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At least 973 records · Page 54Linked to original sources
PubMed2000

Assessment of sperm cryodamage and strategies to improve outcome.

Sperm cryopreservation still represents a valuable clinical aid in the management of infertility. Its current principal indications include (1) donor sperm insemination; (2) freezing before cancer therapy to maintain reproductive capacity; (3) patient's convenience; and (4) because of the outstanding success with ICSI, even patients with different degrees of oligo-asthenoteratozoospermia can now be offered the use of frozen/thawed sperm for oocyte micromanipulation. Although sperm cryopreservation/thawing and results of insemination and IVF have been consistently good using donor semen, results of infertile men (with or without various degrees of oligoasthenoteratozoospermia) have yielded remarkably lower rates of survival and pregnancy. Freezing/thawing techniques have not been subjected to major changes in the last years, Furthermore, the exact nature of sperm cryodamage still remains to be elucidated. Various aspects of sperm freezing are revisited here (1) development of new technical approaches for cryopreservation; (2) analysis of the stimulatory effect of putative cryoprotectant additives; (3) the use of intrauterine insemination-ready processed samples; and (4) selection and optimization of end-points for analysis of cryodamage. It is expected that advances in such areas will improve significantly the cryopreservation/thawing outcome particularly as related to semen samples of subfertile men.

Cryopreservation↗
PubMed2000

Impact of different clinical variables on pregnancy outcome following embryo cryopreservation.

In our program's 13 years of experience, more than 9000 embryos have been cryopreserved in gonadotropin-stimulated IVF cycles. Over 1500 thaw and transfer cycles have yielded a pregnancy rate of approximately 25%. Different ovarian stimulation regimens (various preparations of FSH. alone or in combination with hMG, with or without concomitant use of a GnRH agonist) did not influence embryo survival or pregnancy rate. Likewise, the application of oocyte/embryo micromanipulation techniques for assisted fertilization (ICSI for male infertility) or assisted hatching (performed selectively) did not have an impact on pregnancy results. Survival and transfer rates of embryos cryopreserved at pronuclear or cleaving stage did not differ significantly. However, implantation and pregnancy rates were higher with pronuclear embryo freezing (day-2 transfers) when compared to embryos frozen at the cleavage stage (day-3 transfers). This may be the result of patient selection and transfer policies. Similar implantation and pregnancy results were achieved in natural and estrogen progesterone supplemented transfer cycles. Initial experience with pronuclear freezing followed by transfer at the blastocyst stage appears to offer a very successful alternative for selected patients.

Adult↗
PubMed2001

Intracellular injection of synapsin I induces neurotransmitter release in C1 neurons of Helix pomatia contacting a wrong target.

The contact with the postsynaptic target induces structural and functional modifications in the serotonergic cell C1 of Helix pomatia. In previous studies we have found that the presence of a non-physiological target down-regulates the number of presynaptic varicosities formed by cultured C1 neurons and has a strong inhibitory effect on the action potential-evoked Ca(2+) influx and neurotransmitter release at C1 terminals. Since a large body of experimental evidence implicates the synapsins in the development and functional maturation of synaptic connections, we have investigated whether the injection of exogenous synapsin I into the presynaptic neuron C1 could affect the inhibitory effect of the wrong target on neurotransmitter release. C1 neurons were cultured with the wrong target neuron C3 for three to five days and then injected with either dephosphorylated or Ca(2+)/calmodulin-dependent protein kinase II-phosphorylated Cy3-labeled synapsin I. The subcellular distribution of exogenous synapsin I, followed by fluorescence videomicroscopy, revealed that only synapsin I phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II diffused in the cytoplasm and reached the terminal arborizations of the axon, while the dephosphorylated form did not diffuse beyond the cell body. Evoked neurotransmitter release was measured during C1 stimulation using a freshly dissociated neuron B2 (sniffer) micromanipulated in close contact with the terminals of C1. A three-fold increase in the amplitude of the sniffer depolarization with respect to the pre-injection amplitude (190+/-29% increase, n=10, P<0.006) was found 5 min after injection of Ca(2+)/calmodulin-dependent protein kinase II-phosphorylated synapsin I that lasted for about 30 min. No significant change was observed after injection of buffer or dephosphorylated synapsin I. These data indicate that the presence of synapsin I induces a fast increase in neurotransmitter release that overcomes the inhibitory effect of the non-physiological target and suggest that the expression of synapsins may play a role in the modulation of synaptic strength and neural connectivity.

