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Biomedical subjects

R M Winston

Publications and source records attributed to R M Winston.

At least 109 records · Page 6Linked to original sources
PubMed1991

Microsurgical salpingostomy is not an obsolete procedure.

OBJECTIVE: A review of the results of microsurgery for bilateral distal tubal blockage. DESIGN: A retrospective review. SETTING: Hammersmith Hospital London and local private hospitals. SUBJECTS: 388 patients with bilateral ampullary occlusion treated between 1971 and 1988 by microsurgery. INTERVENTIONS: Full investigation for other causes of infertility followed by abdominal microsurgical salpingostomy. Repeated meticulous follow-up was essential with check laparoscopy one year after surgery. MAIN OUTCOME MEASURE: Successful pregnancy in relation to tubal damage. RESULTS: In 65 women microsurgery followed tubal reocclusion after failed conventionally performed salpingostomy. 74 women (23%) had one term pregnancy after primary salpingostomy and 12 women (18%) after repeat salpingostomy. Over half the women having a term pregnancy subsequently had a second infant. The tubal damage was classified in four stages according to the degree of mucosal damage and tubal fibrosis, the presence of isthmic disease and the quality of tubal and ovarian adhesions. Approximately one quarter of patients had stage I disease and amongst these 39% had babies after primary salpingostomy and 25% after repeat salpingostomy. CONCLUSION: Microsurgical salpingostomy is a specialized procedure. Proper selection of patients, competent microsurgical technique and adequate follow-up appear crucial to success. In selected patients treatment by salpingostomy gives better results than multiple cycles of in vitro fertilization.

Adult↗
PubMed1991

Profiles of hypoxanthine guanine phosphoribosyl transferase and adenine phosphoribosyl transferase activities measured in single preimplantation human embryos by high-performance liquid chromatography.

The profiles of hypoxanthine guanine phosphoribosyl transferase (HGPRT) and adenine phosphoribosyl transferase (APRT) activities were examined in normally fertilized human embryos developing at the normal rate in vitro between the 2-4-cell stage on Day 2 and the blastocyst stage on Day 6 after insemination. The activities of both enzymes were assayed simultaneously in extracts of single embryos by measuring the rate of production of the reaction products, inosine monophosphate (IMP) and adenine monophosphate (AMP), separated by high-performance liquid chromatography (HPLC). The activity profiles of the two enzymes over this period showed marked differences. The activity of HGPRT, coded by the X chromosome, increased between Days 2 and 4 (P less than 0.01) but declined sharply by Day 6 (P less than 0.001), whereas autosome-coded APRT activity remained low between Days 2 and 5, but increased on Day 6 (P less than 0.05). The profile of HGPRT activity may reflect a combination of decreasing levels of maternal enzyme inherited from the oocyte and the initiation of embryonic gene expression followed by X inactivation at the blastocyst stage on Day 6.

Adenine Phosphoribosyltransferase↗
PubMed1990

Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification.

Over 200 recessive X chromosome-linked diseases, typically affecting only hemizygous males, have been identified. In many of these, prenatal diagnosis is possible by chorion villus sampling (CVS) or amniocentesis, followed by cytogenetic, biochemical or molecular analysis of the cells recovered from the conceptus. In others, the only alternative is to determine the sex of the fetus. If the fetus is affected by the defect or is male, abortion can be offered. Diagnosis of genetic defects in preimplantation embryos would allow those unaffected to be identified and transferred to the uterus. Here we report the first established pregnancies using this procedure, in two couples known to be at risk of transmitting adrenoleukodystrophy and X-linked mental retardation. Two female embryos were transferred after in vitro fertilization (IVF), biopsy of a single cell at the six- to eight-cell stage, and sexing by DNA amplification of a Y chromosome-specific repeat sequence. Both women are confirmed as carrying normal female twins.

Base Sequence↗
PubMed1990

Non-invasive measurement of pyruvate and glucose uptake and lactate production by single human preimplantation embryos.

