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Biomedical subjects

R M Winston

Publications and source records attributed to R M Winston.

At least 91 records · Page 5Linked to original sources
PubMed1993

Preimplantation diagnosis of aneuploidy using fluorescent in-situ hybridization: evaluation using a chromosome 18-specific probe.

Fluorescent detection of in-situ hybridization (FISH) with a chromosome 18-specific probe (P5041 B.5 D18Z1) has been used to assess the use of this method for preimplantation diagnosis of aneuploidy. Interphase nuclei (n = 802) have been analysed from 59 normally fertilized embryos developing in vitro at the normal rate between days 2 and 7 postinsemination. The efficiency of hybridization in control cells, as assessed by the proportion with two signals in normal female lymphocytes was 88.9% (n = 353) and with three signals in a trisomic (48,XXX+18) fibroblast cell line 74.0% (n = 290). Fifty-four of the human embryos were considered to be diploid on the basis that the majority of nuclei had two signals. Some nuclei in these embryos had one or no signal, especially on day 2, and tetraploid nuclei were also widespread. Among the remaining five embryos, one 5-cell embryo on day 2 had three hybridization signals in 4/5 nuclei and was trisomic for chromosome 18, one 4-cell embryo on day 2 had only one signal in 4/4 nuclei and was monosomic, and the three other embryos were aneuploid mosaics and/or had multi-nucleated blastomeres. Analysis of the incidence of interphase nuclei with more or less than the diploid number of hybridization signals indicates that more than a single nucleus will be necessary for accurate preimplantation diagnosis of aneuploidy.

Aneuploidy↗
PubMed1993

Insulin-like growth factor-I (IGF-I) inhibits production of IGF-binding protein-1 while stimulating estradiol secretion in granulosa cells from normal and polycystic human ovaries.

We have investigated the actions of FSH and insulin-like growth factor-I (IGF-I) on the production of IGF-binding protein-1 (IGFBP-1) by granulosa cells from unstimulated normal and polycystic (PCO) human ovaries and related these effects to those on estradiol (E2). IGFBP-1 concentrations were measured in granulosa cell-conditioned medium (48-h culture with 10(-7) M testosterone) and follicular fluid. IGF-I (50 ng/mL), in the absence of FSH, stimulated E2 production by granulosa cells from both normal and polycystic ovaries, and there was a synergistic action between IGF-I and FSH. Granulosa cells secreted IGFBP-1 in concentrations ranging from 20-500 pg/1000 cells.48 h, with cells from two of four normal and three of four polycystic ovaries showing a dose-related increase in IGFBP-1 production in response to FSH. In contrast, the addition of as little as 100 pg/mL IGF-I to cells incubated with testosterone or testosterone plus FSH, caused complete inhibition of IGFBP-1 production. FSH treatment produced the expected dose-related increase in E2 accumulation. IGFBP-1 was detectable in fluid from all sizes of follicle tested, but there was no correlation of IGFBP-1 concentrations with follicle size and no difference between normal and polycystic ovaries. These data indicate that IGFBP-1 and E2 are differentially regulated by IGF-1 in the human ovary.

Carrier Proteins↗
PubMed1993

Binucleate blastomeres in preimplantation human embryos in vitro: failure of cytokinesis during early cleavage.

The nuclei of disaggregated blastomeres from two hundred preimplantation human embryos were examined between days 2 and 4 after insemination in vitro by vital labelling with a polynucleotide-specific fluorochrome. Although the majority of blastomeres had a single nucleus, binucleate blastomeres containing two nuclei of equal size were common and other blastomeres had fragmented nuclei or were anucleate. Seventeen per cent of normally fertilized embryos at two- to four-cell stage had at least one binucleate blastomere, and this increased to 65% at the nine- to 16-cell stage when individual embryos had between one and six binucleate blastomeres. The proportion of binucleate blastomeres in normally fertilized embryos increased from 5 to 10% over this period, whereas in abnormally fertilized, polyspermic or parthenogenetic, embryos the proportion was significantly higher during early cleavage stages but decreased at the nine- to 16-cell stage when the majority of these embryos arrest (25 and 6%, respectively). The incidence of anucleate blastomeres in normally fertilized embryos was also high, especially in those of poor morphology. In contrast, blastomeres with fragmented nuclei were relatively uncommon and the incidence was variable among classes and stages of development. Estimates of the volume of binucleate blastomeres based on measurement of their diameters and comparison with mononucleate blastomeres at various cleavage stages indicated that these blastomeres arise from a failure of cytokinesis between the second and fourth cleavage divisions. On this basis, assignment of binucleate blastomeres to particular cleavage stages in normally fertilized day 4 embryos suggests that at least some of these blastomeres arising during early cleavage persist without further cell division for up to 48 h. At the cellular level, therefore, blastomeres with either binucleate or abnormal nuclei contribute to cleavage stage arrest in vitro.