Actins↗
PubMed2003

[Microbeads, nanobeads and cytometry: applications to the analysis and purification of cells and biomolecules].

Nano and microspheres are important tools in cytometry. They have been used in first to optimize fluorescent signals detected by flow cytometry and to evaluate phagocytosis. Some antigens were also detected by using nanospheres covalently coupled to antibodies. Specifically dedicated microspheres are now widely used for antigenic quantitation by flow cytometry, and magnetic nano and micropheres are very usefull for cellular and molecular purifications. To date, analytical methods based on the use of microspheres are developed to detect proteins, nucleic acids, and ions. To this end, antibodies, oligonucleotides, or chelating agents are bound to microspheres characterized by different fluorescences. The applications of these multiplexed microspheres assays allow to identify and quantify simultaneously some macromolecules and ions, but they also permit to analyze enzymatic activities and to perform polymorphism analyses. With microspheres used as reactive support, molecular analyses are therefore possible by flow cytometry. Nano and microspheres are also usefull tools for calibration in confocal microscopy as well as for micromanipulations of biomolecules and of living cells. Inovative methods based on the use of nano and microspheres are expected in the fields of biology, medicine, food industry, and environmental sciences.

Antibodies↗
PubMed1997

Effect of encapsulation on development of mouse pronuclear stage embryos in vitro.

Pronuclear stage embryos with intact (ZI), slit (ZS) or completely removed (ZF) zona pellucida were encapsulated with an artificial zona pellucida (AZP) made of 1.5% sodium alginate. Embryos were cultured in KSOM medium with or without protein and their development in vitro to the blastocyst stage was recorded. AZP significantly (P < 0.05) improved the development of embryos to the blastocyst stage regardless of the presence of the natural zona pellucida. The encapsulated embryos developed at a higher rate (P < 0.05) in the absence of protein as compared with non-encapsulated embryos. Furthermore, the cell contacts at the 4-cell stage were significantly improved (P < 0.05) with encapsulation. AZP improved (P < 0.05) the development of pronuclear stage embryos with a slit zona pellucida to morula and blastocyst stages as compared with ZS embryos. It is concluded that AZP improves the in vitro development of pronuclear stage embryos with intact or completely removed zona pellucida as well as micromanipulated embryos to the blastocyst stage.

Alginates↗
PubMed2002

Mechanical strength of microcapsules made of different wall materials.

The mechanical strength of microcapsules made of three different wall materials, including melamine-formaldehyde resin, urea-formaldehyde resin and gelatin-gum arabic coacervate, were measured by a micromanipulation technique. Single microcapsules were compressed to large deformations or rupture and the force being imposed on them were measured simultaneously. Melamine-formaldehyde and urea-formaldehyde microcapsules showed clear bursting under compression, and their bursting force, deformation at bursting and deformation at a pesudo yield point were determined. Gelatin microcapsules did not show clear bursting under compression, and their mechanical strength was characterized by the force required to cause their deformation to 50%. The mechanical strengths of these three types of microcapsules are compared in this paper.

Capsules↗
PubMed2001

Effects of anterior inferior cerebellar artery occlusion on cochlear blood flow--a comparison between laser-Doppler and microsphere methods.

The effects of anterior inferior cerebellar artery (AICA) occlusion on blood flow were investigated in rats using the laser-Doppler and microsphere methods. A specially designed microclamp was held in a micromanipulator and positioned to occlude the left AICA. After the middle ear mucosa had been removed, a 1.0-mm laser-Doppler probe was placed on the basal turn of the left cochlea. Non-radioactive microspheres were injected intracardially during the AICA occlusion and the numbers of microspheres in various parts of the cochlea were counted, including in the bone surrounding the cochlea. Upon AICA clamping, the blood flow measured by laser-Doppler flowmetry decreased to 46.9+/-11.9% of the baseline value, and the number of microspheres trapped in the cochlea was 17.2+/-13.3% compared with the contralateral side in 15 animals. The number of microspheres in the bone surrounding the cochlea in the AICA-clamped side was 81+/-15% of that of the contralateral side. In animals in which there were few if any microspheres in the cochlea, laser-Doppler output was 30-40% of the baseline value. From these findings, we infer that during complete interruption of cochlear blood flow in rats, residual laser-Doppler output was essentially attributable to blood flow in the bone surrounding the cochlea.

Animals↗
PubMed2000

The filamentous bacterial morphotype 'Nostocoida limicola' I contains at least two previously described genera in the low G+C gram positive bacteria.

