Biomedical subjects

C D Matthews

Publications and source records attributed to C D Matthews.

At least 55 records · Page 3Linked to original sources

PubMed1994

Discontinuous Percoll gradients enrich X-bearing human spermatozoa: a study using double-label fluorescence in-situ hybridization.

The aim of this study was to evaluate the efficacy of enriching X-bearing human spermatozoa using 12-step (25-80%) discontinuous Percoll gradients. X- and Y-bearing spermatozoa were simultaneously identified in neat semen (controls) and in 80% Percoll fractions from the same samples using double-label fluorescence in-situ hybridization and chromosome-specific DNA probes. Hybridization and labelling efficiencies of 95-99% were obtained in all samples. The mean ratio of X- to Y-bearing spermatozoa in the controls was 49.0:48.2, whereas there was a significant enrichment (P < 0.0001) of X-bearing spermatozoa in the Percoll fractions (mean X:Y ratio was 55.1:41.1 or 1.35). The ratio varied from 1.1-1.5 in individual Percoll samples. There were no significant differences in the proportions of aneuploid spermatozoa (XX, YY, XY) between the control and Percoll fractions. We conclude that 12-step discontinuous Percoll gradients enrich X-bearing spermatozoa, but the degree of enrichment is insufficient for use in preconceptional sex selection.

Aneuploidy↗

PubMed1994

The influence of sperm morphology and the acrosome reaction on fertilization outcome after sub-zonal injection (SZI) of human spermatozoa.

The usefulness of sub-zonal injection (SZI) for the treatment of severe male factor infertility has been restricted by low and unpredictable fertilization rates and the high risk of polyspermy after the injection of multiple spermatozoa. In this prospective study, we have evaluated whether sperm morphology and the percentage of acrosome-reacted spermatozoa at the time of injection can be used to predict SZI fertilization outcomes. Populations of motile spermatozoa equivalent to those injected were collected from the medium/oil interface immediately after SZI of each cohort of oocytes. Morphology was assessed using the World Health Organization 1987 criteria and the acrosomal status of spermatozoa was determined after staining with rhodamine-conjugated Pisum sativum agglutinin. A fertilization index (FI) was calculated to express the actual fertilizing potential of the spermatozoa injected. In all, 67 patients underwent 72 SZI cycles. The overall fertilization and polyspermy rates were 36 and 47% respectively, and a clinical pregnancy rate per transfer of 22% was achieved. Linear regression analysis demonstrated a statistically significant relationship between morphology and the FI (r = 0.506, P < 0.0001). Patients with < or = 10% normal morphology always had a FI < or = 10%, and this was reflected by low fertilization and polyspermy rates and the high number (32%) of cycles with complete failure of fertilization in this group. In patients with > 10% normal morphology, there were two patterns: low (< or = 10% FI) or high (> or = 10% FI) fertility. This was evident in the fertilization (23 and 85%, respectively), and polyspermy (25 and 68%, respectively) rates of these two patient sub-groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrosome↗

PubMed1994

A fluorescent in situ hybridization analysis of the chromosome constitution of ejaculated sperm in a 47,XYY male.

Two semen samples from a 47,XXY male were examined using chromosome-specific DNA probes and fluorescent in situ hybridization (FISH) to determine the distribution of sex chromosomes and an autosome (chromosome 17) in the sperm. A motile population of sperm was also prepared from one sample using the swim-up technique to compare the motile and total sperm populations. Chromosomes were localized using single FISH and a biotinylated chromosome 17 probe (TR17), or double FISH using a biotinylated X chromosome probe (TRX) and a digoxigenin-labelled Y chromosome probe (HRY). Labelling efficiencies were 95-98%. Ploidy levels were estimated by measurement against a microscope eye-piece graticule. The overall ratio of X- to Y-bearing sperm was 47% to 48.4% in the neat samples, and 48.4% to 45.3% in the swim-up fraction. Neither of the ratios was significantly different from 1:1. The frequencies of monosomic and disomic (but otherwise haploid sperm) were not different from the frequencies we observed in normal donors. In contrast, the frequencies of both diploid and tetraploid cells were increased in the neat samples of the XYY male. In the swim-up fractions, however, none of these parameters differed from those of ten normal semen donors. These results support the hypothesis that the extra Y chromosome in XYY men is eliminated during spermatogenesis.

Adult↗

PubMed1994

Effect of exogenous melatonin and extending the dark period at dusk before the summer solstice on the onset of oestrus in Romney Marsh ewes.