The consumption of pyruvate and glucose and the production of lactate by 40 single human preimplantation embryos has been measured using a non-invasive technique. Twelve of the embryos showed abnormal fertilization. Of the 28 normally fertilized embryos, nine (32%) developed to the blastocyst stage in culture while the remainder degenerated or arrested during cleavage. In the normal embryos, pyruvate uptake exceeded that of glucose in the early developmental stages (days 2-5 post-insemination) before glucose became the predominant substrate in the blastocyst (day 6). Considerable quantities of lactate were formed throughout development, rising from a value of 43.6 pmol/embryo/h on day 2.5 to 95.4 pmol/embryo/h on day 5.5. The values of pyruvate and glucose uptake and lactate production of those embryos which arrested were below those which developed normally. On the basis that one mole of glucose can give rise to two moles of lactate, only 50% of the lactate produced could be accounted for in terms of glucose uptake from the medium. This figure rose to 90% in the blastocyst. The remaining lactate must be derived from endogenous sources, most probably glycogen. It is proposed that the high production of lactate by human preimplantation embryos in vitro is an adaptation to the conditions of culture.

Blastocyst↗
PubMed1990

Human preimplantation development in vitro is not adversely affected by biopsy at the 8-cell stage.

Normally fertilized human embryos biopsied 3 days after in-vitro fertilization (IVF) have been examined for effects on viability and development in vitro after removal of one or two cells at the 8-cell stage (1/8 and 2/8) from each embryo. A high proportion of 7/8 and 6/8 biopsied and unmanipulated embryos developed to the blastocyst stage between days 5 and 6 (79, 71 and 59%, respectively), and many biopsied embryos (56%) hatched from the zona pellucida in vitro. The viability of biopsied embryos which developed to the blastocyst stage was assessed by daily non-invasive measurement of the uptake of two energy substrates, glucose and pyruvate. Uptake of both substrates was generally lower in 7/8 and 6/8 biopsied embryos but only in proportion to the reduced cellular mass. The total cell number and the numbers of both trophectoderm (TE) and inner cell mass (ICM) cells in biopsied embryos at the blastocyst stage, counted by differential labelling of their nuclei, were also reduced in proportion but the ratio of ICM to TE cells was maintained in both 7/8 and 6/8 biopsied embryos. We conclude that removal of one or two cells at the 8-cell stage, while reducing the cellular mass, does not adversely affect the preimplantation/development of biopsied embryos in vitro and suggest that this approach could be used for preimplantation diagnosis of genetic defects.

Biopsy↗
PubMed1990

Inhibition of oestradiol production by epidermal growth factor in human granulosa cells of normal and polycystic ovaries.

Anovulation in women with polycystic ovary syndrome results from a disorder of FSH-mediated follicular maturation which may involve paracrine modulation of FSH action by intra-ovarian factors. Epidermal growth factor (EGF) is a potent inhibitor of FSH-stimulated oestradiol production in the rat and has also been shown to inhibit aromatase activity in human granulosa cells obtained after superovulation. The purpose of this study was to investigate the action of EGF on granulosa cell function in human ovaries which had not been previously exposed to treatment with exogenous gonadotrophins and to compare the responses in tissue obtained from normal and from polycystic ovaries. Granulosa cells were obtained from antral follicles less than 10 mm in diameter after dissection of unstimulated normal or polycystic ovaries (PCO). Cells were pooled, washed, plated and incubated for 48h in the presence of 10(-7) M testosterone and various doses of human FSH. FSH dose responses were obtained with or without the addition of purified EGF (50 pg/ml). Testosterone in the absence of FSH resulted in a fourfold (range 2-7.5) increase in oestradiol accumulation in the medium after incubation of granulosa cells from both normal and polycystic ovaries. This increase was reversed by addition of EGF. FSH treatment stimulated a dose-related increase in oestradiol regardless of the origin of the granulosa cells. The peak E2 response to FSH, obtained at a dose of 1-2.5 ng/ml was a 20-fold increase above testosterone alone (range 4-55) in cells from PCO compared to sixfold (2.5-13) in cells from normal ovaries.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1989

Embryo biopsy.

Explore the source record for details and available documents.

Biopsy↗
PubMed1989

Biopsy of human preimplantation embryos and sexing by DNA amplification.

