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At least 991 records · Page 55Linked to original sources
PubMed1999

Carbon dioxide laser evaporation of leukoplakia of the lower lip: a retrospective evaluation.

The purpose of this study was the retrospective evaluation of the treatment results of CO2 laser evaporation for 27 cases of leukoplakia of the lip. The data were derived from 23 patients who presented with leukoplakia of the lower lip during the period 1978-96. Four patients developed a second primary leukoplakia of the lip resulting in 27 cases of leukoplakia. All lesions were treated with a CO2 laser equipped with an operation microscope and micromanipulator. Short-term evaluation showed complete epithelialisation 4 weeks after CO2 laser evaporation; there was minimal scar formation and no subsequent interference with normal lip function. During long-term evaluation, four recurrences (14.8%) were diagnosed which developed between 5 and 31 months after treatment, these were retreated with CO2 laser evaporation. There was no development of squamous cell carcinoma in the CO2 laser-treated area. Selective removal of affected epithelium with minimal damage to surrounding structures is possible using CO2 laser evaporation, followed by excellent wound healing and good functional result. Treatment can be performed under local anaesthesia on an outpatient basis. The recurrence rate is low compared with the recurrence rate after surgical excision. Therefore, CO2 laser evaporation is considered a reliable and effective treatment modality for leukoplakia of the lip.

Aged↗
PubMed1999

Mass mating method in combination with G418- and aureobasidin A-resistance markers for efficient selection of hybrids from homothallic strains in Saccharomyces cerevisiae.

We have developed a mass mating method using the spore suspensions of homothallic yeasts of Saccharomyces cerevisiae in combination with dominant selective drug resistance markers, Tn601(903) against geneticin and AUR1-C against aureobasidin A for the selection of the hybrids. To examine the effectiveness of these markers in the mass mating method, each marker was introduced into a homothallic wine yeast. Using a mixed culture of spore suspensions from the resultant transformants, many hybrids were screened by the drug resistance markers. This method is more practical than the spore-to-spore mating method because it does not require the use of a micromanipulator and many hybrids are obtained at one time. The resultant hybrids could be utilized for industrial brewing because plasmids, which are used to confer resistance markers, are easily eliminated from the hybrids by cultivation in a medium without drugs. We propose that the mass mating method using spore suspensions in combination with dominant selective geneticin- and aureobasidin A-resistance markers is useful for the selection of hybrids from industrial homothallic yeasts.

Journal Article↗
PubMed1999

Structural analysis of a biofilm which enhances carbon steel corrosion in nutritionally poor aquatic environments.

Carbon steel coupons were exposed to nutritionally-poor synthetic wastewater inoculated with activated sludge from a municipal waste water plant. Biofilm formation was observed after one day incubation, and the thickness of the film increased proportionally with the incubation period. Mass loss of the coupons was also proportional to the incubation time, and reached 70.4 (mg/cm2) after incubation for 140 d. The observed mass loss was 5 times as much as that under sterile conditions. To characterize the microbiologically influenced corrosion (MIC) of carbon steel, structural analysis of the biofilm was performed. Rapid decrease in the dissolved oxygen (DO) concentration in the zone near the surface of the biofilm was observed by a microelectrode mounted on a micromanipulator. Heterogeneous distribution of the DO concentration on the surface of the steel plate was observed after multiple analyses. The heterogeneous structure of the biofilm composed of viable cells, inanimate objects, voids and pores was elucidated by confocal scanning laser microscopy. Concentrations of both aerobic bacteria and sulphur-reducing bacteria in the biofilm decreased with the incubation time, indicating that the increase in the biofilm thickness reflected an increase in the density of dead microbial cells or in extracellular polymer accumulation by the microbes. The average roughness of the metal surface observed after 112 d of incubation was +/-7.14 microm, which was 14.1% of the average thickness of the coupons. These observations indicated that uneven distribution of the DO profile and the cell concentration were critical for MIC of the carbon steel.