Groups of Romney Marsh ewes (n = 24) were injected with exogenous melatonin or subjected to extended darkness or a combination of both at dusk from the winter to the summer solstice before being re-exposed to the natural photoperiod at 35 degrees S. The animals were at all times exposed to natural dawn. The onset of subsequent oestrus was delayed when compared with controls subjected to the natural photoperiod. Either melatonin or darkness or a combination were equally effective at delaying oestrus. The results are consistent with a hypothesis based on an external coincidence model of seasonal breeding involving the coincidence of a light-sensitive phase set close to dusk that, when exposed to light in spring and summer, prepares the reproductive axis to respond correctly to the direct effects of the inductive photoperoid in autumn. The offset of the time of oestrus was not affected by the experimental treatments nor was the timing of the subsequent reproductive season. Since melatonin was as effective as darkness at influencing the assumed photosensitive signal, even in the presence of natural light, melatonin may act through the light reception system.

Animals↗

PubMed1994

Anatomy of adult female common marmoset (Callithrix jacchus) reproductive system.

Better appreciation of the female reproductive anatomy of the common marmoset (Callithrix jacchus) should improve the prospects for nonsurgical embryo transfer in this model. Vaginal measurements were performed in 8 female adult marmoset monkeys. Four monkeys were measured at laparotomy for gross internal anatomy, and 1 monkey was analysed at autopsy. The vagina of the marmoset monkey was found to be divided into a lower and upper vagina with a marked vaginal isthmus between them. The mean lengths of the lower and upper vagina were 17 mm (34 mm in total vagina). The mean uterine size was 8.4 (length) x 10.0 (width) x 6.4 (thickness) mm, with the ovary measuring 5.3 x 4.3 x 3.8 mm. The mean length of the fallopian tube was 10.5 mm with a width of 1.5 mm. Nonsurgical embryo transfer in this model appears to be feasible, but the proportionally long vagina and short uterine cavity needs to be recognised.

Animals↗

PubMed1993

X larger than Y.

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Animals↗

PubMed1993

Simultaneous detection of X- and Y-bearing human sperm by double fluorescence in situ hybridization.

Double fluorescence in situ hybridization (FISH) was used to detect sex chromosomes in decondensed human sperm nuclei. Biotinylated X chromosome specific (TRX) and digoxigenin-labeled Y chromosome specific (HRY) probes were simultaneously hybridized to sperm preparations from 12 normal healthy donors. After the hybridization, the probes were detected immunocytochemically, using two different and independent affinity systems. Ninety-six percent of the 12,636 sperm showed fluorescent labeling, of which 47.4% were haploid X and 46.8% were haploid Y. A frequency of 0.46% of XX-bearing sperm (0.28% disomic, 0.18% diploid) and 0.38% YY-bearing sperm (0.21% disomic, 0.17% diploid) was found. The overall proportions of X- and Y-bearing sperm in the ejaculates were 47.9% and 47.2%, respectively, which was not significantly different from the expected 50:50 ratio. In addition 0.21% of cells appeared to be haploid XY-bearing sperm, 0.62% were diploid XY-bearing cells, and 0.05% of cells were considered to be tetraploid cells. The application of double FISH to human sperm using X-chromosome and Y-chromosome probes has allowed a more accurate assessment of the sex chromosal complements in sperm than single FISH method and quinacrine staining for Y-bodies.

Adult↗

PubMed1993

Hatching rate: an optimal discriminator for the assessment of single-blastomere biopsy.

PURPOSE: In order to determine an optimal marker to discriminate embryo injury following single-blastomere embryo biopsy, mouse embryos were examined for rates of blastocyst formation, hatching, implantation, and fetal development following single-blastomere biopsy. RESULTS: Early studies of single-blastomere biopsy (1-8 series) resulted in similar rates of blastocyst formation (P > 0.05) but a lower rate of hatching of biopsied (n = 140) versus control (nonbiopsied) (n = 145) embryos (78.6 vs 95.2%; p < 0.01). Subsequent experience (9-13 series) eliminated this difference between biopsied (n = 145) and control embryos (n = 133) (95.9 vs 94.0%; P > 0.05). Embryo transfer of hatching blastocysts of biopsied (n = 100) and nonbiopsied control (n = 100) groups resulted in equivalent rate of fetal development (70.0 vs 68.0%; p > 0.05). CONCLUSIONS: The hatching rate appeared to be a simple, sensitive, and reliable method to evaluate the single-blastomere biopsy technique.

Animals↗

PubMed1993

Transgenic systems for in vivo mutation analysis.

