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W T Garside

Publications and source records attributed to W T Garside.

10 recordsLinked to original sources
PubMed1997

Sequential analysis of zona thickness during in vitro culture of human zygotes: correlation with embryo quality, age, and implantation.

Zona pellucida thickness was measured daily in zygotes and cleavage stage embryos. Measurements were performed on a Nikon inverted microscope equipped with Hoffman modulation optics, using an ocular micrometer. Zona thickness of each zygote/embryo was measured four times, the zygote/embryo was then "rolled over," and four more measurements were repeated for a total of eight. The zygotes/embryos were photographed daily and the measurements repeated on the prints. Subsequently, the mean zona thickness for each stage was calculated. A total of 81 patients (mean age 33.8 +/- 4.2) participated in the study. A total of 427 embryos were evaluated. Categorical data differences between groups were evaluated by ANOVA and multiple linear regression. For nominal data, the Kruskal-Wallis test was applied; when P < 0.05 the differences were considered to be significant. We found that the average zona thickness on day 1 of in vitro culture was 17.7 +/- 0.14 microns; 16.3 +/- 0.14 microns on day 2 and 14.9 +/- 0.14 microns on day 3 (P < .0001). When the zona thickness was analyzed in relation to the number of blastomeres on day 3 of culture, there was a highly significant correlation with blastomere number (P < .0001). Similarly, there was a highly significant correlation with embryo grade (P < .005) and fragmentation (P < .001). The data were also analyzed for embryos transferred that resulted in a successful pregnancy, revealing that embryos in a pregnancy cycle had significantly thinner zonae pellucidae (P < .0001), when compared to embryos that were not transferred or from nonconceptual cycles. The average zona thickness also decreased with age, and was most apparent after 35 years. Changes in zona thickness correlated with the number of blastomeres, grade, fragmentation, age and were more evident in embryos transferred from cycles resulting in successful pregnancies. Therefore, zona pellucida measurements should be included in the overall assessment of embryo quality, since this information may be useful in the selection of optimal embryos for transfer.

Adult↗
PubMed1997

Analysis of the human zona pellucida during culture: correlation with diagnosis and the preovulatory hormonal environment.

PURPOSE: The objective of this study was to analyze sequentially the human zona pellucida changes in an in vitro fertilization program as it relates to several variables. METHODS: The zona pellucida thickness was measured daily in zygotes and cleavage-stage embryos on a Nikon inverted microscope equipped with Hoffman modulation contrast optics, using an ocular micrometer. A total of 512 embryos from 96 patients was evaluated. RESULTS: There was a highly significant direct correlation between zona thickness and preovulatory estradiol and basal day 3 FSH levels (P < 0.02 and P < 0.0006, respectively). This relationship showed a rapid reversal following 48 hr of culture; embryos from patients with the highest FSH levels had thinner zonae prior to transfer (P < 0.0007). The zonae from patients with unexplained infertility were thicker (19.4 +/- 2.7 microns) than those from patients with endometriosis (17.7 +/- 2.2 microns), tubal (17.5 +/- 2.4 microns), or male-factor infertility (16.4 +/- 2.7 microns) (P < 0.0001) on the first day of culture. CONCLUSIONS: We hypothesize that the thickness of the human zona pellucida is influenced by the preovulatory hormonal environment and diagnosis. These factors should be considered as part of the embryo quality evaluation prior to transfer or when assessing the possibility of using assisted hatching. More studies are needed to understand the factors regulating the thickness of the human zona pellucida.

Adult↗
PubMed1997

Micromachined analytical devices: microchips for semen testing.

Micromachined devices (microchips) have been designed and tested for a range of clinically important assays. In this study we compare sperm motility determined using disposable glass microchips and a conventional Makler chamber. The 17 x 14 mm glass microchips contained three etched test structures each comprising either duplicate or quadruplicate analytical microchannels. Semen samples with sperm counts ranging from 21 to 78 million sperm per ml and forward progression scores of from 1+ to 3+ were evaluated and swimming times ranging from 360 s (3.3+ progression) to 770 s (1+,2 forward progression) observed in the microchips. Motility determined by the time taken for sperm to swim to the end of a microchannel (100 microns wide x 40 microns deep x 10 mm long) in the microchip correlated with forward progression of the sperm determined by the conventional Makler chamber method. This study demonstrates the feasibility of microchips for sperm motility testing and suggests that this technique would be applicable to the study of other types of motile cells.

