A retrospective study of preimplantation embryos diagnosed with monosomy by fluorescence in situ hybridization (FISH).

This report is a retrospective study of preimplantation embryos diagnosed with monosomy for chromosomes 13, 15, 16, 18, 21, 22, X and Y on day 3 to determine the rate of true positives, false positives and/or mosaicism and to assess if these embryos are suitable for in vitro fertilization (IVF) transfer. In a one year period, 80 patients went through preimplantation genetic diagnosis for aneuploidy screening (PGD-AS). Monosomy was diagnosed in 51 embryos. Fluorescence in situ hybridization (FISH) was then performed on the blastomeres at day 5-7 with commercially available probes using the same probe set that initially identified monosomy for chromosomes 13, 16, 21 and 22 or chromosomes 15, 18, X and Y. Based on FISH analysis, the monosomy diagnosed during routine PGD-AS analysis was confirmed in 17 of the 51 embryos. A euploid result for the specific chromosomes tested was observed in 16 of the 51 embryos while mosaicism was found in the remaining 18 embryos. This results in an estimated false positive rate of 3.8% for a diagnosis of monosomy. Reanalysis of these embryos demonstrates that the majority of monosomy diagnoses represents true monosomy or mosaicism and should be excluded for transfer in IVF. Furthermore, improved understanding from recent emerging data regarding the fate of oocytes in women with advanced maternal age undergoing IVF to the development of early embryos may provide a valuable insight into the mechanism of chromosome mosaicism.

Probing the spatial dependence of the emission spectrum of single human retinal lipofuscin granules using near-field scanning optical microscopy.

The emission spectra of single lipofuscin granules are examined using spectrally resolved confocal microscopy and near-field scanning optical microscopy (NSOM). The emission spectrum varies among the granules examined revealing that individual granules are characterized by different distributions of fluorophores. The range of spectra observed is consistent with in vivo spectra of human retinal pigment epithelium cells. NSOM measurements reveal that the shape of the spectrum does not vary with position within the emissive regions of single lipofuscin granules. These results suggest that the relative distribution of fluorophores within the emissive regions of an individual granule is homogeneous on the spatial scale approximately 150 nm.

Imaging of monolayers composed of palmitic acid and lung surfactant protein B.

Near-field scanning optical microscopy and atomic force microscopy are used to probe the sub-micrometre phase structure in palmitic acid monolayers containing the 25 peptide amino terminus of lung surfactant protein B (SP-B(1-25)). Monolayers deposited onto mica substrates at a surface pressure of 15 mN m-1 exhibit a two-phase coexistence across a broad range of SP-B(1-25) concentrations. Monolayers containing 5 wt.% SP-B(1-25) or less exhibit an expanse of liquid condensed phase in which elliptical liquid expanded (LE) domains with areas of approximately 25 microm2 coexist. By contrast, monolayers containing 20 wt.% SP-B(1-25) exhibit an expanse of liquid expanded phase in which circular liquid condensed domains coexist. The phase distribution dependence on SP-B(1-25) concentration suggests that the peptide induces disorder in the monolayer.

Mapping the distribution of emissive molecules in human ocular lipofuscin granules with near-field scanning optical microscopy.

Several high resolution imaging techniques are utilized to probe the structure of human ocular lipofuscin granules. Atomic force microscopy reveals typical granule sizes to be about one micrometre in diameter and hundreds of nanometres in height, in agreement with previous electron microscopy results. For issues concerning the role of lipofuscin in age-related macular degeneration, recent attention has focused on the orange-emitting fluorophore, A2E. Confocal microscopy measurements are presented which reveal the presence of a highly emissive component in the granules, consistent with the presence of A2E. It is shown, however, that the interpretation of these results is complicated by the lack of structural details about the particles. To address these issues, near-field scanning optical microscopy (NSOM) measurements are presented which measure both the lipofuscin fluorescence and topography, simultaneously. These measurements reveal distinct structure in the fluorescence image which do not necessarily correlate with the topography of the granules. Moreover, direct comparison between the NSOM fluorescence and topography measurements suggests that A2E is not the major component in lipofuscin. These measurements illustrate the unique capabilities of NSOM for probing into the microstructure of lipofuscin and uncovering new insights into its phototoxicity.