Transgenic mice carrying shuttle vectors containing the lacI gene as the target permit the in vivo measurement of mutations in multiple tissues and have been used to test the mutagenic effects of several compounds. Tissue-specific and time-dependent responses have been observed, and the spectrum of mutations determined by sequencing allows analysis of the role of expression time in mutagenesis. The results obtained from sequencing analysis have demonstrated spectra paralleling those observed in alternative in vivo assays. In addition to color screening, modifications to this system have permitted direct selection for mutations in the lacI target by a variety of methods. Transgenic rats containing the same lambda/lacI shuttle vector have been developed for inter-species comparison of mutagenesis testing results, which may offer a better understanding of the specific mechanisms involved in mutagenesis at the molecular level in vivo.

Animals↗

PubMed1993

Inhibin and relaxin concentrations in early singleton, multiple, and failing pregnancy: relationship to gonadotropin and steroid profiles.

OBJECTIVE: To examine the inter-relationships between inhibin, relaxin, steroid concentrations, estradiol (E2), progesterone (P), and gonadotropins in early pregnancy. DESIGN: Hormone concentrations in plasma were measured during the luteal phase of subjects who became pregnant (n = 58) or failed to become pregnant (n = 47) after ovarian hyperstimulation for in vitro fertilization-gamete intrafallopian transfer (IVF-GIFT) (group 1). A further group of subjects became pregnant (n = 7) or failed to become pregnant (n = 8) during endocrinology tracking of a natural cycle (group 2). Blood was obtained every 3 days in the luteal phase from day 5 in group I (day 0 was oocyte recovery) and from day 0 (first increase in luteinizing hormone [LH]) in group II. RESULTS: Progesterone and E2 were increased over nonpregnant values by day 11 (P) and day 16 (E2) in group I and by day 11 (E2 and P) in group II. Inhibin and relaxin concentrations were significantly increased by day 16 in group I (often by day 11) and by day 14 in group II pregnancy subjects. A direct relationship existed between inhibin, P, relaxin, and human chorionic gonadotropin (hCG). Subjects who had twin pregnancies demonstrated higher concentrations of all hormones and often exhibited increases earlier (by day 11 in group I) than singleton pregnancy subjects. Pregnancies that ended in miscarriages tended to have lower concentrations of P and inhibin. None of the hormones reliably discriminated between the clinical conditions of blighted ovum and of spontaneous abortion, and the predictive value of any hormone measured for miscarriage was not high. CONCLUSIONS: The trend of inhibin and relaxin concentrations closely parallels rises in P during early pregnancy. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels are suppressed very early in pregnancy. The suppression of LH and FSH in hyperstimulated cycles is more governed by E2 than inhibin in stimulated cycles. Some subjects destined to miscarry exhibit abnormal endocrine changes very early in the luteal phase.

Abortion, Spontaneous↗

PubMed1993

Prediction of in vitro fertilization rates from semen variables.

OBJECTIVE: To assess the value of semen variables for predicting fertilization rates. DESIGN: Measures of the fresh semen and the motile sperm fraction used for insemination were related to the fertilization rate by multiple regression analysis. The regression model was then used to construct a two-dimensional clinical chart. SETTING: University-affiliated reproductive medicine unit. PATIENTS: The results of 294 IVF cycles were analyzed retrospectively. Selection criteria were: [1] first cycle of IVF; [2] tubal and/or male factor infertility; and [3] four or more oocytes inseminated. INTERVENTIONS: None. MAIN OUTCOME MEASURES: The fertilization rate was related to measured variables of the fresh semen and the motile sperm fraction used for insemination. Fertilization rate was categorized as poor (< 35%) or acceptable (> or = 35%). RESULTS: Multiple regression analysis demonstrated a strong correlation between the fertilization rate and the combined indexes of percentage normal morphology and grade of motility in the fresh semen and percentage progressive motility in the motile sperm fraction. A two-dimensional chart that expressed these relationships was constructed. Its accuracy of prediction was 77% for poor fertilization and 95% for acceptable fertilization. CONCLUSIONS: The fertilization rate is strongly correlated with percentage normal sperm morphology in the fresh semen and the percentage progressive motility in the motile sperm fraction used for insemination. The clinical chart provides a simple but powerful tool for predicting fertilization outcome.

Confidence Intervals↗

PubMed1993

Detection of X- and Y-bearing human spermatozoa after motile sperm isolation by swim-up.