Isolates of eight bacterial filaments fitting the published morphological description of 'Nostocoida limicola' I were obtained from the mixed liquor of four different Australian and one Czech Republic activated sludge plants by micromanipulation. On the basis of their near complete (Ben 200 and Ben 201), or partial (Ben 77, Ben 78, Ben 202, Ben 203, Ben 204 and Ben 205) 16S rRNA gene sequences, six of these isolates were 99.3-100% similar to Lactosphaera pasteurii and Trichococcus flocculiformis, a bulking filament only reported previously in Germany. The other two (Ben 203 and Ben 204) were 99.9% similar to Streptococcus suis. Hence, all are in the low mol % G+C gram-positive bacteria division of the Bacteria. On this evidence 'N. limicola' I is phylogenetically unrelated to 'Nostocoida limicola' II, which is now known to be in the Actinobacteria, even though these two filamentous bacteria appearing in activated sludge systems have been considered to be closely related to each other historically.

Actinobacteria↗
PubMed1994

A prospective, in vivo study of the relationship between blood flow hemodynamics and atherosclerosis in a hyperlipidemic swine model.

PURPOSE: This is the first prospective evaluation of the relationship between shear stress and atherosclerotic plaque formation in a pulsatile, in vivo model. METHODS: A 50% aortic stenosis was created in six Yucatan micropigs by placing a suture line across the left half of the aorta. A specially modified 20 MHz Doppler ultrasound probe mounted at a 45 degree angle on a micromanipulator was used to measure blood flow velocity along the lateral aspects of the aorta. Shear stress at these locations was calculated with linear regression. The pigs were then fed a specially formulated hyperlipidemic diet for 7 months, after which their aortas were harvested. Plaque thickness was measured and correlated with mean, maximum, and minimum shear stress at each site and side on or away from the aortic plication and distance from the level of the aortic plication with general linear modeling. RESULTS: Plaque thickness was negatively correlated with the mean shear stress during the cardiac cycle (correlation coefficient -0.3972 p = 0.0164) and with the maximum shear stress during the cardiac cycle (correlation coefficient -0.4581 p = 0.0050). Plaque thickness was correlated with a multivariate model of mean shear stress, maximum shear stress, and their interactive effects with other parameters in the model with a correlation coefficient of 0.6733, (p = 0.0048). CONCLUSIONS: This is the first prospective, in vivo study with a pulsatile, hyperlipidemic model, which clearly demonstrates that low shear stress is associated with plaque formation.

Animals↗
PubMed1999

Drag reducing polymers may decrease atherosclerosis by increasing shear in areas normally exposed to low shear stress.

PURPOSE: Drag reducing polymers (DRPs) have been shown to decrease plaque formation. Their mechanism of action is unknown. Atherosclerosis tends to develop in areas of low shear stress. This study investigates whether DRPs increase shear stress in areas normally exposed to low shear stress. METHODS: Six dogs underwent surgical plication of the left half of the aorta. A specially modified 20-MHz Doppler ultrasound probe mounted at a 45-degree angle on a micromanipulator was used to measure blood flow velocity at six 4-mm intervals along both lateral sides of the aorta starting at the aortic wall and then at subsequent 0.1-mm depths moving into the lumen before and after administering DRP. Shear rates were calculated using linear regression and then compared using the paired t test. The blood viscosity remained constant at 0.04 poise during infusions of this amount of DRP. RESULTS: The maximum shear rate occurring during the cardiac cycle on the side of the aortic stenosis (plication) was 9.96 +/- 1.52/sec before the administration of the DRP and 14.27 +/- 2.01/sec after the administration of the DRP (P =.0240). The maximum shear rate on the side of the unstenosed aortic wall was 57.25 +/- 7.93/sec before the administration of the DRP and 44.80 +/- 6.23/sec after the administration of the DRP (P =. 0081). CONCLUSION: One of the ways that DRPs inhibit the development of atherosclerosis appears to be by increasing shear stress in areas normally exposed to low shear stress. Understanding this mechanism may lead to the development of pharmaceutical agents that inhibit the development of atherosclerosis.

Animals↗
PubMed1995

The effect of mechanical deformation on the distribution of ions in fibroblasts.