Blastomeres↗
PubMed1993

Effects of pyruvate and glucose on the development of human preimplantation embryos in vitro.

Although human embryos will develop in vitro for six days or more, little is known about the effects of the primary nutrients, pyruvate and glucose, on development. Because the nutrient requirements of embryos change throughout preimplantation development, the effects of altering substrate concentrations in the culture medium were examined, using 'surplus' human preimplantation embryos cultured from the two-four-cell stage to the blastocyst stage in medium containing various concentrations of pyruvate and glucose. Between the one-cell stage and the two-four-cell stage all of the embryos were exposed to 0.47 mmol pyruvate l-1 and 5.5 mmol glucose l-1. Pyruvate as sole substrate in the medium could support blastocyst development to an extent of 59% (10 of 17). Conversely, culture of embryos in pyruvate-free medium resulted in the developmental arrest of 84% (21 of 25) of embryos, and for the 16% (4 of 25) that did reach the blastocyst stage there was a significant decrease in metabolic activity on day 4-5, during the morula to blastocyst stage transition. Embryos could not use glucose to compensate for the lack of pyruvate in the medium. Pyruvate uptake was related to exogenous concentration and optimal development occurred at the highest concentration tested, 0.47 mmol l-1. Embryo development to the eight-cell stage was slightly enhanced 82% (14 of 17) versus 60% (24 of 40) when no glucose was added to the medium, and the resulting blastocysts had significantly more cells (99.1 +/- 13.5 versus 58.4 +/- 8.2; P < 0.02) than did embryos grown in the presence of 1 mmol glucose l-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Blastocyst↗
PubMed1993

Activity of enzymes of energy metabolism in single human preimplantation embryos.

A method was developed to measure the activities of enzymes in extracts from single human preimplantation embryos. The method permits the analysis of two enzymes plus appropriate controls in an extract from a single embryo, and was used to investigate the control of energy metabolism during the development of human embryos from the two-cell to the blastocyst stage. Hexokinase (HK), 6-phosphofructokinase (PFK), pyruvate kinase (PK), fructose-1,6-diphosphate aldolase (ALD), glucose phosphate isomerase (GPI), lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G6PDH) and 2-oxoglutarate dehydrogenase (ODH) were all detectable, whereas glycogen phosphorylase (GP) was not. The enzyme activities of ODH, PFK, LDH, PK, GPI and G6PDH, averaged over all stages of development from the two-cell to blastocyst stage (days 2-6 after insemination), were 3.5, 6.6, 15, 69, 73 and 87 times greater than HK, respectively. The activity of ALD was very similar to that of HK. The activities of ALD, GPI, PFK, PK and LDH showed no significant variation with stage of development, although the activity of GPI fell significantly from the four-eight cell to the eight-sixteen cell stage (P < 0.05). HK activity decreased from the two-eight cell to the eight-sixteen cell (P < 0.05), and increased significantly from the eight-sixteen cell to the blastocyst stage (P < 0.01). The overall relationship between hexokinase activity and stage approached significance (P = 0.059, one-way analysis of variance). The activity of G6PDH decreased significantly with development (P < 0.001, one way analysis of variance).(ABSTRACT TRUNCATED AT 250 WORDS)

Blastocyst↗
PubMed1992

Birth of a normal girl after in vitro fertilization and preimplantation diagnostic testing for cystic fibrosis.

BACKGROUND: Cystic fibrosis is a common, severe autosomal recessive disease caused in a majority of cases by a three-nucleotide deletion (delta F508) in the cystic fibrosis transmembrane regulator gene. Current methods of prenatal diagnosis involve chorionic-villus sampling or amniocentesis. In vitro fertilization and diagnosis during embryonic development before implantation would allow only unaffected embryos to be selected for transfer to the uterus, thereby avoiding the need to terminate a pregnancy. METHODS: Preimplantation diagnosis of cystic fibrosis was attempted in the cases of three couples, both members of which carried the delta F508 deletion. In vitro fertilization techniques were used to recover oocytes from each woman and fertilize them with her husband's sperm. Three days after insemination, embryos in the cleavage stage underwent biopsy and removal of one or two cells for DNA amplification and analysis. RESULTS: Only two oocytes from one woman were fertilized normally; DNA analysis of one of the embryos failed and cystic fibrosis was diagnosed in the other (i.e., it was homozygous for delta F508), so neither was transferred. The oocytes of each of the other two women produced noncarrier, carrier, and affected embryos. Both couples chose to have one noncarrier embryo and one carrier embryo transferred. One woman became pregnant and gave birth to a girl free of the deletion in both chromosomes. CONCLUSIONS: Preimplantation diagnosis of the delta F508 deletion causing cystic fibrosis is possible through in vitro fertilization, biopsy of a cleavage-stage embryo, and amplification of DNA from single embryonic cells. This approach should be equally applicable to other single-gene diseases in which the defect has been identified. Analysis of a series of pregnancies, however, will be required to assess the method adequately.