OBJECTIVE: To assess the ratio of X- to Y-bearing human spermatozoa in motile fractions isolated by the swim-up technique. DESIGN: The proportions of X- and Y-bearing sperm were determined in neat semen samples (control) and in motile fractions isolated from the same samples by swim-up. X- and Y-bearing sperm were simultaneously identified using chromosome-specific DNA probes and double fluorescence in situ hybridization. SETTING: Hospital-based university department. PARTICIPANTS: Ten healthy donors with normal semen characteristics. MAIN OUTCOME MEASURES: The distribution of haploid cells (X or Y), normal size cells with two sex chromosome (XX, YY, or XY), and large cells containing two (XX, YY, or XY) or four (XXYY) sex chromosomes were measured in neat semen samples and in motile fractions prepared by swim-up. RESULTS: Overall, 95% of sperm in the neat semen and swim-up fractions were labeled with the probes. The ratios of X- to Y-bearing sperm were 47.3:46.9 (neat semen) and 48.4:47.1 (swim-up fractions), which were not significantly different from a 1:1 ratio. The frequencies of sperm with normal size nuclei and two sex chromosomes (XX, YY, or XY) in the swim-up fractions were not significantly different from the controls, but there was a significant reduction in the proportion of cells with large nuclei and two (XX, YY, or XY) or four (XXYY) sex chromosomes in the swim-up fractions. CONCLUSIONS: The swim-up technique does not selectively enrich either X- or Y-bearing sperm. Because the isolation of motile spermatozoa is an important procedure for routine IUI, IVF-ET, and GIFT, the results of this study are important reassurance that the sex ratio is not altered by this method of sperm preparation.

Cell Nucleus↗

PubMed1993

Induction of hepatic mutations in lacI transgenic mice.

Transgenic B6C3F1 and C57BL/6 mice containing a lambda shuttle vector that carries a lacI target and an alpha lacZ reporter gene have been constructed for use in in vivo mutagenesis assays. After chemical treatment of mice carrying the lacI target gene, genomic DNA is isolated and the shuttle vector is recovered by exposing the DNA to lambda phage packaging extracts in vitro. Mutations in the lacI target gene that inactivate the repressor gene allow expression of the alpha lacZ reporter gene, resulting in blue mutant plaques. We have examined the ability of two genotoxic agents, dimethylnitrosamine (DMN) and methylmethane sulfonate (MMS), to induce mutations in these transgenic mice. Both compounds induce a variety of DNA adducts in mouse liver; DMN is a hepatocarcinogen that induces significant hepatic cell proliferation, but MMS is not hepatocarcinogenic and does not induce hepatic cell proliferation. The effects of animal age, differences in strain and dosing regimen, and length of expression time were evaluated. Mice were treated for 5, 14 or 21 days and were sacrificed 1, 8 or 22 days after the final dose to evaluate the effects of increased expression time on mutant frequency in liver. In 3 week old mice, DMN (2 mg/kg/day) produced 10- to 20-fold elevations in mutant frequency that increased with expression time and the number of treatments. In contrast, MMS (20 mg/kg/day) failed to increase the mutant frequency. DMN failed to induce mutations in 6 week old mice at 2 mg/kg/day, but 4 mg/kg/day yielded significant elevations in hepatic mutations. Sequencing results indicate that treatment of mice with DMN produced predominantly C:G-->T:A transitions.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1993

Blood analysis of mice born following single-cell embryo biopsy.

Single-cell embryo biopsy is an important technique in preimplantation diagnosis. The development of the mouse embryo and fetus, and the results of some analyses after birth following embryo biopsy, have been demonstrated to be normal. Histopathological analysis of mice born following single-cell embryo biopsy with a physical method (zona puncture) also showed normal organ and cell structure, thus demonstrating the safety of embryo biopsy. This experiment analysed the parameters of blood cells and the blood chemistry of 28 mice born following single-cell embryo biopsy. White cell count, red cell count, haemoglobin level and platelet count of the blood, and plasma sodium, potassium, chloride, bicarbonate, calcium, glucose, albumin, protein, creatinine, urea, bilirubin, aspartate transaminase, creatine kinase and lactate dehydrogenase of the blood were not significantly different between the mice from the biopsied and the control embryo groups, which again demonstrated the safety of single-cell embryo biopsy. The remaining totipotent cells in the biopsied embryos would thus be expected to develop to the correct cell counts and normal organic functions according to the intact hereditary messages. Further studies on the safety of embryo biopsy (including long-term observation after birth) and the improvement of the different biopsy techniques and skills for preimplantation diagnosis are necessary.