Biotechnology↗
PubMed1996

Classification of male factor infertility relevant to in-vitro fertilization insemination strategies using mannose ligands, acrosome status and anti-cytoskeletal antibodies.

Polyvalent mannose ligands in the presence of free mannose act as zona pellucida agonists which rapidly induce acrosome exocytosis in competent motile human sperm from fertile donors following in-vitro capacitation. Quantification of the binding patterns of fluorescein isothiocyanate-labelled mannosylated albumins and of specific antisera which recognize mannose receptors and other related integral sperm membrane proteins as well as the incidence of induced acrosome exocytosis after capacitation has allowed us to identify three categories of male infertility. Category 1 males have normozoospermic semen parameters, their spermatozoa have elevated sperm cholesterol values and fail to fertilize oocytes in vitro after standard short-term incubations. These spermatozoa do not bind mannose ligands and do not show spontaneous or induced acrosome reactions, but treatments to remove cholesterol from the spermatozoa (e.g. prolonged incubation in the presence of sterol acceptors) confer the ability to fertilize. Cholesterol loading and unloading experiments have demonstrated the reversible character of sperm membrane properties in category 1 male infertility. Category 2 males have normal-appearing spermatozoa in semen which express mannose ligand receptors on incubation, but fail to undergo acrosome reactions in response to mannose treatment. Interestingly, all category 2 males identified in this study have clinical varicocele. Category 3 males have semen which may be normozoospermic or teratozoospermic with, in some cases, high percentages of tapering spermatozoa in the absence of clinical varicocele. Spermatozoa from category 3 men are deficient in a superfamily of integral membrane proteins whose cytoplasmic tails have myosin motors as identified by amino acid sequence analysis and anti-myosin antibody reactivity. Their spermatozoa do not express mannose ligand receptors or undergo induced acrosome reactions. Fertilization with category 2 and 3 semen is only achieved by micromanipulation procedures. These findings illustrate the practical application of basic research for infertility classification.

Acrosome↗
PubMed1994

Protein databases for compacted eight-cell and blastocyst-stage mouse embryos.

High-resolution two-dimensional sodium dodecyl sulfate-polyacrylamide (2D-SDS) gel electrophoresis combined with computerized analysis of gel images was used to construct and analyze protein data-bases for two stages of preimplantation mouse embryogenesis, the compacted eight-cell stage and the fully expanded blastocyst stage. These stages were chosen for their ease in identification of multiple synchronous embryos. Synchronous cohorts of 30-50 embryos were labelled with L-[35S]methionine for 2 hr. The embryos were then lysed in 30 microliters hot SDS sample buffer, and the lysates were stored at -80 degrees C until the gels were run. Five replicates were run for eight-cell embryos, and four for blastocyst-stage embryos. The samples were processed for 2D gel electrophoresis and fluorography; multiple exposures were made. Gel images were analyzed using the PDQUEST system, and databases were constructed. Analysis of the databases for both developmental stages showed high reproducibility of protein spots in multiple gel images. Of 1,674 total spots in eight-cell embryo standards, > 79% of spots had a percentage error (S.E.M./average) < 50%, and > 45% had a percentage error < 30%. Similarly, of 1,653 total spots in blastocyst-stage embryo standards, 74% of spots had a percentage error < 50%, and approximately 47% of spots had a percentage error < 30%. Forty-three spots (approximately 3% of the total spots) were found to be detected only in the eight-cell stage, while 75 spots were detected solely in the blastocyst stage. Sixty-nine proteins showed a greater than threefold increase in isotope incorporation from the eight-cell to the blastocyst stage, with a percentage error < 50% in both the eight-cell and the blastocyst stages. In contrast, 41 of the proteins showed a decrease during this period. Analysis of the protein databases described in this study has allowed us to document the overall quantitative changes in proteins from the compacted eight-cell stage to the blastocyst stage of mouse preimplantation development. These data-bases provide a valuable tool for further detailed quantitative analysis of specific proteins associated with developmental events. In addition they will permit analysis of the effects of environmental factors, such as growth factors, on early embryo development.

Animals↗
PubMed1994

Insulin family growth factors have specific effects on protein synthesis in preimplantation mouse embryos.

