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Biomedical subjects

R M Winston

Publications and source records attributed to R M Winston.

At least 73 records · Page 4Linked to original sources
PubMed1995

Normal development and metabolic activity of preimplantation embryos in vitro from patients with polycystic ovaries.

Polycystic ovary syndrome (PCOS) is closely associated with high miscarriage rates and, following in-vitro fertilization (IVF), with decreased fertilization rates, suggesting that oocytes and embryos are of poor quality. In this prospective study, we examined the development, metabolic activity and blastocyst cell number of embryos following IVF from 51 patients with either anovulatory PCOS, ovulatory PCOS or tubal disease. The number of oocytes retrieved and the fertilization rates were similar for patients with PCOS and tubal disease. Following embryo transfer, 46% of the patients with PCOS and 36% of patients with tubal disease became pregnant. A similar proportion of surplus embryos from patients with PCOS and tubal disease developed to the blastocyst stage (38% and 43% respectively). Patients with anovulatory PCOS had embryos with less fragmentation which cleaved faster, cavitated earlier and had more cells at the blastocyst stage than embryos from patients with tubal disease. While the profile of glucose uptake and lactate production was similar for all groups throughout preimplantation development, patients with tubal disease who underwent ovulation induction using the 'titrated' regimen optimized for PCOS patients resulted in embryos with reduced pyruvate uptake, in addition to low blastocyst cell numbers. This study demonstrates that with an optimized ovulation induction regimen, embryos from PCOS patients are of good quality and developmental potential.

Adult↗
PubMed1995

Endovaginal ultrasonography in the diagnosis of adenomyosis uteri: identifying the predictive characteristics.

OBJECTIVE: To evaluate prospectively the role of endovaginal ultrasonography in the diagnosis of adenomyosis and to identify predictive characteristics. SETTING: In Vitro Fertilisation Unit, Hammersmith Hospital. SUBJECTS: Fifty-six women with menorrhagia and dysmenorrhea. DESIGN: Endovaginal sonography was performed and uterine body morphometry and myometrial echogenicity were assessed. The sonographic suspicion of adenomyosis was scored high or low depending on the degree of uterine enlargement, uterine asymmetry not due to fibroids and heterogenicity of myometrial echoes. The sonographic diagnosis was compared either with the histological findings after hysterectomy (n = 34) or to the appearances on magnetic resonance imaging (n = 22). RESULTS: Adenomyosis was diagnosed in 28 patients: 15 by histology and 13 by magnetic resonance imaging. Endovaginal ultrasound demonstrated a sensitivity of 86%, a specificity of 50%, a positive predictive value of 86%, and a negative predictive value of 77%. Uterine morphometry alone did not predict adenomyosis: although the mean length of the longitudinal, anteroposterior and transverse axis was larger in uteri with, compared with those without, adenomyosis, this did not reach statistical significance. The uterine asymmetry ratio was 1.43 (SD 0.6) and 1.34 (SD 0.4) (P = 0.26) in uteri with and without adenomyosis, respectively, but in the presence of adenomyosis the mean posterior wall was significantly thicker than the mean anterior wall: 25.6 (SD 6.6) mm compared with 21.8 (SD 5.0) mm, P = 0.02. Therefore, adenomyosis was best predicted on the basis of ill-defined myometrial heterogeneity. However, leiomyomas and various echogenic shadows and artefacts often complicate subjective assessment of the myometrial echogenicity. CONCLUSION: Endovaginal sonography in symptomatic patients can be a sensitive but not a specific procedure for the diagnosis of adenomyosis.

Adult↗
PubMed1995

EGF, TGF-alpha and EGFR expression in human preimplantation embryos.

