upon brain stimulation that has the same threshold as does activation of the tergotrochanteral muscle (TTM) and dorsal longitudinal muscles (DLMs). A consistent time interval occurs between the spike and activation of the TTM. Thus, the spike probably results from activity in the GFs. The time intervals between the spike and activation of the TTM during GF stimulation and visually elicited flight initiation are similar. These results suggest that the GFs coordinate the activation of the TTM and DLMs during the jumping stage of visually elicited flight initiation. A spike is also recorded from the cervical connective during olfactory-induced flight initiations, but its shape and the time interval between it and activation of the TTM is different from that observed during GF stimulation. Although some olfactory-induced flight initiations exhibit a pattern of muscle activation, olfactory-induced flight initiations exhibit a pattern of muscle activation indistinguishable from that evoked by GF stimulation, our results indicate that regardless of the pattern of muscle activation, olfactory-induced flight initiations are not coordinated by the GF circuit. The sterotypic sequence and timing of activation of TTM and DLMs characteristic of the GF pathway can, therefore, be evoked by neurons other than those constituting the GF pathway.

Flight initiations in Drosophila melanogaster are mediated by several distinct motor patterns.

We have monitored the patterns of activation of five muscles during flight initiation of Drosophila melanogaster: the tergotrochanteral muscle (a mesothoracic leg extensor), dorsal longitudinal muscles #3, #4 and #6 (wing depressors), and dorsal ventral muscle #Ic (a wing elevator). Stimulation of a pair of large descending interneurons, the giant fibers, activates these muscles in a stereotypic pattern and is thought to evoke escape flight initiation. To investigate the role of the giant fibers in coordinating flight initiation, we have compared the patterns of muscle activation evoked by giant fiber stimulation with those during flight initiations executed voluntarily and evoked by visual and olfactory stimuli. Visually elicited flight initiations exhibit patterns of muscle activation indistinguishable from those evoked by giant fiber stimulation. Olfactory-induced flight initiations exhibit patterns of muscle activation similar to those during voluntary flight initiations. Yet only some benzaldehyde-induced and voluntary flight initiations exhibit patterns of muscle activation similar to those evoked by giant fiber stimulation. These results indicate that visually elicited flight initiations are coordinated by the giant fiber circuit. By contrast, the giant fiber circuit alone cannot account for the patterns of muscle activation observed during the majority of olfactory-induced and voluntary flight initiations.

The motor neurons innervating the direct flight muscles of Drosophila melanogaster are morphologically specialized.

The anatomy of the motor neurons innervating six direct flight muscles in Drosophila melanogaster has been investigated by using a horseradish peroxidase backfilling technique. The somata of these motor neurons are arranged in two distinct clusters ipsilateral to the muscle they innervate. One cluster of cell bodies is located in the ventrolateral region between the prothoracic neuromere and the mesothoracic leg-related neuropil and the other is situated dorsally and posteriorly to the mesothoracic leg-related neuropil. Axons from somata in the ventrolateral cluster run in the anterior dorsal mesothoracic nerve, while axons from somata in the other cluster run in the mesothoracic accessory nerve. This distribution of somata and axons is discussed in the light of the morphological similarity and proximity of these functionally related muscles. On the basis of the branching patterns of their neurites, direct flight muscle motor neurons can be classified as stubbly, fibrous or tufted. The terminal arborizations of the motor neurons over the direct flight muscles are also morphologically specialized. Both the central and the peripheral morphological specializations of the direct flight muscle motor neurons correlate with the activity patterns exhibited by their associated muscles during flight and courtship song.