Search

Background: There is minimal data on the influence of pre-analytical variables on the use of calibrated automated thrombography (CAT), to measure thrombin generation. Objectives: To evaluate the impact of centrifugation methods, time after collection, and contact activation inhibition on the CAT assay performed using two commercial reagents. Methods and Results: Six different methods of plasma separation were examined. Thrombin generation triggered by a 5 pM tissue factor reagent was not greatly affected by plasma separation method,with similar results obtained with all methods apart from single centrifugation and membrane filtration. Membrane filtration increased APTT and is not recommended. Extended double centrifugation at higher speed was required to minimise the impact of residual phospholipid with 1 pM tissue factor trigger, particularly with inhibition of contact activation. The effect of a delay of up to 24 hours in preparing plasma was assessed. No significant difference in results was observed among samples processed between 0.5 and 6 hours after blood collection into plastic Vacuette® tubes. The presence or absence of corn trypsin inhibitor had a significant impact on all parameterswith 1 pM tissue factor trigger, with minor differences seen on Peak and ttPeak results using 5 pM tissue factor. Conclusions: The impact of pre-analytical variables on thrombin generation results is dependent on the concentration of tissue factor in the trigger reagent used. Results with 1 pM tissue factor are particularly sensitive to centrifugation method and contact activation, and standardisation is required to allow large collaborative studies to be performed. Refereed/Peer-reviewed

In a recent report in Reproductive Biomedicine Online by Ebner et al., a comprehensive multi-centre study was presented on the use of a calcium ionophore, A23187, to artificially activate oocytes from patients who had poor fertilization rates in previous cycles. Under physiological conditions, the calcium increase in oocytes at activation is caused by influx and release from specific stores and ion channels, and has precise temporal, quantitative and spatial patterns. Calcium ionophores may release Ca2+ in an uncontrolled fashion from intracellular stores that would not normally be involved in the activation process. Ionophores, including A23187, have a multitude of effects on cell homeostasis, not yet defined in oocytes, that may have long-term effects, for example on gene expression. We suspect that the successful births reported by Ebner et al. are a result of the overriding influence of the injected spermatozoa, rather than the effect of the ionophore; nevertheless, such an invasive non-physiological approach to assisted reproduction techniques is worrying, especially as epigenetic effects may result in future generations.

Objective To apply preimplantation genetic diagnosis (PGD) for the treatment of patients with a history of failed IVF-ET or habitual aborters. Design Prospective clinical study. Setting Tertiary center for assisted reproduction. Patient(s) Ninety-four couples with failed IVF-ET after >2 IVF cycles and 64 couples with >2 spontaneous abortions. Intervention(s) Patients were prepared for oocyte retrieval using standard controlled ovarian hyperstimulation protocols after standard laboratory techniques. Blastomeres from 6- to 8-cell embryos were analysed using fluorescence in situ hybridization with commercial chromosomal probes, and normoploid embryos were transferred on day 3 after fertilization. Main outcome measure(s) Pregnancy and implantation rates and live births. Result(s) Both 3- and 5-probe PGD resulted in a significantly higher outcome than controls for failed IVF-ET. Five-probe PGD appeared to be more suitable for habitual aborters. Conclusion(s) This pilot study suggests that 3-probe PGD is a valid option for failed IVF-ET patients. The use of five or more probes is indicated for habitual aborters.

In the preovulatory follicle, the LH surge stimulates progesterone production, reduces estradiol synthesis, and scales up the permeability of the blood-follicle barrier. The purpose of this study was to investigate whether the extent of these changes is correlated with the levels of estradiol, estradiol esters, and cholesteryl esters in the follicular fluid. The follicular levels of progesterone, estradiol, estradiol linoleate, cholesterol, and cholesteryl linoleate were measured by HPLC. The estradiol linoleate/estradiol ratio, which reflects the efficiency of in vivo estradiol esterification, and the cholesteryl linoleate/cholesterol ratio were calculated and found negatively correlated. The estradiol level was positively correlated with the cholesteryl linoleate/cholesterol ratio while negatively correlated with the estradiol linoleate/estradiol ratio. The in vitro activity of lecithin-cholesterol acyltransferase, the enzyme esterifying both cholesterol and estradiol, was assayed by incubating the fluid with labeled substrates. This activity was not correlated with either the estradiol linoleate/estradiol or the cholesteryl linoleate/cholesterol ratio. The enzyme K m and V max values were lower with estradiol than with cholesterol. Higher estradiol linoleate/estradiol ratios and lower cholesteryl linoleate/cholesterol ratios were associated with higher level of Haptoglobin penetration into the follicle. This level, which was determined by ELISA, was found increased with increased progesterone concentration and, therefore, used as a marker of the LH-stimulated permeability of the blood-follicle barrier. Our data suggest that early preovulatory follicles contain more cholesteryl esters and less estradiol esters than follicles closer to ovulation.