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) through their common receptor, epidermal growth factor receptor (EGFR) are known to enhance mitogenesis, development and implantation in several species. In the mouse, co-culture of grouped embryos in microdrops increases the cell number and proportion developing to the blastocyst stage. A similar effect is observed with culture of single embryos in medium supplemented with EGF or TGF-alpha highlighting their embryotrophic effects. To study the role of EGF, TGF-alpha and EGFR in early human development, two methods applicable for analysis of expression at the single embryo level have been employed. In the first method, reverse transcription-polymerase chain reaction has been used to examine the presence of transcripts. Following reverse transcription, strategically designed nested primers, optimised for specificity, were used for amplification from the cDNA equivalent of a single embryo. The products were then verified by restriction enzyme digestion and sequence analysis. In the second method, immunocytochemistry has been used to colocalise the expressed proteins. Individual embryos were paraffin embedded and serial sectioned, allowing adjacent sections to be examined with different antibodies and controls. Monoclonal TGF-alpha and polyclonal EGF and EGFR primary antibodies were used. Staining was performed by peroxidase-conjugated avidin-biotin immunocytochemistry with the appropriate controls. The combination of these two methods can potentially be used for simultaneous analysis of several growth factors and/or their receptors in the same human embryos. Transcripts for EGF, TGF-alpha and EGFR were detected in unfertilized oocytes and embryos between 8-cell and blastocyst stages on day 3 to 6 post-insemination.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1995

Ultrasonographic growth measurements in triplet pregnancies.

The incidence of triplet pregnancies is increasing due to the widespread use of ovulation induction agents and assisted conception treatments. The aim of this study was to acertain the normal ultrasonographic measurements for fetal growth parameters in triplet pregnancies. The ultrasonographic measurements of all triplet pregnancies managed in two major hospital centres were reviewed retrospectively and those in which there was less than 25% discordance in birth weight were included in the study. Triplet 50th centile for fetal biparietal diameter, whilst falling through the normal singleton centile range from 27 weeks gestation, did not fall below the 10th centile value of singletons. Triplet 50th centile for head circumference was equivalent to the singleton 10th centile from 23 weeks gestation. Triplet 50th centile for abdominal curcumference was persistently below that of the singleton 50th centile, falling below the singleton 10th centile from 29 weeks gestation. Normal growth rate of triplet gestations in the third trimester of pregnancy varies from that of singletons. An awareness of this altered growth rate is necessary to avoid the inappropriate diagnosis of asymmetrical triplet fetal growth retardation. We suggest that the growth curves presented here may be used to monitor triplet fetal growth.

Birth Weight↗
PubMed1995

Increased number of cells and metabolic activity in male human preimplantation embryos following in vitro fertilization.

The number of cells and metabolic activity of male and female human preimplantation embryos were examined to determine whether male embryos are more advanced than female embryos following in vitro fertilization (IVF). The metabolic activity of embryos fertilized normally was assessed daily by non-invasive measurement of pyruvate and glucose uptake and lactate production between days 2 and 6 after insemination. On day 6, the numbers of nuclei from the trophectoderm and inner cell mass of blastocysts were counted by differential labelling and fluorescence microscopy. Nuclei were then recovered and the sex of the embryos identified using nested primers to amplify the amelogenin gene and pseudogene sequences on the X and Y chromosomes, respectively. Development of male and female embryos were then compared retrospectively. From 69 of 178 (39%) embryos that developed to the blastocyst stage, the sex of 57 was determined; 21 (37%) were male and 36 (63%) female. The number of cells in male embryos was significantly greater on day 2 (P < 0.005), and this difference was maintained up to the blastocyst stage (in both the trophectoderm and the inner cell mass), although differences were not always significant. Pyruvate uptake was significantly higher by male embryos between days 2 and 5 (P < 0.05). Glucose uptake and lactate production were significantly higher in male embryos on days 4-5 (P < 0.05); this difference was not significant on days 5-6. Extrapolation from differences in the number of cells indicates that female embryos are approximately 4.5 h delayed in their development from day 2 onwards compared with male embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

Blastocyst↗
PubMed1994

Transcription of paternal Y-linked genes in the human zygote as early as the pronucleate stage.