Combining AFM and FRET for high resolution fluorescence microscopy.

Here we demonstrate a new microscopic method that combines atomic force microscopy (AFM) with fluorescence resonance energy transfer (FRET). This method takes advantage of the strong distance dependence in Förster energy transfer between dyes with the appropriate donor/acceptor properties to couple an optical dimension with conventional AFM. This is achieved by attaching an acceptor dye to the end of an AFM tip and exciting a sample bound donor dye through far-field illumination. Energy transfer from the excited donor to the tip immobilized acceptor dye leads to emission in the red whenever there is sufficient overlap between the two dyes. Because of the highly exponential distance dependence in this process, only those dyes located at the apex of the AFM tip, nearest the sample, interact strongly. This limited and highly specific interaction provides a mechanism for obtaining fluorescence contrast with high spatial resolution. Initial results in which 400 nm resolution is obtained through this AFM/FRET imaging technique are reported. Future modifications in the probe design are discussed to further improve both the fluorescence resolution and imaging capabilities of this new technique.

Scanning near-field fluorescence resonance energy transfer microscopy.

A new microscopic technique is demonstrated that combines attributes from both near-field scanning optical microscopy (NSOM) and fluorescence resonance energy transfer (FRET). The method relies on attaching the acceptor dye of a FRET pair to the end of a near-field fiber optic probe. Light exiting the NSOM probe, which is nonresonant with the acceptor dye, excites the donor dye introduced into a sample. As the tip approaches the sample containing the donor dye, energy transfer from the excited donor to the tip-bound acceptor produces a red-shifted fluorescence. By monitoring this red-shifted acceptor emission, a dramatic reduction in the sample volume probed by the uncoated NSOM tip is observed. This technique is demonstrated by imaging the fluorescence from a multilayer film created using the Langmuir-Blodgett (LB) technique. The film consists of L-alpha-dipalmitoylphosphatidylcholine (DPPC) monolayers containing the donor dye, fluorescein, separated by a spacer group of three arachidic acid layers. A DPPC monolayer containing the acceptor dye, rhodamine, was also transferred onto an NSOM tip using the LB technique. Using this modified probe, fluorescence images of the multilayer film reveal distinct differences between images collected monitoring either the donor or acceptor emission. The latter results from energy transfer from the sample to the NSOM probe. This method is shown to provide enhanced depth sensitivity in fluorescence measurements, which may be particularly informative in studies on thick specimens such as cells. The technique also provides a mechanism for obtaining high spatial resolution without the need for a metal coating around the NSOM probe and should work equally well with nonwaveguide probes such as atomic force microscopy tips. This may lead to dramatically improved spatial resolution in fluorescence imaging.

An analysis of transvection at the yellow locus of Drosophila melanogaster.

Studies of a wide variety of organisms have shown that homologous sequences can exert a significant impact on each other, resulting in changes in gene sequence, gene expression, chromatin structure, and global chromosome architecture. Our work has focused on transvection, a process that can cause genes to be sensitive to the proximity of a homologue. Transvection is seen at the yellow gene of Drosophila, where it mediates numerous cases of intragenic complementation. In this article, we describe two approaches that have characterized the process of transvection at yellow. The first entailed a screen for mutations that support intragenic complementation at yellow. The second involved the analysis of 53 yellow alleles, obtained from a variety of sources, with respect to complementation, molecular structure, and transcriptional competence. Our data suggest two ways in which transvection may be regulated at yellow: (1) a transcriptional mechanism, whereby the ability of an allele to support transvection is influenced by its transcriptional competency, and (2) a structural mechanism, whereby the pairing of structurally dissimilar homologues results in conformational changes that affect gene expression.

Domain formation in thin lipid films probed with near-field scanning optical microscopy.