Although the spermatozoon is 500,000 times smaller in volume than the oocyte, it induces rapid and dramatic changes in oocyte physiology that lead to meiosis re-initiation. These oocyte activation events are described here, as is the evidence for a soluble activating factor in the spermatozoon. Since changes in plasma membrane conductance, calcium ion release and maturation-promoting factor inactivation are common to all animal oocytes at activation, it is expected that the sperm-borne trigger is also ubiquitous. One likely candidate, phospholipase C (PLC) zeta 1, induces calcium release in mammalian oocytes; however, work on other deuterostomes suggests that the sperm factor is non-specific and multifactorial, regulating several activation events. Human, sea urchin and ascidian gametes are remarkably similar and comparative studies across the deuterostomes may help in elucidating basic principles in fertilization. Questions to be answered include the identification of PLC zeta 1 in invertebrate spermatozoa and the characterization of other targets in mammalian oocytes, such as the adenosine diphosphate ribose/nitric oxide pathway.

Using the whole-cell voltage-clamp technique we have studied electrical coupling and dye coupling between pairs of blastomeres in 16- to 128-cell-stage sea urchin embryos. Electrical coupling was established between macromeres and micromeres at the 16-cell stage with a junctional conductance ( G j ) of 26 nS that decreased to 12 nS before the next cleavage division. G j between descendants of macromeres and micromeres was 12 nS falling to 8 nS in the latter half of the cell cycle. Intercellular current intensity was independent of transjunctional voltage, nondirectional, and sensitive to 1-octanol and therefore appears to be gated through gap junction channels. There was no significant coupling between other pairs of blastomeres. Lucifer yellow did not spread between these electrically coupled cell pairs and in fact significant dye coupling between nonsister cells was observed only at the 128-cell stage. Since 1-octanol inhibited electrical communication between blastomeres at the 16- to 64-cell stage and also induced defects in formation of the archenteron, it is possible that gap junctions play a role in embryonic induction.

Ascidian oocytes are blocked in metaphase (M) of the first meiotic division. Fertilization triggers the completion of meiosis without any further arrest. In this review, we have analyzed the mechanisms that regulate the progression through meiosis in these oocytes. A primary signal from the fertilizing spermatozoon, probably soluble sperm factor(s), induces intracellular calcium release by activating the IP3and CICR pathways and gates the fertilization current by triggering the generation of ADP ribose (ADPr). The calcium oscillations are not required for the inactivation of MPF observed at M–I release; however, ADPr may be indirectly involved in the activity of MPF associated kinase, Cdc2. MPF activity reaches a second peak at M–II followed by subsequent inactivation. Progression to M–II is dependent on the intracellular calcium oscillations. MAP kinase (MAPK) activity decreases at M–I exit and remains low during the completion of meiosis. Finally, although Cdc2, Cyclin B and MAPK–like proteins have been identified in ascidian oocytes, components of CSF still remain to be identified.

In this paper we use the nitric oxide (NO) donor sodium nitroprusside to examine the response of the unfertilised oocyte of the ascidian Ciona intestinalis to nitric oxide. We show that the release of NO triggers an inward current that displays similar properties to the ascidian fertilisation current. Furthermore, the production of NO causes the release of intracellular calcium through a ruthenium-red sensitive mechanism. Our data suggest that these effects are due to the stimulation of nicotinamide nucleotide metabolism, but the active second messenger is not cyclic adenosine diphosphate ribose (cADPr). Finally, we show that NO production increases at fertilisation. The results suggest that ascidian sperm trigger the release of NO and this second messenger causes the breakdown of nicotinamide nucleotides leading to the production of a second messenger which induces the fertilisation current and may assist in the production of the increase in calcium.

Recently, differences in DNA methylation patterns in placental and umbilical blood samples taken from children born after IVF and children conceived naturally have been reported. Since this may have an effect on gene expression, we highlight some of the biochemical/metabolic pathways in oocytes and embryos that might be relevant to methylation and imprinting during the process of human IVF. First, ovarian stimulation leads to elevated concentrations of follicular homocysteine which may have an effect on methylation. This should be compensated for by the systematic administration of folic acid and other B vitamins to patients. Second, there has been a trend to culture early human embryos in culture medium lacking essential amino acids. Consequently, methionine is not available during the first 3 days of in-vitro culture, a time when methylation is of major importance. We strongly recommend the use of culture medium with essential amino acids in human fertilization and early developmental stages. Finally, although all animals are, to some extent, a model for others, great caution should be exercised in extrapolating data; in particular, data from the mouse should not be assumed to be applicable to human embryology.