Base Sequence↗
PubMed1992

Dual fluorescent in situ hybridisation for simultaneous detection of X and Y chromosome-specific probes for the sexing of human preimplantation embryonic nuclei.

Dual fluorescent in situ hybridisation has been used for the simultaneous detection of X and Y chromosome-specific probes in single cleavage nuclei from disaggregated 4- to 7-cell human embryos. Based on the presence of a Y signal or 2 X signals in the absence of a Y, 89% of poor quality metaphases and 72% of interphase nuclei could be classified as male or female. With further refinements, this technique will offer a credible alternative to the polymerase chain reaction for the diagnosis of sex in human preimplantation embryos in families segregating for X-linked genetic disease.

Blastomeres↗
PubMed1992

Human embryo biopsy on the 2nd day after insemination for preimplantation diagnosis: removal of a quarter of embryo retards cleavage.

OBJECTIVE: To assess any reduction in viability and development in vitro after biopsy of a quarter of the cells of human embryos on day 2 after insemination. DESIGN: A prospective study in which normally fertilized surplus embryos of good morphology with two to eight cells approximately 48 hours after insemination were randomly allocated to a control or biopsied group, respectively. SETTING: In vitro fertilization (IVF) unit and laboratories of the Hammersmith Hospital, Institute of Obstetrics and Gynaecology, London University. PATIENTS, PARTICIPANTS: One hundred twenty-nine embryos from 28 infertile IVF patients. INTERVENTIONS: Follicular aspiration by ultrasound-guided transvaginal puncture and embryo biopsy by micromanipulative procedures. MAIN OUTCOME MEASURE(S): Pyruvate uptake and cell number at the blastocyst stage. RESULTS: Embryo biopsy did not have an adverse effect on either the proportion developing to the blastocyst stage (50% [32 of 64] and 47.7% [31 of 65] for the control and biopsied groups, respectively) or embryo viability, measured indirectly through pyruvate uptake. However, the proportion of embryos that reached the morula stage after day 4 (retarded embryos) was significantly higher (44%, 11 of 25 versus 8.7%, 2 of 23) in the biopsied group. The total number of cells (29.6 +/- 3.1 versus 62.4 +/- 4.7), numbers of inner cell mass (7.7 +/- 2.2 versus 24.5 +/- 1.4) and trophectoderm (24.0 +/- 5.2 versus 45.0 +/- 6.4) cells, and the inner cell mass:trophectoderm ratio (34.7 +/- 7.9 versus 59.5 +/- 11.7) were strikingly reduced at the blastocyst stage in the biopsied group. This reduction was greater in embryos that reached the morula stage after day 4. CONCLUSIONS: More investigation is needed to assess whether the detrimental effects observed were because of the biopsy method used in this study or to a high sensitivity of human embryos at early stages to manipulation in vitro.

Biopsy↗
PubMed1992

Energy metabolism of the human fallopian tube.

The consumption of oxygen (QO2), the production of lactate and the profile of four key metabolic enzymes were measured in small samples of human oviductal mucosa (endosalpinx) removed at surgery. The QO2 in the absence of substrate was 3.4 microliters O2 (mg dry wt)-1 h-1, a value typical of quiescent tissue. The QO2 was stimulated by glucose, but diminished by glutamine and acetoacetate. Tissue lactate production was low and not increased by glucose. Hexokinase had the highest activity of the enzymes measured, followed by 2-oxoglutarate dehydrogenase; 6-phosphofructokinase and glycogen phosphorylase had low activities. The data are consistent with the proposition that glucose is a major metabolic fuel for human endosalpinx.

Acetoacetates↗
PubMed1991

Programming in vitro fertilization for a 5- or 3-day week.