Journal Article↗
PubMed2004

Partial hydatidiform mole following the transfer of single frozen-thawed embryo subsequent to ICSI.

Hydatiform mole is a gestational trophoblastic disease characterized by the dominance of dispermic fertilization. Micromanipulation techniques in assisted reproduction technologies have enabled direct evaluation of the zygotes and the formation of pronuclei in the zygote. Intracytoplasmic sperm injection (ICSI) of oocytes ensures that only a single spermatozoon enters the ooplasma. This study reports a case of partial hydatiform mole following the transfer of day 3 frozen-thawed embryo. ICSI was used as the assisted fertilization method because there was male factor infertility due to severe oligoasthenoteratozoospermia. Possible predisposing factors for partial hydatidiform mole after ICSI are also discussed.

Adult↗
PubMed2005

Microtubule turnover in ooplasm biopsy reflects ageing phenomena in the parent oocyte.

Oviductal oocytes retrieved from superovulated B6D2F1 mice at 13.5, 16 and 19 h after human chorionic gonadotrophin (HCG) (groups A, B and C respectively, n = 382) were micromanipulated to obtain 12-20 mum sized ooplasm biopsy fragments. Experiments were divided into three sets. Ooplasmic microtubule dynamics were studied in ooplasm biopsy specimens and parent oocytes (set 1) and ooplasm biopsy specimens (set 2), whilst zona pellucida dissolution time, cortical granule loss and spindle/chromatin morphology using confocal microscopy were also studied in parent oocytes (set 2). Oocytes withstood oocyte biopsy with a high survival rate (98.2%) and the biopsied oocytes underwent successful fertilization and development (set 3). An absolute one-to-one correlation was seen between the oocyte biopsy specimens and the parent oocytes in terms of ooplasmic microtubule dynamics (set 1), and increased ooplasmic microtubule dynamics in oocyte biopsy specimens paralleled ageing phenomena in the parent oocytes (set 2). Zona pellucida dissolution time was significantly lower in parent oocytes from group A versus groups B (P = 0.032), and C (P < 0.001). (Groups A, B, C include minimal, moderate, increased ooplasmic microtubule dynamics in oocyte biopsy specimens respectively.) Oocyte cortical granule loss and spindle/chromatin abnormalities were mainly seen in group C (P < 0.001). Oocyte biopsy can thus be applied to judge age-related changes in the parent oocytes.

Animals↗
PubMedDate not supplied

Laboratory techniques for human embryos.

This review is concerned with laboratory techniques needed for assisted conception, particularly the handling of gametes and embryos. Such methods are being increasingly refined. Successive stages of fertilization and embryogenesis require especial care, and often involve the use of micromanipulative methods for intracytoplasmic sperm injection (ICSI) or preimplantation genetic diagnosis. Embryologists must take responsibility for gamete collection and preparation, and for deciding on the means of insemination or ICSI. Embryos must be assessed in culture, during the 1-cell, cleaving and morula/blastocyst stages, and classified according to quality. Co-culture methods may be necessary. The best embryos for transfer must be selected and loaded into the transfer catheter. Embryos not transferred must be cryopreserved, which demands the correct application of current methods of media preparation, seeding and the correct speed for cooling and warming. Before too long, methods of detecting abnormal embryos and avoiding their transfer may become widespread.

Blastocyst↗
PubMed2005

Changing genetic world of IVF, stem cells and PGD. A. Early methods in research.