Objective: To compare the potency, reversibility, and perioperative bleeding risk of Hepalean with those of PPC heparin. Conclusions: PPC heparin use was associated with greater heparin and protamine dose requirements than Hepalean. These findings indicate that heparin preparations are not interchangeable and suggest that a direct comparison of the potency with the brand in use is needed if a change is made to ensure that the agents exert similar anticoagulant effects in vivo. Methods: Because in vitro testing failed to detect differences in the potency or protamine reversibility of the 2 heparin preparations, we conducted a parallel group, single-center, double-blind, randomized, controlled trial to compare the anticoagulant effects of Hepalean to those of PPC heparin in patients undergoing coronary artery bypass grafting with cardiopulmonary bypass. Results: From June 1, 2011, to June 30, 2011, we randomly assigned 11 patients to receive PPC heparin and 10 to receive Hepalean. Despite similar initial doses of heparin, the median initial activated clotting time was numerically lower in the PPC heparin group than in the Hepalean group (median, 516.0 seconds; interquartile range, 481.0-633.0; vs median, 584.0 seconds, interquartile range, 520.0-629.0; P = .418). Those given PPC heparin required a greater total heparin dose (median, 46,000.0 U; interquartile range, 39,500.0-60,000.0 vs median, 34,500.0 U; interquartile range, 32,250.0-37,000.0; P = .011) and a greater dose of heparin per kilogram than those given Hepalean (median, 572.9 U/kg; interquartile range, 443.0-659.7 vs median, 401.1 U/kg; interquartile range, 400.0-419.4; P = .003). The key secondary results included an increased median total protamine dose (median, 600.0 mg; interquartile range, 550.0-700.0; vs median, 500.0 mg; interquartile range, 425.0-542.5; P = .026) and a trend toward increased chest tube output within 24 hours (median, 830.0 mL; interquartile range, 425.0-1135.0; vs median, 702.5 mL; interquartile range, 550.0-742.5; P = .324). Refereed/Peer-reviewed

Fragments of ascidian eggs, but at random in any plane and ranging in size from 10 to 90% of the total egg volume, displayed the electrical characteristics of the intact egg, having a resting potential of −86 mV and giving rise to an action potential upon stimulation by electrical current injection. Following insemination, the fragments generated fertilization potentials, comparable to those of intact eggs, although the repolarization phase was shorter. Our data show that there are sufficient ion channels throughout the egg surface to generate action potentials and fertilization potentials in excised egg fragments, irrespective of their global origin. Furthermore, the fertilizing spermatozoon is capable of activating fertilization channels in areas of the egg plasma membrane not destined for sperm entry.

The fertilization potential of the ascidian oocyte has been studied using two intracellular electrodes. Two classes of oocyte were observed; low resting potential (RP) oocytes of −20 to −35 mV and high RP oocytes of −80 to −90 mV. The two types have comparable membrane resistance, falling in the range of 100–300 MΩ, and both may be fertilized and develop normally, although the fertilization potential (FP) is different in the two cases. High RP oocytes give rise to step-like regenerative potentials which attain positive values, whereas low RP oocytes give rise to slower depolarizations that reach zero level. In both cases the FP was sometimes preceded by a small-step depolarization as normally observed in the sea urchin. Irrespective of the original RP, the membrane resistance always decreased to 1–10% of its initial value during the first few minutes of the FP plateau. In contrast when the membrane was depolarized to a comparable potential by current injection the membrane resistance did not decrease. Polyspermic fertilization was induced by removing the extracellular coats, aging the oocytes and using high densities of sperm. The FP in monospermic and polyspermic oocytes were comparable and we could not correlate additional sperm interactions with additional electrical events. Our results suggest that the plasma membrane in ascidian oocytes lacks intrinsic mechanisms, electrical or otherwise, to prevent polyspermic fertilization.

Two early events of activation in the ascidian egg, the surface contraction and the fertilization current, were studied. Ca ionophore induces contraction without generating a fertilization current, whereas microinjection of IP3 or soluble fractions of homogenized spermatozoa trigger both a contraction and a current. This suggests that the primary trigger of activation in ascidian eggs is a soluble component of spermatozoa that may be released into the egg subsequent to gamete fusion. IP3, or other intermediates in phosphoinositide metabolism, is a putative second messenger that activates fertilization channels directly (probably a Ca-independent process), and subsequently induces surface contraction by releasing Ca from intracellular stores.