The extracellular and intracellular sodium, potassium and chloride concentrations were determined in fibroblast cells located in the rat calvarium. The ionic values were determined by fluorescence microscopy after incubation with the fluorescent probes, sodium-binding benzofuran isophthalate (SBFI), potassium-binding benzofuran isophthalate (PBFI) and 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) (Dyes were supplied by CALBIOCHEM, Nottingham, England). After determination of the resting membrane potential, the calvaria were placed under tension by retraction of a micromanipulator. The fluorescence was measured again. A statistically significant difference was found in the calculated potassium ion concentration (Mann-Whitney; p < 0.05). This affected the resting cell membrane potential by an average of 5.2 mV. This effect was blocked by the addition of a potassium channel blocker, tetraethylammonium (TEA).

Animals↗
PubMed2000

Advances in assisted reproductive technologies.

Several clinical advances in the field of assisted reproductive technology have improved the success rates of IVF. These advances include improvements in ovulation induction protocols, the introduction of recombinant gonadotropins, GnRH agonists, and, most recently, GnRH antagonists. ICSI has proved to be the most successful technique for the treatment of male infertility. The micromanipulation techniques developed in the embryology laboratory have facilitated advances in the field of preimplantation genetic diagnosis. Years of research in embryology laboratories have enabled the successful culture of embryos to the blastocyst stage. In the future, blastocyst transfer may have even more impact on overall success rates of IVF and multiple pregnancy rates. The field of assisted reproductive technologies has come a long way since the first successful IVF pregnancy. Future developments are expected to be equally dynamic as efforts continue to help couples conceive healthy pregnancies.

Embryo Transfer↗
PubMed2001

Physical and chemical stability of marine lipid-based liposomes under acid conditions.

Liposomes made from a marine lipid extract containing a high polyunsaturated fatty lipid ratio were submitted to large pH variations, ranging from 1 to 8. Shape transformations were followed by video microscopy using giant liposomes and micromanipulation experiments. Acidification induced a decrease of the vesicle size simultaneous to the appearance of invaginations. These pH-dependent structural rearrangements were interpreted in terms of osmotic shocks and chemical modifications of the membranes. Liposomes produced by direct filtration were studied using turbidity measurements and optical microscopy observations. A low pH led to an instantaneous vesicle aggregation and to complex supramolecular and/or morphological changes as a function of time. The subsequent buffer neutralization of the liposome suspensions induced a partial reversion of the aggregation phenomenon while the structural membrane rearrangements were persisting. Furthermore, weak chemical degradations (oxidation and hydrolysis) were evidenced when the vesicles were incubated at low pH up to a 24-h incubation time. Thus, although acidification revealed liposome size and shape changes, the bilayer structure was maintained indicating that marine lipid-based liposomes could be used as oral administration vectors.

Journal Article↗
PubMed2000

DNA topology: feeling the pulse of a topoisomerase.

The action of individual type II DNA topoisomerases has been followed in real time by observing the elastic response of single DNA molecules to sequential strand passage events. Micromanipulation methods provide a complementary approach to biochemical studies for investigating the mechanism of DNA topoisomerases.

Adenosine Triphosphate↗
PubMed2000

A dual electrospray ionization source combined with hexapole accumulation to achieve high mass accuracy of biopolymers in Fourier transform ion cyclotron resonance mass spectrometry.

A dual electrospray ionization (ESI) source employed with hexapole accumulation and gated trapping provides a novel method of using an internal standard to achieve high mass accuracies in Fourier transform ion cyclotron resonance mass spectrometry. Two ESI emitters are sequentially positioned in front of the heated metal capillary inlet by a solenoid fitted to an XYZ micromanipulator; one emitter contains the analyte(s) of interest and the other an internal standard. A 5 V transistor-transistor logic pulse from the data station controls the solenoid by means of a solid-state relay so that matching of spectral peak intensities (i.e., analyte and internal standard intensities) can be accomplished by adjusting the hexapole accumulation time for each species. Polythymidine, d(pT)18, was used as the internal standard for all studies reported here. The absolute average error for an internally calibrated 15-mer oligonucleotide (theoretical monoisotopic mass = 4548.769 Da) was -1.1 ppm (external calibration: 41 ppm) with a standard deviation of +/-3.0 ppm (external calibration: +/-24 ppm) for a total of 25 spectra obtained at various hexapole accumulation time ratios. Linear least squares regression analysis was carried out and revealed a linear dependence of the magnitudes of the peak height ratios (analyte/internal standard) vs. hexapole accumulation time ratios (analyte/internal standard) which is described by the following equation: y = 0.45 x - 0.02. The fitted line had a %RSD of the slope of 28% with an R2 of 0.93. The applicability of this methodology was extended to a polymerase chain reaction product with a theoretical average molecular mass of 50,849.20 Da. With the internal standard, d(pT)18, an absolute average error of -8.9 ppm (external calibration: 44 ppm) based on five measurements was achieved with a standard deviation of 11 ppm (external calibration: +/-36 ppm), thus illustrating this method's use for characterizing large biomolecules such as those encountered in genomics and proteomics related research.