OBJECTIVE: To schedule oocyte retrievals on either 5 or 3 days per week in in vitro fertilization (IVF) cycles. DESIGN: Human chorionic gonadotropin (hCG) administration was delayed by at least 24 hours in patients undergoing superovulation to avoid egg collections on weekends (group 1). Encouraged by the results, a further prospective study in which oocyte retrievals were programmed for only 3 days a week was undertaken (group 2). SETTING: Hammersmith Hospital, a tertiary referral unit. PATIENTS: All patients undergoing IVF treatment were included. MAIN OUTCOME MEASURES: To schedule oocyte retrievals on either 5 or 3 days per week. RESULTS: Only 4.0% of egg collections (12/303) occurred on weekends compared with 12.6% (22/175) before delaying hCG. In group 2 (n = 215), only four egg collections (2.1%) had to be performed out of schedule. Delaying administration of hCG had no detrimental effects. CONCLUSIONS: Delayed administration of hCG allows scheduling oocyte retrievals on either 5 or 3 days per week, leading to a substantial decrease in out-of-hours oocyte retrievals, reducing cycle costs, and facilitating efficiency. The method is applicable to assisted reproduction specialized units as well as district general hospitals that use gonadotropin-releasing hormone analogs in their superovulation regimens before IVF, gamete intrafallopian transfer, or intrauterine insemination.

Chorionic Gonadotropin↗
PubMed1991

Resources for infertility treatment.

Infertility is common, a serious medical problem in both advanced and underdeveloped countries. At present, medical resources are often used very haphazardly and frequently extravagantly to combat fertility problems. Whilst it seems very unlikely that society will be able to prevent infertility effectively, there is no doubt that the resources available could be better organized for greater benefit. There is evidence of poor co-ordination of services, of inadequate financial planning by health managers, and squandering of limited resources by professionals. Better organization of in vitro fertilization (IVF), with the promotion of larger regional services, would be an effective use of finances. IVF is too often only available to the wealthy and the selection of patients for treatment is frequently arbitrary; many fertility treatments are of unproved value and are wasteful. It is argued, for example, that many patients receiving gamete intrafallopian transfer (GIFT) for so-called 'unexplained infertility' would be better treated by other, less expensive, methods which are often more effective. Many useful treatments, such as tubal surgery, are being disregarded or misused and there is need for better education of specialists who treat infertile patients. Better primary care of infertile patients should include non-medical counselling, and investigation and firm diagnosis before treatment is commenced. The heavy accent on high technology in the treatment of infertility is often misplaced, and we need to strike a careful balance if resources are to be properly allocated.

Adaptation, Psychological↗
PubMed1991

Fluorescent in-situ hybridization to interphase nuclei of human preimplantation embryos with X and Y chromosome specific probes.

Fluorescent in-situ hybridization (ISH) to interphase nuclei of human preimplantation embryos has been demonstrated with the X and Y chromosome-specific DNA probes, pBamX7 and pHY2.1, respectively. Assigning the sex on the basis of the number of hybridization signals in the majority of nuclei, the efficiencies with both probes to nuclei from male embryos were considerably higher than those previously reported for pHY2.1 detected by isotopic or conventional non-isotopic methods. Only approximately 15% of nuclei from male embryos failed to hybridize with these probes. With pBamX7, a high incidence (18%) of nuclei with two (or more) signals in embryos classified as males and four signals in a female embryo was observed. In some cases, the double spot nuclei were larger than those with single spots, providing evidence of tetraploidy. The feasibility of using fluorescent ISH for sexing biopsied embryos in couples at risk of X-linked disease and for the preimplantation diagnosis of chromosome abnormalities is discussed.

Blastocyst↗
PubMed1991

Beneficial effects of a 24 h delay in human chorionic gonadotrophin administration during in-vitro fertilization treatment cycles.

We used a gonadotrophin-releasing hormone agonist (buserelin) and human menopausal gonadotrophin (HMG) for superovulation for in-vitro fertilization (IVF) in 143 patients. The patients were prospectively allocated to two balanced groups. In one group (47 patients) human chorionic gonadotrophin (HCG) was given when the three largest follicles were greater than or equal to 17 mm in diameter, with consistent levels of plasma oestradiol (standard group). In the second group (96 patients), HCG injection was delayed by 24 h (delayed group). In the delayed group of patients, proportionately more had clinical pregnancies (52.1% versus 34.0%). These results suggest that IVF patients will benefit from delayed administration of HCG. The traditional criteria for HCG administration should be changed when buserelin is used.

Adult↗
PubMed1991

Attitudes towards gamete donation among couples undergoing in vitro fertilization.

The attitudes of 234 anonymous couples undergoing in vitro fertilization toward sperm and oocyte donation were explored by questionnaire. All the questionnaires were returned of which 222 (95%) were complete and analysed. A high proportion of couples found the use of donor sperm acceptable for therapeutic, diagnostic and treatment purposes (77%, 90% and 97% respectively) and 72%, 84% and 90% respectively were willing to donate oocytes for these purposes. Of potential oocyte donors 41% would agree to nonanonymous donation, 12% would wish to meet the recipient couple and although only 4% wanted to choose the recipient, a quarter of the couples would prefer a relative or friend as the recipient. Provision of nonidentifying information about the donor to the recipient couple was acceptable to almost 70% whereas 40% found giving the same information to the child acceptable.

Adult↗