In the early embryos of ascidians and sea urchins, blastomeres are in electrical communication; however, the type and extent of interaction is related to the basic characteristics of the embryo. In the mosaic-like structure of the ascidian embryo, blastomeres have a coupling ratio of about 1 throughout the division cycle. Coupling is facilitated by the extremely low conductance of the non-junctional membrane and possibly mediated via specialized low resistance junctions. Sea urchin embryos do not have specialized low resistance junctions; however, blastomeres are electrically coupled, probably via cytoplasmic bridges, during the first half of the division cycle. The coupling ratio in sea urchins, initially about 0.3, progressively decreases, together with the conductance of the nonjunctional membrane. During the latter half of the division cycle blastomeres are uncoupled; however, a structural junction appears at the periphery, which may play a role in their destiny.

Some electrical properties of the sea urchin oocyte during germinal vesicle breakdown (GVBD) and fertilization have been studied using two intracellular electrodes. Oocytes with distinct germinal vesicles have resting potentials of −70 to −90 mV and the specific membrane resistance may range from 3 to 10 kΩ·cm 2 . Around rest the I–V relationship is concave toward the axis origin and the membrane is K + selective. A second electrical state, of lower potential and higher resistance, preexists in the membrane. Following GVBD, the K + -selective system is lost and the oocyte attains the characteristics of the second state with a resting potential of −10 to −50 mV and specific membrane resistance of 10–50 kΩ·cm 2 . At rest the I–V relationship tends to be convex toward the axis origin. The majority of sea urchin eggs (which have undergone GVBD and completed meiosis) have a resting state of −10 to −30 mV; 10–50 kΩ·cm 2 . The I–V relationship around rest is convex toward the axis origin and the resting potential is sensitive to changes of Na + , Cl − , and K + in the external medium. There is probably no major change in the electrical properties of the oocyte during the completion of meiosis. A small percentage of eggs from suboptimal animals have high resting potentials of −70 to −90 mV and specific membrane resistance of 5–50 kΩ·cm 2 . Such eggs have predominantly K + -selective membranes and we suggest that they are either underripe or aged. The first electrical event across the egg plasma membrane during fertilization is a step-like depolarization which occurs about 2 sec after the attachment of the fertilizing spermatozoon to the vitelline layer. There is no change—at the level of the light microscope—either in the egg surface or in the behavior of the spermatozoon until the second event, the fertilization potential (FP), is initiated 11 sec later. The cortical reaction occurs simultaneously with the FP and during the rising phase of the FP the spermatozoon stops gyrating around its point of attachment. Oocytes, which do not have cortical granules, upon insemination exhibit step events but no FP; in contrast artificially activated eggs, either spontaneous or induced by the ionophore A23187, give rise to only the FP. We suggest that the FP is the electrical result of the modification of the egg plasma membrane during cortical exocytosis.

Using the whole-cell voltage clamp technique, experiments were carried out on ascidian eggs to determine the role of intracellular Ca in the gating of fertilization channels. Raising the level of Ca by adding Ca to the intracellular perfusion medium or by loading the egg cortex (greater than 50 microM) with Ca through voltage gated channels did not lead to the activation of fertilization channels. Alternatively, eggs exposed to low-Ca seawater, perfused with the chelator K-EGTA or Ca channel blocking agents to prevent the release of Ca from intracellular organelles, and subsequently inseminated generated fertilization currents. This argues against Ca as a second messenger in the activation of fertilization channels in the ascidian egg and alternative mechanisms are discussed.

We have studied the electrical and morphological changes in the egg of the anuran Discoglossus pictus during fertilization and activation by pricking. Despite its atypical structure the fertilization potential (FP) in this egg resembles that of other anurans, consisting of a rapid, Cl− dependent, depolarization of some 40 mV, accompanied by a 10- to 100-fold increase in conductance. Pricking eggs at the predetermined site of sperm entry, the dimple, causes an immediate depolarization, similar to the FP, and a wave of contraction can be seen to spread from the dimple to the antipode. When eggs are pricked outside the dimple a contraction wave spreads from the puncture site to the antipode, however, the activation potential (AP) is not generated until the wave reaches the dimple. Thus the farther the puncture site from the dimple the longer the delay in the generation of the AP. Our results suggest that the contraction wave reflects a propagating Ca2+ wave and the channels activated during the AP are localized within the dimple. As there is no obvious ultrastructural change in the dimple region until several seconds after the generation of the AP it is probable that the ion channels preexist in the egg plasma membrane and are not inserted by membrane fusion.