High-resolution near-field scanning optical microscopy (NSOM) fluorescence and topographic images of L-alpha-dipalmitoylphosphatidylcholine (DPPC) monolayers doped with a fluorescent dye are presented. DPPC monolayers are deposited onto mica substrates from the air-water interface at several surface pressures using the Langmuir-Blodgett technique. Sub-diffraction limit phase domain structures are observed in both fluorescence and topographic NSOM images of the lipid films. The morphology of the resulting monolayers depends strongly on the surface pressure and composition of the subphase used in the film transfer. Mechanisms for lipid domain formation and growth are discussed.

Near-field scanning optical microscopy studies of L-alpha-dipalmitoylphosphatidylcholine monolayers at the air-liquid interface.

The phase structure in L-alpha-dipalmitoylphosphatidylcholine-20 mol% fluorescent 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate Langmuir monolayers dispersed on a 2 M sucrose solution subphase is studied with near-field scanning optical microscopy (NSOM). Cantilevered NSOM probes operating in a tapping-mode feedback or an optical interferometric feedback mode are capable of tracking the air-sucrose solution interface. At the micrometre scale, the NSOM fluorescence images reveal lipid domain features similar to those observed previously in supported Langmuir-Blodgett (LB) monolayers. At the submicrometre scale, the small nanometric lipid islands seen in LB films are not observed at the air-sucrose interface. This supports a mechanism in which domain formation in LB films can be induced by means of the transfer process onto the solid support. Progress towards extending these studies to films at the air-water interface using the optical interferometric feedback method is also discussed.

Low blastocyst formation rates in day-2 fertilized oocytes.

PURPOSE: To examine the blastocyst formation rates of day-2 fertilized oocytes. METHODS: A retrospective study of the outcomes/blastocyst formation of day-2 fertilized oocytes was undertaken. RESULTS: Fertilization rates of day-1 and -2 oocytes by intra-cytoplasmic sperm injection were similar. The development frequencies to four cells were similar. However, the blastulation rates were significantly lower from the day-2 fertilized eggs. The fertilization rates from day-2 conventional in vitro fertilization reinsemination were lower than the fertilization rates of day-1 oocytes. The blastulation rates from day-2 fertilized eggs were also lower than the rates from day-1 fertilized eggs in the in vitro fertilization group. CONCLUSIONS: Fertilization is not a good indicator to predict the viability of fertilized oocytes. Day-2 fertilized oocytes had significantly lower blastocyst formation rates than the rates from day-1 fertilized oocytes.

Submicron structure in L-alpha-dipalmitoylphosphatidylcholine monolayers and bilayers probed with confocal, atomic force, and near-field microscopy.

Langmuir-Blodgett (LB) monolayers and bilayers of L-alpha-dipalmitoylphosphatidylcholine (DPPC), fluorescently doped with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diIC18), are studied by confocal microscopy, atomic force microscopy (AFM), and near-field scanning optical microscopy (NSOM). Beyond the resolution limit of confocal microscopy, both AFM and NSOM measurements of mica-supported lipid monolayers reveal small domains on the submicron scale. In the NSOM studies, simultaneous high-resolution fluorescence and topography measurements of these structures confirm that they arise from coexisting liquid condensed (LC) and liquid expanded (LE) lipid phases, and not defects in the monolayer. AFM studies of bilayers formed by a combination of LB dipping and Langmuir-Schaefer monolayer transfer exhibit complex surface topographies that reflect a convolution of the phase structure present in each of the individual monolayers. NSOM fluorescence measurements, however, are able to resolve the underlying lipid domains from each side of the bilayer and show that they are qualitatively similar to those observed in the monolayers. The observation of the small lipid domains in these bilayers is beyond the spatial resolving power of confocal microscopy and is complicated in the topography measurements taken with AFM, illustrating the utility of NSOM for these types of studies. The data suggest that the small LC and LE lipid domains are formed after lipid transfer to the substrate through a dewetting mechanism. The possible extension of these measurements to probing for lipid phase domains in natural biomembranes is discussed.