Previously constructed protein databases for two stages of preimplantation mouse embryogenesis, the compacted eight-cell stage and the fully expanded blastocyst stage, have been used to analyze the effects of insulin, IGF-I, and IGF-II on protein synthesis in these developmental stages. Proteins were labeled by placing, for 2 hr, synchronous cohorts of 35-50 embryos into human tubal fluid (HTF) medium containing L-[35S]-methionine (1 mCi/ml) in the presence or absence of one of the growth factors. The embryos were then washed with medium and lysed. Samples were processed for 2-D gel analysis. For each embryonic stage and each growth factor, four or five experimental replicates were done and the gel images were compared using the PDQUEST system. Using the computer-assisted analysis, we were able to identify proteins that showed a statistically significant (P < 0.05) change in synthesis. At the eight-cell stage of development insulin caused increased synthesis of two proteins and decreased synthesis in three proteins. Insulin-treated blastocyst stage embryos exhibited an increased synthesis in eight proteins and decreased synthesis for one protein. The effect of IGF-I at the eight-cell stage of development was mostly inhibitory; the synthesis of only one protein increased and the synthesis of five proteins showed a decrease. Similar results were obtained with blastocyst stage embryos; four proteins demonstrated an increase in synthesis while 14 proteins showed a decrease. Eight-cell stage embryos incubated with IGF-II had seven proteins with a decreased synthesis, although in blastocyst stage embryos, nine proteins showed increased synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Functions of the IGFs in early mammalian development.

The identification of growth factors and/or receptors produced by mammalian embryos or present in the maternal reproductive tract is of basic interest, as well as having practical application. Early studies established that receptors binding insulin and the insulin-like growth factors (IGFs) are expressed by preimplantation mouse embryos. These studies have been confirmed at the molecular level using RT-PCR techniques. In addition, high resolution electron microscopy has shown that insulin is internalized by the cells of the blastocyst stage mouse embryo, and that immunologically intact insulin is detectable in the cells of the trophectoderm and inner cell mass. Similar studies with gold labelled IGF-I have shown that this ligand is also bound and internalized by mouse blastocysts. However, although all blastocysts express receptors that bind IGF-I on the basolateral cell surface of the trophectoderm, only 30% exhibit apically located receptors. In order to elucidate the functions of IGFs in early mouse development, we are in the process of constructing protein data-bases for embryos at the eight-cell and blastocyst stage. By the use of the database, it should prove possible to elucidate targets of growth factor action.

Animals↗
PubMed1993

Mouse preimplantation embryos exhibit receptor-mediated binding and transcytosis of maternal insulin-like growth factor I.

High-resolution microscopy in conjunction with colloidal gold-labeled insulin-like growth factor I (IGF-I) has been used to provide evidence that the IGF-I receptor is first detected in 8-cell-stage mouse embryos, confirming the results of previous reverse transcriptase polymerase chain reaction (RT-PCR) studies. Specificity for the IGF-I receptor was demonstrated by displacement with unlabeled IGF-I and dual-labeling experiments with colloidal gold-labeled or unlabeled insulin. Labeled IGF-I ligand is internalized by means of receptor-mediated endocytosis following its concentration in coated pits, and it can be visualized within cytoplasmic organelles. Immunocytochemical analyses at the blastocyst stage, using gold-labeled antibodies to the receptor, confirmed the expression of IGF-I receptors on all cells of the embryo. Similar studies with antibodies directed against the ligand demonstrated that IGF-I internalized by the embryo in vivo is maternally derived. Approximately 40% of blastocysts showed apical plasma membrane binding of gold-labeled ligand ("responders"), while approximately 60% did not demonstrate binding ("nonresponders"); however, both classes of embryo expressed receptors on basolateral membranes of trophectoderm cells and on the surface of inner masses. Functional studies show that incubating embryos in physiological levels of IGF-I (40 ng/ml) results in increased numbers of cells in the inner cell mass (p < 0.05), but not the trophectoderm, as compared to controls.

Animals↗
PubMed1993

Applications of a microfabricated device for evaluating sperm function.

Mesoscale structures (microns dimensions, nL-pL volumes) have been designed and fabricated in silicon for use in various analytical tasks. We studied sperm motility and performed sperm selection in channels (80 microns wide x 20 microns deep), branching structures (40 microns wide x 20 microns deep, eight bifurcations), and channels containing barriers (7 microns feature size). Sperm-cervical mucus and sperm-hyaluronic acid interactions were assessed by using appropriate microchannel-chamber structures filled with either cervical mucus or hyaluronic acid. Simultaneous assessment of the potency of different spermicides (e.g., nonoxynol-9, C13G) and spermicide concentrations was achieved with structures comprising chambers containing spermicide connected via channels to a central chamber into which semen was introduced. Semen was also tested for the presence of sperm-specific antibodies by using microchannels filled with human anti-IgG antibody-coated microbeads.

Humans↗