Genetics proved essential to introduce IVF, preimplantation diagnosis (PGD) and embryo stem cells in the 1960s. Its small input in early years was confined to aspects such as timing follicle growth and ovulation. Modest understanding in the mid- to late 1980s, mostly on studies in mice, involved the actions of single genes and the balance between maternal and zygotic transcripts in preimplantation stages. Human IVF began after human oocytes were matured in vitro, and their meiotic chromosomes analysed. Their fertilization in vitro led to PGD and embryo stem cells. Unlike mouse embryos, most human embryos failed to implant, so the best had to be selected to improve IVF pregnancy rates. Initially, faster-growing embryos proved superior. Later, patterns of polarized nucleoli in pronuclei, the degree of blastomere fragmentation and growth of embryos in vitro to blastocysts provided excellent markers. Single cells could be isolated from embryos using micromanipulation. Stem cells from inner cell mass, a branch of IVF, differentiated into immortal stem cell lines in vitro if disaggregated. They formed virtually all body tissues in blastocysts cultured intact or when injected singly into recipient blastocysts. Later, the genetic controls of ES cell differentiation were assessed, together with factors switching them along specific differentiation pathways. Marker genes identified ES cells differentiating into various tissues.

Animals↗
PubMedDate not supplied

Oocyte-induced haploidization.

This paper describes the technical approach to treatment of age-related oocyte aneuploidy. Although one solution can be oocyte/embryo selection, another is represented by the nuclear transplantation procedure. The efficiency of nuclear transplantation into immature oocytes is described as a way of generating embryos, and the possibility that viable female gametes can be constructed by transfer of diploid somatic cell nuclei into enucleated oocytes. Germinal vesicle (GV)-stage mouse oocytes were collected from unstimulated ovaries and somatic nuclei were obtained from mouse cumulus cells obtained after ovarian stimulation. Spare human GV-stage oocytes were donated from consenting patients undergoing intracytoplasmic sperm injection (ICSI) treatment, and human somatic cells were stromal cells coming from uterine biopsies performed on consenting patients undergoing endometrial cell co-culture. GV ooplasts, prepared by enucleation, were transplanted with either GV or somatic nuclei by micromanipulation. Grafted oocytes were electrofused and cultured to allow maturation, following which they were selected at random for insemination or cytogenetic analysis. GV transplantation was accomplished with an overall efficiency of approximately 80 and 70% in the mouse and the human respectively. The maturation rate of 96% (mouse) and 62% (human) following reconstitution was comparable to that of control oocytes, as was the incidence of aneuploidy among the reconstituted oocytes. The reconstituted human oocytes were successfully fertilized by ICSI at a rate of 52%. After the transfer of mouse cumulus or human endometrial cell nuclei into enucleated immature oocytes, a polar body was extruded in >40%. In a limited number of observations where the nucleus of an aged oocyte was transferred into a younger ooplasm, the chromosomes segregated normally at the time of polar body extrusion. The technique of nuclear transplantation itself did not increase the incidence of chromosomal anomalies in the mouse or human, since their oocytes reconstituted with homologous donor GV resumed meiosis to metaphase II and maintained a normal ploidy. In addition, immature mouse ooplasts induced haploidization of transplanted somatic cell nuclei. Although further evaluation of their genetic status is needed, the procedure may offer a realistic way of producing normal oocytes in cases of aged-related infertility. While the procedure is technically similar to cloning, it would generate a unique individual as a result of the contribution of both parental genomes.

Aneuploidy↗
PubMedDate not supplied

Novel use of laser to assist ICSI for patients with fragile oocytes: a case report.