Global activation of the embryonic genome occurs at the 4- to 8-cell stage in human embryos and is marked by continuation of early cleavage divisions in the presence of transcriptional inhibitors. Here we demonstrate, using reverse transcriptase-polymerase chain reaction (RT-PCR), the presence of transcripts for two paternal Y chromosomal genes, ZFY and SRY in human preimplantation embryos. ZFY transcripts were detected as early as the pronucleate stage, 20-24 h post-insemination in vitro and at intermediate stages up to the blastocyst stage. SRY transcripts were also detected at 2-cell to blastocyst stages. The expression of SRY and ZFY at these early stages and the faster cleavage rate of male embryos observed in many mammalian species focuses attention on the role of events in sex determination prior to gonad differentiation.

Base Sequence↗
PubMed1994

Identification of the sex of human preimplantation embryos in two hours using an improved spreading method and fluorescent in-situ hybridization (FISH) using directly labelled probes.

Dual fluorescent in-situ hybridization (FISH) using X and Y chromosome specific probes has been used to identify the sex of human embryos for preimplantation diagnosis of X-linked disease. With a modified spreading method and directly labelled fluorescent DNA probes, we have examined the possibility of reducing the time of the FISH procedure from 7 to 2 h. A total of 17 normally fertilized human embryos were disaggregated and 98 intact blastomeres obtained. The spreading efficiency was 96% and FISH signals were obtained from 97% of nuclei. In all cases, sibling blastomeres from the same embryo were the same sex. Mosaicism was observed in some embryos. Five cells which lysed during the disaggregation process were spread to determine whether FISH was possible in these cells, but in all cases the morphology of the nuclei was poor and multiple signals were observed so that reliable diagnosis of sex was not possible. The data reported here confirm that by using an improved spreading method in combination with directly labelled DNA probes, we have increased the efficiency and reduced the time required for sexing embryos for preimplantation diagnosis of X-linked disease.

Blastomeres↗
PubMed1994

Detection of fertilization in embryos with accelerated cleavage by fluorescent in-situ hybridization (FISH).

Embryos from a couple undergoing routine in-vitro fertilization for unexplained infertility had shown cleavage by day 1 in two consecutive cycles. The response of the woman to fertility drugs had been normal, and there were no known sperm abnormalities. In a subsequent cycle, accelerated cleavage occurred again and we used a modified method of spreading whole embryos and dual fluorescent in-situ hybridization (FISH) with directly labelled probes for chromosomes X and Y to determine if fertilization had occurred in these embryos. Nine oocytes were collected, five of which had cleaved when examined for pronuclei early on day 1 following late insemination. Pronuclei were observed in one of the remaining oocytes, but in this case, three were present. On day 2, all of the oocytes/embryos had cleaved and two were transferred to the patient. The remaining seven were spread for FISH analysis but nuclei were only obtained from five. In three, a Y signal was detected, indicating that fertilization had occurred. In all five embryos, a wide range of X chromosome signals were observed. These data suggest that the embryos had undergone abnormal fertilization and accelerated cleavage.

Adult↗
PubMed1994

Effects of oestrogen on progesterone synthesis and arachidonic acid metabolism in human luteal cells.