Biopolymers↗
PubMed2002

Production of mitochondrial DNA transgenic mice using zygotes.

Several animal models of human disease, which have been developed by random or targeted modifications of genomic DNA sequences, have furthered our understanding of pathogenesis and the development of therapeutics. However, these models have not facilitated studies on mitochondrial diseases, since modifications to mitochondrial DNA (mtDNA) sequences are not possible using current recombination techniques. Consequently, information on human mitochondrial diseases is relatively sparse, and issues related to mitochondrial pathogenesis and inheritance remain unresolved. Recently, we reported the development of a new technique to generate mice carrying mutant mtDNA from a mouse cell line. In this report, we describe our techniques in detail, with emphasis on the preparation of donor cytoplasts and the micromanipulative procedures for electrofusion of cytoplasts and recipient zygotes. These steps are critically important for the successful introduction of exogenous mtDNA into embryos, and thereby into animals, so that the mutant mtDNA is efficiently propagated in subsequent generations.

Animals↗
PubMedDate not supplied

Infertility update: use of assisted reproductive technology.

OBJECTIVE: To describe assisted reproductive technology (ART) and use of medications during these procedures. DATA SOURCES: Recent clinical literature. STUDY SELECTION: Not applicable. DATA EXTRACTION: Not applicable. DATA SYNTHESIS: ARTs are procedures used in treatment of infertility that involve removal of oocytes and their manipulation outside the woman's uterus. The simplest form of ART, in vitro fertilization, involves aspirating eggs from the ovaries, fertilizing them outside the body, and transferring the embryos into the uterus at the four- to eight-cell stage. Experimental regimens for in vitro fertilization include use of various medications (gonadotropin-releasing hormone agonists, human menotropins, follicle-stimulating hormone, growth hormone) at varying points in the menstrual cycle and after introduction of the embryo into the uterus. Human chorionic gonadotropin has been used to increase implantation of embryos during the woman's luteal phase. Gamete intrafallopian transfer (GIFT) involves transfer of oocytes and sperm into the fallopian tubes, where fertilization takes place. This technique has the advantage of causing the zygote to enter the uterus at the time it would during natural conception. Zygote intrafallopian transfer is similar to GIFT, except that fertilization occurs in vitro, with embryos placed in the fallopian tubes at the two-cell stage. Various micromanipulation techniques and innovative sperm aspiration procedures are currently under development. CONCLUSION: Many advancements have been made in ART, and pharmacists who understand these procedures can serve patients by providing medication information in an empathetic and supportive manner.

Female↗
PubMedDate not supplied

[Molecular detection of Y chromosome microdeletions: a new approach based on the denaturing gradient gel electrophoresis].

Ten percent of couples trying for a child fail to conceive. In approximately 50% of cases, infertility is due to the inability of the male partner to produce spermatozoa in sufficient numbers to effect conception. Over the past 5 years, molecular studies have suggested that interstitial microdeletions in Yq11 represent an etiological factor for male infertility. Y-microdeletions have been detected in 12% of non-obstructive azoospermia and 6% of severe oligozoospermia. In general, microdeletions were detected by separate multiplex-polymerase chain reaction (PCR) reactions using primer pairs for single tagged sites (STSs) of all three azoospermia factor (AZF) regions (AZFa, AZFb and AZFc). This review describes the molecular methods and laboratory guidelines for molecular diagnosis of Y-chromosomal microdeletions. The diagnostics of Y-chromosomal microdeletions should be performed in two steps: in any case, the routine diagnostic should include six STS loci, two STS loci localised in each AZF region, and once a deletion is detected, the analysis can be extended to STS loci known to cross the proximal and the distal borderlines of each AZF region. Other molecular techniques such as DGGE, Southern blot should be performed to detect partial deletions of gene copies or mosaicism. These different molecular approaches should allow explaining 10% of male infertility, to evaluate the risk to pass the defect onto their male offspring (by intracytoplasmic sperm injection) and improve the genetic counselling of couples undergoing micromanipulative assisted reproduction.

Blotting, Southern↗