Calcium regulation of nuclear pore permeability.

The nuclear envelope is an integral part of the structural framework of the nucleus, and is involved in organizing intranuclear events. It serves as a selective barrier, actively transporting proteins required for normal nuclear function and exporting RNA. The movement of molecules across the nuclear envelope is critical for cellular homeostasis, and it allows cells to respond to external events. The only known pathway for direct communication between the cytoplasm and the nucleoplasm of a cell is through the nuclear pore complex. In the past decade, rapid advances have been made in elucidating the structure and function of the nuclear pore complex. Yet, researchers are just beginning to identify some of the regulatory mechanisms controlling transport through the pore complex. The nucleus is surrounded by a Ca2+ storage compartment, which sequesters and releases Ca2+ in response to intracellular second messengers, Recent evidence suggests that the nuclear Ca2+ store may indirectly regulate passive diffusion through the nuclear pore complex. The evidence for Ca2+ regulation of the nuclear pore complex will be discussed, along with the introduction of the simplest, testable model to describe the observations.

Sterilization with fibrin sealant in a rabbit uterine horn model.

OBJECTIVE: This study investigated the effects of a fibrin sealant with or without coagulation in obliterating the tubal lumen at the uterotubal junction in rabbits. STUDY DESIGN: Twenty-three female rabbits were subjected to laparotomy and hysterotomy. They were randomly divided into four groups, and the fallopian tubes underwent different treatment: In group 1 (n = 14) both bipolar coagulation and the fibrin sealant were used. In group 2 (n = 10) only fibrin sealant without coagulation was used. In group 3 (n = 11) only coagulation was used. In group 4 (n = 10) no treatment was performed (control group). Two to three rabbits were euthanized on days 3 to 4, 7 to 11, 14 to 17, and 21 to 24 postoperatively. Both uterotubal junctions were excised, and step cross sections were obtained. The morphologic features including denuded epithelium with debris, submucosal fibrosis, hemorrhage, and fibrinoid matter in the tubal lumen and occluded lumen were observed. RESULTS: Tubal lumina were successfully occluded in 10 of 14 tubes (71.4%) in group 1. In groups 2 and 4 none of the tubes were occluded, and in group 3 only three tubes were partially occluded. The difference between group 1 and other groups was significant (p < 0.05). CONCLUSIONS: The effects of bipolar coagulation in enhancing the inflammatory reaction on the mucosa and scar formation induced by fibrin sealant at the uterotubal junction in the rabbit were demonstrated. This sterilization method with the use of fibrin sealant may be developed into an outpatient procedure in humans and may be a new method for female sterilization in the future.

Optimal insemination concentration for human in vitro fertilization (IVF).

This study is looking for optimal insemination concentration to achieve optimal IVF pregnancy. Sperm lateral head displacement, total abnormal form, Kruger morphology and index, hypo-osmotic swelling test were significantly correlated with fertilization in vitro. Based on those parameters, logistic regression models were formulated. These models predict either fertilization probability provided with an insemination concentration or insemination concentration assigned with a definite fertilization percentage. These models showed that increased insemination concentration can increase fertilization percentage. The increase of fertilization didn't compensate for the significant loss of implantation by increasing insemination concentration.

Blastocyst formation is a good indicator for attainment of assisted reproduction.

Blastocyst formation is a late stage of embryogenesis before implantation. The examination for the percentage of blastocyst formation (PBF) can predict the viability/pregnancy of the assisted reproduction trials. The PBF significantly correlates with age and pregnancy. The PBF is significantly lower in the intracytoplasmic sperm injection (ICSI) treatment than the conventional IVF treatment. The zygotes from immature oocytes give less blastocyst formation than the zygotes from mature oocytes. One pronucleus "zygotes" have significantly less chance to blastocyst than the normal 2 pronuclei zygotes. A mathematical model is proposed, verified, and predicts the hatching/hatched is the rate limiting step for the outcome of pregnancy.