An inadvertent consequence of intracytoplasmic sperm injection (ICSI) is the degeneration of some of the microinjected oocytes. Most patients may not suffer any disadvantage through losing oocyte(s) during micromanipulation; however, in some circumstances, this can result in a reduction of the chances for pregnancy. This study reports a clinical pregnancy obtained by a novel approach using laser-assisted micro-opening of the zona pellucida prior to ICSI to secure a non-traumatic microinjection that avoids degeneration of oocytes. A total of 12 oocytes were obtained from the 36 year old patient in her third IVF treatment cycle, following two previously failed attempts where very high degeneration rates of oocytes after ICSI were recorded, together with suboptimal embryo quality. Five of the 11 matured (MII) oocytes were submitted to conventional ICSI and the other six MII oocytes first underwent laser-assisted opening of the zona pellucida (5-7 microm hole size was created with a 1.48 microm diode laser) before microinjection (LA-ICSI). Three of the five conventionally microinjected oocytes degenerated while one oocyte fertilized normally and developed to a good quality embryo. After the LA-ICSI procedure, one of the six oocytes degenerated and four oocytes fertilized normally; of these, two developed to excellent quality embryos, one to a good quality embryo and one to a poor quality embryo. The three best embryos (LA-ICSI group) were transferred to the patient on day 3. Rising serum human chorionic gonadotrophin concentrations were measured 12 days after transfer and on week 7 two implantation sites were detected, together with regular heart activity. The results of the present report suggest that laser-assisted ICSI may provide a safer approach to non-traumatic microinjection of oocytes than conventional ICSI, thereby minimizing the risk of degeneration and possibly also improving embryo quality. Therefore, it is suggested that laser-assisted ICSI might be applied in all cases associated with difficult zona pellucida penetration or/and fragile oolemma, or where patients have very few oocytes available, to improve the chances for pregnancy.

Adult↗
PubMed2004

Single sperm cryopreservation on cryoloops: an alternative to hamster zona for freezing individual spermatozoa.

Methodology enabling cryopreservation of individual spermatozoa in extreme cases of oligozoospermia would tremendously benefit patients. This study explores the use of a nylon loop for cryopreservation of small quantities of spermatozoa, and also describes a novel technique for freezing individual spermatozoa with this cryoloop. Experiments were conducted to compare sperm recovery and viability after cryopreservation in conventional vials versus on the cryoloops. Discarded human sperm specimens with varying parameters were utilized. The study also examines two different glycerolbased cryoprotectants, with and without test yolk buffer. For single sperm cryopreservation, 5-10 spermatozoa were selected and loaded onto cryoloops with the aid of a microscope and micromanipulation equipment. Sperm function testing was performed on both human and bovine spermatozoa frozen on cryoloops. Microquantities of spermatozoa frozen on cryoloops exhibited overall motility and viability parameters similar to control samples frozen in cryovials. Individually selected spermatozoa cryopreserved on loops were easily recovered and post-thaw motility was generally good. Sperm function testing demonstrated that both human and bovine spermatozoa cryopreserved on loops were able to undergo sperm head decondensation when injected into oocytes. Cryoloops may be an excellent alternative to hamster zonae for cryopreserving small numbers of human spermatozoa.

Animals↗
PubMed2001

Preimplantation diagnosis for long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency.

Long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency is a fatal autosomal recessive metabolic disorder, presenting during infancy. Preimplantation genetic diagnosis (PGD) provides an option for establishing an unaffected pregnancy, avoiding the risk for termination of pregnancy following prenatal diagnosis. The method for pre-selection of mutation-free oocytes for LCHAD deficiency was developed by testing the first and second polar body removed from oocytes by micromanipulation techniques in the framework of in-vitro fertilization. To avoid misdiagnosis, testing was done using hemi-nested polymerase chain reaction (PCR), with outer primers designed to lie outside the pseudogene, eliminating false priming. Four of 12 tested oocytes were predicted to be unaffected, based on the heterozygous first and mutant second polar body. The embryos resulting from these mutation-free oocytes were replaced, yielding a singleton clinical pregnancy and birth of a healthy child following confirmation by prenatal diagnosis.

Journal Article↗
PubMed2001

Methods in preimplantation genetic diagnosis.

Preimplantation genetic diagnosis (PGD) is a new strategy, orientated toward primary prevention of congenital anomalies in couples with reproductive risk, such as advanced maternal age, carriers of chromosomal abnormalities, and carriers of monogenic conditions. For these patients, PGD is an acceptable alternative to prenatal diagnosis, mainly in those countries where pregnancy interruption is forbidden by law. PGD effectively avoids the implications linked to traditional prenatal diagnosis. Centres that provide medical servicies on reproductive biomedicine are responsible for the development and improvement of this new prevention strategy. Thanks to advances in micromanipulation techniques, associated with recent progress in molecular genetics, PGD may be employed for any genetic condition in the future.