A single cell was removed, through a hole made in the zona pellucida, from each of 30 human embryos at the 6-10 cell cleavage stage three days after in-vitro fertilisation. A normal proportion of the embryos (37%) developed to the blastocyst stage by day six in culture and 6 hatched from the zona. Each male embryo was sexed from the DNA by amplification of a repeated sequence specific for the Y chromosome. In 15 embryos with the normal two pronuclei the sex was determined also by in-situ hybridisation with a Y specific probe or fluorescent chromosome staining to detect metaphase Y chromosomes; the results of Y specific amplification were confirmed. This approach may be valuable for couples at risk of transmitting X-linked disease.

Biopsy↗
PubMed1989

Rapid sexing of human embryos by non-radioactive in situ hybridization: potential for preimplantation diagnosis of X-linked disorders.

Sixty spare human embryos at various stages of preimplantation development were prepared for cytogenetic analysis. Fluorescent staining of those with metaphases allowed scoring for the presence of a Y chromosome. In situ hybridization was then performed using a biotinylated Y-specific sequence, and the probe was detected by a standard streptavidin-linked alkaline phosphatase system. This enabled comparison of the chromosomal sex with that obtained after in situ hybridization in 28 embryos, and the sexing result obtained by the two methods was concordant in all cases. A further 21 embryos in which no metaphase chromosomes were obtained were sexed by biotinylated in situ hybridization only. Overall, 66 per cent of male interphase nuclei demonstrated a Y-specific hybridization signal. Results were obtained in under 24 h, which may permit the sexing of an embryo biopsied during cleavage and the transfer of sexed embryos at the blastocyst stage to the mother's uterus in the same cycle as oocytes are collected for in vitro fertilization.

Blastocyst↗
PubMed1989

Non-invasive measurement of glucose and pyruvate uptake by individual human oocytes and preimplantation embryos.

Pyruvate and glucose uptake by 73 individual human oocytes and preimplantation embryos was measured non-invasively, using an ultramicrofluorescence assay to analyse changes in substrate levels in microdroplets of culture medium. The uptake of both substrates was measured over successive daily incubations between days 1 (unfertilized oocytes) or 2 ('spare' embryos which were not transferred) and day 6 (day 0 = day of insemination). Under these conditions, 58% (25/43) of fertilized embryos with two pronuclei on day 1 developed to the blastocyst stage by day 6. The pyruvate uptake of these embryos increased from approximately 28 to a maximum of 40 pmol/embryo/h between days 2.5 and 4.5. Similarly, glucose uptake increased from approximately 8 to 14 pmol/embryo/h between days 2.5 and 4.5, but then increased further to 24 pmol/embryo/h on day 5 at the blastocyst stage. [corrected] The pyruvate uptake of fertilized embryos which arrested at cleavage stages was significantly lower than for those which developed to the blastocyst stage. Polyspermic and parthenogenetic embryos, and unfertilized oocytes also had lower pyruvate uptakes at later stages. The glucose uptake of unfertilized oocytes and abnormal embryos never reached the level of fertilized embryos at the blastocyst stage on day 5.5. Non-invasive measurement of pyruvate uptake before embryo transfer may provide a valuable functional criterion for the selection of viable embryos capable of developing to the blastocyst stage.

Blastocyst↗
PubMed1989

The human blastocyst: cell number, death and allocation during late preimplantation development in vitro.

The development of 181 surplus human embryos, including both normally and abnormally fertilized, was observed from day 2 to day 5, 6 or 7 in vitro. 63/149 (42%) normally fertilized embryos reached the blastocyst stage on day 5 or 6. Total, trophectoderm (TE) and inner cell mass (ICM) cell numbers were analyzed by differential labelling of the nuclei with polynucleotide-specific fluorochromes. The TE nuclei were labelled with one fluorochrome during immunosurgical lysis, before fixing the embryo and labelling both sets of nuclei with a second fluorochrome (Handyside and Hunter, 1984, 1986). Newly expanded normally fertilized blastocysts on day 5 had a total of 58.3 +/- 8.1 cells, which increased to 84.4 +/- 5.7 and 125.5 +/- 19 on days 6 and 7, respectively. The numbers of TE cells were similar on days 5 and 6 (37.9 +/- 6.0 and 40.3 +/- 5.0, respectively) and then doubled on day 7 (80.6 +/- 15.2). In contrast, ICM cell numbers doubled between days 5 and 6 (20.4 +/- 4.0 and 41.9 +/- 5.0, respectively) and remained virtually unchanged on day 7 (45.6 +/- 10.2). There was widespread cell death in both the TE and ICM as evidenced by fragmenting nuclei, which increased substantially by day 7. These results are compared with the numbers of cells in morphologically abnormal blastocysts and blastocysts derived from abnormally fertilized embryos. The nuclei of arrested embryos were also examined. The number of TE and ICM cells allocated in normally fertilized blastocysts appears to be similar to the numbers allocated in the mouse. Unlike the mouse, however, the proportion of ICM cells remains higher, despite cell death in both lineages.