Insulin-like growth factor binding proteins in peritoneal fluid of women with minimal and mild endometriosis.

This prospective cohort study was carried out in a university-based infertility clinic to determine the profile of insulin-like growth factor binding proteins (IGFBPs) in patients with mild endometriosis and no obvious mechanical factor contributing to infertility. A total of 26 patients with minimal and mild endometriosis and 10 controls contributed peritoneal fluid at surgery. The variety, expression and levels of IGFBPs were determined by radio-immunoassay and Western ligand blots (WLBs) with quantitation by laser densitometer. A 27 kDa species was significantly lower and 31 kDa species tended to be lower in patients with endometriosis as determined by quantitative laser densitometer. The levels of IGFBP-3 detected by radioimmunoassay and by WLB were correlated in the control group and in the patients with endometriosis in the follicular phase but not in patients with endometriosis in the luteal phase. The level of 27 kDa species seen on WLBs did not appear to correspond to IGFBP-1 determined by radioimmunoassay and IGFBP-3 levels in luteal phase endometriosis patients also departed from values determined by radioimmunoassay. These discrepancies suggest a complex system to control levels of IGF in the peritoneum involving multiple binding proteins and proteases. The IGFBPs of patients with endometriosis may contribute to reproductive dysfunction and be able to serve as markers.

Efficacy of adhesion barriers. Resorbable hydrogel, oxidized regenerated cellulose and hyaluronic acid.

OBJECTIVE: To compare a novel resorbable hydrogel barrier with two previously studied barriers, oxidized regenerated cellulose and hyaluronic acid, for the prevention of postoperative adhesions. STUDY DESIGN: Two models were employed in the rat uterine horn, one of adhesion formation after devascularization and serosal injury and one of adhesion reformation after adhesiolysis RESULTS: In the devascularization model, hydrogel treatment reduced the mean extent of adhesion formation from 73% in the control group to 13% (P < .005). Hyaluronic acid pretreatment reduced the extent of adhesion formation to 44% (P < .05), while oxidized regenerated cellulose failed to reduce formation (P > .25). In the adhesiolysis model, treatment with the hydrogel reduced the mean extent of adhesion formation from 87% in the control group to 20% (P < .005). Neither the oxidized regenerated cellulose nor the hyaluronic acid treatments lowered the extent of adhesion formation from the control group (P > .25). The hydrogel barrier was observed to be resorbed over a five-day period and remained adherent to the tissue during resorption. CONCLUSION: Resorbable hydrogel barriers are highly effective in the reduction of adhesion formation and reformation in the rat. This probably due to the good biocompatibility and retention of these materials upon the site of application.

Local release of fibrinolytic agents for adhesion prevention.

Tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), and streptokinase were evaluated for their ability to reduce postsurgical adhesion formation in a rat uterine horn devascularization and serosal injury model in a blinded, randomized study. Small doses of tPA, uPA, or streptokinase were delivered over approximately a 4-day period either from a biodegradable hydrogel matrix or as four daily intraperitoneal injections. The hydrogel was formed upon the uterine horns by photopolymerization of an aqueous precursor solution containing dissolved drug. A control group that received no treatment had an average extent of adhesion formation of 72 +/- 15% (mean +/- SEM, percentage of the length of the uterine horns involved in adhesions). Application of this formulation of the hydrogel alone reduced the extent of adhesion formation to 22 +/- 10% by functioning as a mechanical barrier. When tPA was released from the hydrogel, adhesion formation was reduced to 4 +/- 3%, while when tPA was given by intraperitoneal injection, adhesion formation was only reduced to 49 +/- 8%. Local delivery of urokinase reduced adhesion formation to 6 +/- 6%, but intraperitoneal injection of urokinase did not reduce adhesion formation. Streptokinase did not reduce adhesion formation when administered by intraperitoneal injection and increased adhesion formation to 45 +/- 9% when locally released relative to the hydrogel alone. These results suggest that both tPA and uPA may be used to prevent adhesion formation when delivered locally.