Journal Article↗
PubMed1985

Sex determination and sex differentiation in coccidia: gametogony and oocyst production after monoclonal infection of cats with free-living and intermediate host stages of Isospora (Toxoplasma) gondii.

Clones of single oocysts, single sporocysts, single sporozoites, single proliferative parasites, single cysts and single cystozoites of Isospora (Toxoplasma) gondii (KB-strain) were made under visual control using a de Fonbrune micromanipulator. Cloning was successful in 28, 32, 21, 8, 54 and 7% of the trials, respectively. All clones were used for monoclonal infection in non-immune conventionally reared (CV) or specified pathogen-free (SPF) cats. Pre-patent and patent periods, sporulation percentages of excreted oocysts, mouse infectivity of sporulated oocysts, antibody response and immunity to reinfection of CV cats were determined. For these parameters almost no differences were observed between monoclonal infections and infections described with the non-cloned KB-strain. In all cats autopsied during the patent period, 5-8 days post-infection, macrogametes, microgametes and oocysts were found. Since meiosis occurs during sporulation and since all free-living and intermediate host stages proved to be bisexual, it is concluded that sex differentiation in I. (T) gondii is not determined by segregation of sex chromosomes or sex genes but is caused by some final host factor(s) that induce(s) differential gene expression in genetically identical cells.

Animals↗
PubMed2000

The effects of cell size and ploidy on cell allocation in mouse chimaeric blastocysts.

In a previous study of mouse tetraploid<-->diploid chimaeric blastocysts, tetraploid cells were found to be more abundant in the trophectoderm than the inner cell mass (ICM) and more abundant in the mural trophectoderm than the polar trophectoderm. This non-random allocation of tetraploid cells to different regions of the chimaeric blastocyst may contribute to the restricted tissue distribution seen in post-implantation stage tetraploid<-->diploid chimaeras. However, the tetraploid and diploid embryos that were aggregated together differed in several respects: the tetraploid embryos had fewer cells and these cells were bigger and differed in ploidy. Each of these factors might underlie a non-random allocation of tetraploid cells to the chimaeric blastocyst. A combination of micromanipulation and electrofusion was used to produce two series of chimaeras that distinguished between the effects of cell size and ploidy on the allocation of cells to different tissues in chimaeric blastocysts. When aggregated cells differed in cell size but not ploidy, the derivatives of the larger cell contributed significantly more to the mural trophectoderm and polar trophectoderm than the ICM. When aggregated cells differed in ploidy but not cell size, the tetraploid cells contributed significantly more to the mural trophectoderm than the ICM. In both experiments the contributions to the polar trophectoderm tended to be intermediate between those of the mural trophectoderm and ICM. These experiments show that both the larger size and increased ploidy of tetraploid cells could have contributed to the non-random cell distribution that was observed in a previous study of tetraploid<-->diploid chimaeric blastocysts.

Animals↗
PubMed2000

Behaviour of mouse primary spermatocyte nuclei after fusion to enucleated metaphase II oocytes.

Primary spermatocytes originating from prepubertal mouse testes were electrofused to metaphase II (MII)-stage oocytes, enucleated either by the conventional micromanipulation method or by chemical treatment with etoposide and cycloheximide. These experiments were followed by assessment of morphological changes in transferred nuclei using light microscopy, by chromosomal analyses and by screening of hybrids for the presence or absence of DNA synthesis using anti-bromodeoxyuridine antibody and immunofluorescence staining of the hybrids. The results show differences between the two types of ooplasts in susceptibility to activation stimuli. However, when activated, both types of ooplasts gave rise to hybrids of similar morphology. From 35.3% to 63% of activated hybrids originating from chemically or microsurgically enucleated oocytes, respectively, contained one large pronucleus in cytoplasm, 62% or 31.6% hybrids from those two groups, respectively, possessed two smaller pronuclei and a few contained three or four pronuclei. No DNA synthesis was detected in any hybrid containing one or more pronuclei. The chromosome spreads of hybrids with premature chromosome condensation (PCC) morphology (before activation) show that most of the hybrids had a diploid (2n) number of chromosomes. The nature and regularity of the cell division cycle in the hybrids are discussed.