OBJECTIVE: Locally produced oestrogens and prostaglandins (PGs) are implicated in the regulation of luteal lifespan in the human ovary. This study (1) assesses direct effects of these factors on progesterone synthesis in isolated luteal cells, and (2) explores interactions between luteal age and treatment with gonadotrophin or oestrogen on the metabolism of arachidonic acid (prostaglandin precursor) by steroidogenic luteal cells in vitro. DESIGN: Primary monolayer cultures of human luteal cells obtained at different stages of the luteal phase were used to investigate the effect of oestradiol, catechol oestrogens (2- and 4-hydroxyoestradiol), diethylstilboestrol, PGE2 and PGF2 alpha on basal and human chorionic gonadotrophin (hCG) stimulated progesterone production in vitro. The role of PGs as modulators of luteal cell function was further investigated by studying the metabolic fate of radioactively labelled arachidonic acid in hormone treated (oestradiol and hCG) and control cultures, assessed by high performance liquid chromatography. PATIENTS: Corpora lutea were enucleated from nine women with regular ovulatory cycles undergoing microsurgical reversal of tubal sterilization. Granulosa cell aspirates were obtained from three patients undergoing in-vitro fertilization treatment. RESULTS: PGE2 and PGF2 alpha at various concentrations did not have a consistent effect, whereas oestradiol, diethylstilboestrol (and 2-hydroxyoestradiol in early luteal cell cultures) significantly inhibited basal and hCG stimulated progesterone biosynthesis. Evidence for direct inhibition of 3 beta-hydroxysteroid dehydrogenase enzymic activity by oestradiol was obtained. Both major metabolic pathways of arachidonic acid (lipoxygenase and cyclo-oxygenase) were operative in steroidogenic luteal cells recovered throughout the luteal phase. The ratio of PGE2 to PGF2 alpha synthesis in vitro by human luteal cells from endogenously incorporated arachidonic acid did not change significantly with corpus luteum age, with PGE2 tending to predominate. Oestradiol treatment shifted arachidonic acid metabolism from the lipoxygenase towards the cyclooxygenase pathway in cells isolated from ageing corpora lutea. CONCLUSIONS: Oestradiol, at relatively high concentrations, is a potent inhibitor of basal and hCG induced luteal cell steroidogenesis in vitro. No support is provided for the concept that luteolysis is mediated by local production of PGF2 alpha. The putative luteolytic effect of oestradiol may entail reduced metabolism of arachidonic acid to lipoxygenase derived products by luteal cells rather than direct stimulation of prostaglandin production by itself.

Arachidonic Acids↗
PubMed1994

Estradiol production by granulosa cells of normal and polycystic ovaries: relationship to menstrual cycle history and concentrations of gonadotropins and sex steroids in follicular fluid.

The underlying cause of anovulation in polycystic ovary syndrome is unknown. Circulating levels of immuno- and bioactive FSH are within the normal range, and the follicles contain measurable levels of bioactive FSH. The aim of this study was to compare estradiol (E2) production in response to FSH by granulosa cells from normal ovaries with those from polycystic ovaries derived from both anovulatory (anovPCO) and ovulatory subjects (ovPCO). Intrafollicular levels of immunoactive FSH, E2, and androstenedione in follicles of less than 12 mm were also measured. Follicular fluid steroid concentrations were obtained from 41 pairs of normal ovaries and 23 pairs of polycystic ovaries (8 anovPCO and 15 ovPCO). In size-matched follicles from each group there were no significant differences in follicular fluid FSH or E2 concentrations, but androstenedione levels were significantly higher in 5- to 11-mm follicles from ovPCO than in corresponding follicles from normal ovaries. Dose responses to FSH were determined in granulosa cells derived from 9 pairs of normal ovaries, 7 anovPCO, and 8 ovPCO. Cells from anovPCO produced 6- to 10-fold more E2 in response to FSH than normal cells, although there was no significant difference in the ED50 values. The response in cells from ovPCO was reduced compared to normal, but this difference did not reach significance. In summary, as judged by their FSH and E2 contents, polycystic ovaries do not have a higher proportion of atretic follicles than normal. Indeed, cells from anovPCO are hyperesponsive to FSH in vitro. This could be explained by stimulation of aromatase in vivo by either paracrine or, more probably, by endocrine factors, of which insulin is an arguable candidate.

Adult↗
PubMed1993

New challenges in human in vitro fertilization.