Blastocyst↗
PubMed1989

Cellular basis of luteal steroidogenesis in the human ovary.

A primary monolayer cell culture system was developed to investigate human corpus luteum (CL) function in vitro. Steroidogenic cells were isolated by collagenase dispersal and Percoll density-gradient fractionation from CLs enucleated at progressive stages of the luteal phase (tubal surgery patients). 'Pure' granulosa-lutein cells were aspirated from ovulatory follicles at mid-cycle (in-vitro fertilization patients). The steroidogenic capacity (progesterone/20 alpha-dihydroprogesterone biosynthesis and aromatase activity) of isolated luteal cells was assessed in relation to CL development. Basal luteal cell steroidogenesis was maximal at around the expected time of ovulation and declined with CL age during the luteal phase. Conversely, human chorionic gonadotrophin (hCG)-responsive steroidogenesis was initially undetectable but developed as the luteal phase progressed. These results show that luteal cell steroidogenesis becomes increasingly dependent upon gonadotrophic support with CL age. This is evidence that functional luteolysis in human ovaries (1) is pre-programmed to occur at the cellular level, (2) is initiated automatically at the time of ovulation and (3) is reversed at the time of CL 'rescue' in early pregnancy by the direct action of trophoblastic hCG on steroidogenic luteal cells. The culture system described should be of value in further defining the control of human CL form and function at the cellular level.

20-alpha-Dihydroprogesterone↗
PubMed1988

Improvement of in vitro fertilisation after treatment with buserelin, an agonist of luteinising hormone releasing hormone.

Treatment with buserelin, an agonist of luteinising hormone releasing hormone, and human menopausal gonadotrophin was compared with the conventional treatment of clomiphene citrate and human menopausal gonadotrophin in the outcome of in vitro fertilisation. Seventy seven infertile women had 83 cycles of treatment with buserelin and human menopausal gonadotrophin, and concurrently another 328 infertile women were treated with clomiphene citrate and human menopausal gonadotrophin. Seven (8%) cycles were cancelled owing to inadequate super-ovulation or ovarian hyperstimulation in the women receiving buserelin and 103 (31%) were cancelled because of poor follicular development in those receiving clomiphene citrate. The mean number of oocytes recovered was significantly higher with buserelin (9.5 (SD 4.5) v 5.5 (2.2)) as was the mean number of embryos obtained (4.3 (2.4) v 2.9 (1.7)). Significantly more women who had an embryo transfer became clinically pregnant after treatment with buserelin (53% (30/57) v 30% (48/159), or 36% v 14% of treatment cycles). Altogether 33% (10) of pregnancies in women treated with buserelin were multiple compared with 23% (11) in those treated conventionally. Of the 17 completed pregnancies in women treated with buserelin, 11 resulted in the birth of live babies (eight singletons, two sets of twins, and one set of triplets) and six failed, five before 12 weeks' gestation and one at 22 weeks. The 13 continuing pregnancies (32 weeks) were eight singletons, two sets of twins, and three sets of triplets. Of the 48 completed pregnancies in women treated with clomiphene citrate, 35 resulted in the birth of live babies (26 singletons, five sets of twins and four sets of triplets) and 13 failed, eleven before 12 weeks' gestation and two by 27 weeks. Buserelin increased the chance of pregnancy after in vitro fertilisation compared with conventional treatment, but the risk of multiple pregnancy may be increased.

Buserelin↗