Animals↗
PubMed2004

Use of mouse oocytes to evaluate the ability of human sperm to activate oocytes after failure of activation by intracytoplasmic sperm injection.

The objective of the present study was to investigate the nuclei of human sperms that failed to fertilize human oocytes after intracytoplasmic sperm injection (ICSI). The sperms were injected into mouse oocytes by a piezo-micromanipulator, and some of these oocytes were artificially activated with strontium chloride (SrCl2) after ICSI. The oocytes were fixed, stained, and subjected to chromosomal analysis. The survival rate of mouse oocytes injected with infertile human sperms was 92.0% (46/50), while that of the control mouse oocytes injected with fertile human sperms was 73.6% (81/110). The rate of two pronuclei (2PN) formation was 0 (0/46) by the infertile sperms and 81.5% (66/81) by the fertile ones, a significant difference (p < 0.01). Sperm chromosomes in non-activated oocytes were present as premature chromosome condensation (PCC). Artificial activation after ICSI increased the 2PN formation rate in the infertile group to 90.3% (28/31). The results of the present study suggest that infertile sperms have a low potential to spontaneously activate oocytes and to form pronuclei. Thus, artificial activation after ICSI may rescue oocytes fertilized with infertile human sperms that do not produce 2PN. The present study proved the usefulness of mouse oocytes as specimens in evaluating the oocyte-activating capacity of objective human sperms prior to ICSI treatment.

Animals↗
PubMed2000

Capillary electrophoresis with an integrated on-capillary tubular detector based on a carbon sol-gel-derived platform

An integrated on-capillary tubular electrochemical detector for capillary electrophoresis systems has been fabricated based on sol-gel technique. It consists of a sol-gel carbon composite tubular electrode attached permanently onto the outlet of the separation capillary. The device greatly eases the setting up of capillary electrophoresis with electrochemical detection (CEEC) as it makes possible electrode/capillary alignment without the aid of a micromanipulator since this integrated unit can be simply immersed in the CE separation buffer in an ordinary three-electrode stationary cell. To improve analytical performance of the integrated unit, the external wall of the exit capillary was etched with HF after the polyimide coating of the capillary had been removed. Influences of the working electrode length and the wall thickness at the outlet of capillary on the separation efficiency and amperometric sensitivity were assessed and optimized. The practical applicability of this configuration is demonstrated with the detection of both catecholamines and carbohydrates. The advantages, namely, versatility, convenience, ease of operation, and low-cost, of the new design combined with an excellent performance lead to high stability and low detection limits.

Journal Article↗
PubMed2000

Tantalum-filament pulling device for fabrication of submicrometer fused-silica tips.

A simple and low-cost pulling device for fused-silica capillaries was developed. By using a tantalum heating filament and the self-tension in a bent capillary, tips and constricted regions with outer diameters of approximately 1 microm and inner diameters of a few hundred nanometers could be reproducibly pulled from 50-microm-i.d., 375-microm-o.d. capillaries. The tips can be used in different applications such as microinjection, micromanipulation, and single-channel patch-clamp, injection ends for CE or as electrospray tips. Constricted capillaries with optimized dimensions to minimize cylindrical lensing effects and to match the size of a diffraction-limited laser focus can be used as optical detection windows in CE and micro-HPLC. Fused silica has several advantages over other glasses such as high melting temperature and superior optical and mechanical properties.

Journal Article↗