Animals↗

PubMed1993

Precise sex selected births of mice following single cell embryo biopsy and Y-linked testis-specific gene analysis.

We report on the birth of 39 mice following single cell embryo biopsy and precise sex determination following in-vitro fertilization. Polymerase chain reaction was used to amplify fragments of the mouse testis-specific gene sequence (pYMT2/B) on the Y chromosome and the ovary-specific gene (ZP3) sequence on chromosome 5 from the single biopsied cell. Embryo biopsy was not associated with any deleterious effects.

Animals↗

PubMed1993

Gamete intra-fallopian transfer: outcome following the elective or non-elective replacement of two, three or four oocytes.

The outcome of 807 gamete intra-Fallopian transfer (GIFT) cycles following the elective or non-elective transfer of two, three or four oocytes has been retrospectively studied. Electively replacing either three or two oocytes did not reduce the clinical or ongoing pregnancy rate when compared with replacing four oocytes. The incidence of high-order multiple gestation (triplet or more) was significantly reduced by replacing fewer oocytes, but the occurrence of twin pregnancy was not altered. Lower pregnancy rates were found when the number of oocytes available for replacement was limited and non-elective replacement was performed. It is suggested, therefore, that a higher number of oocytes available may allow selection of higher quality oocytes for transfer. We conclude that the overall expectation of pregnancy from the GIFT procedure is high (30-40%) and the number of oocytes replaced should be two in order to minimize the risk of high-order multiple pregnancies. It is not clear whether increasing the number of oocytes transferred will benefit subjects who failed to become pregnant previously with GIFT, but limited data suggest that transferring large numbers of oocytes to women > 40 years does not improve the expectation of pregnancy.

Age Factors↗

PubMed1993

Melatonin and photoperiodic time measurement: seasonal breeding in the sheep.

Well-established circadian physiology supports the view that photoperiodic time measurement utilizes the coincidence between the presence of light and a photosensitive phase of a 'biological clock' to alter reproductive status--the so-called external coincidence model of seasonal breeding. In this review, we examine the mechanism whereby photoperiod interacts with presumed suprachiasmatic nuclei activity to allow endogenous melatonin to normally synchronize reproductive activity to the optimal time of year. The Romney Marsh sheep is particularly explored as an experimental model. It is suggested that the on/off activity of seasonal reproduction may be a robust mechanism able to be predictably manipulated by the judicious use of the light/dark cycle and exogenous melatonin, but firmly based on circadian principles.

Animals↗

PubMed1993

Molecular heterogeneity of the beta-core fragment of human chorionic gonadotrophin.

We have analysed the structure and composition of the beta-core fragment of human chorionic gonadotrophin (beta C-hCG) from fresh urine specimens obtained from pregnant women and compared our findings with those previously proposed by other groups using different protocols. SDS-PAGE separation of reduced beta C-hCG demonstrated two major bands with apparent molecular weights of M(r) 8900 and M(r) 7500. The molecular weight of the agalacto beta C-hCG was estimated to be M(r) 10,218 from the amino acid analysis after high-performance liquid chromatography (HPLC) separation. Moreover, HPLC separation of its reduced and S-carboxymethylated peptides resulted in three peaks, but only two of them could be sequenced and demonstrated to be the previously reported beta 6-40 (M(r) 5000) and beta 55-92 (M(r) 5300) peptides of the beta hCG subunit. The results showed that 56-78% of beta C-hCG molecules of molecular weight M(r) 12,800 were able to bind Concanavalin A (Con A). While most were lacking all the peripheral monosaccharides and terminated in mannose, some retained other sugar residues on their antennae. Direct carbohydrate analysis showed the following molar content normalized to six mannose molecules: galactose 2.8, glucosamine 5.3, galactosamine 0.3, fucose 1.7 and sialic acid 3.0. Approximately 22-44% of the beta C-hCG molecules did not bind Con A (Con A non-reactive forms), of which 88% were totally deprived of sugar units and had an apparent molecular weight of approximately M(r) 10,000, and 12% were weakly reactive to Con A and reactive to anion exchange (negatively charged forms), being incompletely trimmed of their oligosaccharide chains. Comparison of our results with those of two other groups have indicated that the differences noted among preparations are due to either the source or the methods used to purify and characterize this fragment. In addition, our results showed significant microheterogeneity on the N-linked oligosaccharide moieties with some molecules apparently having no sugar molecules. These results have implications for the origins of beta C-hCG, suggesting secretion of some molecules without sugar chains and in other cases possible metabolism of hCG in the peripheral tissues.

Amino Acids↗