This review assesses some scientific and ethical problems with human in vitro fertilization. Improved selection of viable embryos, better culture conditions, and greater understanding of the uterine environment will increase success and prevent multiple pregnancy. Further advances will also improve oocyte cryopreservation, in vitro maturation of oocytes, knowledge of sperm function, and sperm microinjection. Preimplantation diagnosis will help avoid genetic diseases and increase understanding of embryonic defects and the viability of zygotes. The greatest ethical problem with all these developments seems to be delivery of these complex treatments when health-care resources are increasingly limited.

Cryopreservation↗
PubMed1993

Selection criteria for human embryo transfer: a comparison of pyruvate uptake and morphology.

PURPOSE: Pyruvate uptake is higher in human embryos developing to the blastocyst stage than those arresting at cleavage stages. To investigate whether pyruvate uptake provides an improved criterion for selecting embryos for transfer, we have measured uptakes by individual embryos noninvasively over 24-hr periods between the first day (day 1) postinsemination and embryo transfer on day 2 to 3 and correlated the levels with implantation and pregnancy outcome. RESULTS: The mean uptake was significantly lower for embryos that implanted than for those which failed to implant: 22.9 +/- 1.0 and 27.1 +/- 0.6 pmol/embryo/hr, respectively on day 2, and 22.4 +/- 1.5 and 26.9 +/- 0.8 pmol/embryo/hr, respectively, on day 3, but the wide range of uptakes by individual embryos was overlapping. CONCLUSION: We conclude that pyruvate uptake as the sole criterion for embryo selection cannot predict which embryos will implant after transfer. Assessment of embryos using morphological and developmental criteria, therefore, remains the most consistent, though inefficient, indicator of pregnancy potential.

Adult↗
PubMed1993

Detection of aneuploidy and chromosomal mosaicism in human embryos during preimplantation sex determination by fluorescent in situ hybridisation, (FISH).

Five couples at risk of producing offspring with X-linked recessive disease underwent in vitro fertilisation with a view to preimplantation determination of embryo sex and selective transfer of females. On day three postinsemination, one or two blastomeres were removed by embryo biopsy, and used for dual fluorescent in situ hybridisation with X and Y chromosome-specific DNA probes. In two cases, two female embryos were transferred and one pregnancy, (sex confirmed), is ongoing at 19 weeks. All eight embryos from one couple were of such poor quality that diagnosis was possible in one only. In the remaining two cases no embryos were transferred due to the detection of an abnormal number of X chromosome signals. Investigation of the biopsied embryos that were not transferred revealed evidence of mitotic non-disjunction in one and of complete X monosomy in a second. A surviving fetus with this latter constitution would have developed Turner syndrome and would also have been at high risk of X-linked disease. The use of fluorescent in situ hybridisation rather than the polymerase chain reaction allowed the detection of abnormal copy numbers of X chromosomes thus preventing the transfer of potentially abnormal zygotes.

Adult↗
PubMed1993

Variability of human sperm response to immediate and prolonged exposure to pentoxifylline.

Pentoxifylline improves some motility characteristics of human spermatozoa, but the variability of response to this drug has not been clearly defined. We used computer-assisted sperm motion analysis to examine the in-vitro response of spermatozoa to pentoxifylline. Individuals (n = 31) with normal sperm counts were randomly selected and their spermatozoa exposed to different concentrations of pentoxifylline. Further tests on a subgroup of individuals examined the longevity of spermatozoa in response to this agent. Straight line velocity (VSL) was only improved at 0.1 mM and the major effect of the drug was on curvilinear velocity (VCL) and lateral head displacement (ALH). Prolonged exposure to pentoxifylline enhanced sperm motion only at 0.1 mM. Higher concentrations produced dose-dependent detrimental effects on all the motion characteristics. There was considerable inter-individual variability in both VCL and ALH response ranging from little or no detectable response to a 40% increase above control value. The maximum response was most commonly seen at a concentration of 2 mM pentoxifylline.

Humans↗