PubMed Health⌕ Search

Biomedical subjects

R M Winston

Publications and source records attributed to R M Winston.

At least 19 recordsLinked to original sources
PubMed2002

Preimplantation genetic diagnosis: patients' experiences and attitudes.

BACKGROUND: This study aims to report the experiences and attitudes of patients who have undergone preimplantation genetic diagnosis (PGD). The extent to which this technique is acceptable to the individuals for whom it is intended is relatively unexplored, and remains a crucial issue that may ultimately determine the value of PGD as an alternative to prenatal diagnosis in high-risk couples. METHODS: An information sheet and questionnaire was distributed to 67 couples who had been treated at the Hammersmith Hospital, London and the Dexeus Institute, Barcelona. RESULTS: One-third of patients had an affected child, over half had previous experience of conventional prenatal diagnosis and over one-third had had terminations of pregnancy because of a genetic risk. Patients perceive the main advantage of PGD to be that only unaffected embryos are transferred to the uterus and thus therapeutic termination of pregnancy can be avoided; the main disadvantage is the low success rate. A total of 41% of patients found the treatment cycle extremely stressful, and, of the 20 patients who had experienced both prenatal diagnosis and PGD, 40% of patients found PGD less stressful, although 35% experienced more stress. Of those couples who contemplated a further pregnancy 76% would choose PGD, 16% would opt for prenatal diagnosis, and 8% no tests at all. CONCLUSIONS: The experience of prenatal diagnosis and termination of pregnancy can be an unwelcome memory and this leads to a demand for an alternative approach. Our data suggest that PGD is acceptable to patients and is a valuable alternative to prenatal diagnosis.

Abortion, Induced↗
PubMed2001

MUC 1: a genetic susceptibility to infertility?

In man and some animals regulation of embryo implantation by endometrial expression of the highly polymorphic MUC 1 mucin has been suggested. We assessed the polymorphism of MUC 1 in women known to be fertile and those with infertility due to suspected failure of embryo implantation. The median of the lower allele size in the infertile group was only 2.5 kb compared with 3.4 kb in the fertile group (p=0.0029, difference 0.9, [95% CI 0.1-1.3]). Women with unexplained infertility might have a genetic susceptibility to failure of embryo implantation due to small MUC 1 allele size.

Adult↗
PubMed2001

From cell death to embryo arrest: mathematical models of human preimplantation embryo development.

Human preimplantation embryos exhibit high levels of apoptotic cells and high rates of developmental arrest during the first week in vitro. The relation between the two is unclear and difficult to determine by conventional experimental approaches, partly because of limited numbers of embryos. We apply a mixture of experiment and mathematical modeling to show that observed levels of cell death can be reconciled with the high levels of embryo arrest seen in the human only if the developmental competence of embryos is already established at the zygote stage, and environmental factors merely modulate this. This suggests that research on improving in vitro fertilization success rates should move from its current concentration on optimizing culture media to focus more on the generation of a healthy zygote and on understanding the mechanisms that cause chromosomal and other abnormalities during early cleavage stages.

Apoptosis↗
PubMed2001

Follicular growth in fresh and cryopreserved human ovarian cortical grafts transplanted to immunodeficient mice.

OBJECTIVE: To investigate follicle growth in fresh and cryopreserved human ovarian cortical grafts transplanted to immunodeficient mice. STUDY DESIGN: Fresh or frozen-thawed human ovarian cortex was grafted subcutaneously or under the kidney capsule of 43 mice (35 nude mice and eight SCID mice), 14 of which were non-stimulated controls, 21 injected intra-peritoneally with gonadotrophins during 2 weeks and eight injected during 3 months. Follicle count was compared by Chi-square. RESULTS: Proportions of primordial follicles were significantly lower in grafts than in the tissue before transplantation in gonadotrophin-stimulated mice (37% versus 79%), but not in non-stimulated mice (51% versus 74%). Proportions of primary and secondary follicles were increased after transplantation indicating early follicular growth. One antral follicle was observed in a graft in a mouse stimulated for 3 months. CONCLUSION: Primordial follicles in fresh or frozen-thawed human ovarian cortex transplanted under the kidney capsule or subcutaneously can grow and are responsive to hormonal stimulation. CONDENSATION: Primordial follicles in fresh and cryopreserved human ovarian cortical grafts can initiate growth after transplantation to immunodeficient mice

Adult↗
PubMed2001

Quantitative measurement of transcript levels throughout human preimplantation development: analysis of hypoxanthine phosphoribosyl transferase.

We have developed a competitive reverse transcription-polymerase chain reaction (RT-PCR) sensitive enough to detect and quantify as little as 2-fold differences in gene expression in individual oocytes and embryos throughout human preimplantation development. This RT-PCR assay can be tailored for the examination of any specific gene and so will give a unique insight into human preimplantation development. This technique was used to quantify the level of hypoxanthine phosphoribosyl transferase (HPRT) expression during preimplantation development and to correlate this with embryo sex. The amount of HPRT transcripts present in the unfertilized oocyte was equivalent to 7.7 fg of competitor cDNA. At the 4-cell stage there is a significant drop (P: = 0.0006) to approximately 1.2 fg. There was no detectable difference in the HPRT levels between female and male embryos following 2 days of in-vitro culture. In contrast HPRT gene expression was higher in day 3 female embryos than in males. This is the first study to quantify gene transcripts throughout each stage of human preimplantation development and it indicates that the accumulated HPRT transcripts present in the unfertilized human oocyte undergo extensive destruction following fertilization. This work also suggests that X-inactivation occurs beyond the 8-cell stage of human preimplantation development.

Base Sequence↗
PubMed2000

The significance of delayed suppression using buserelin acetate and recombinant follicle-stimulating hormone in a long protocol in vitro fertilization program.

OBJECTIVE: To determine whether the time taken to achieve ovarian suppression has an impact on ovarian responsiveness and the outcome of IVF-ET. DESIGN: Retrospective analysis. SETTING: An assisted reproduction unit at a university center. PATIENT(S): Patients undergoing a long protocol of IVF-ET that included buserelin acetate therapy initiated on day 2 of the cycle and recombinant FSH. INTERVENTION(S): Patients were divided into two groups according to the duration of buserelin acetate therapy required to achieve pituitary and ovarian suppression (group 1 = 2 weeks, n = 172; group 2 = > or =3 weeks, n = 337). MAIN OUTCOME MEASURE(S): Number of recombinant FSH ampules administered, duration of ovarian stimulation (days), ovarian response, and IVF outcome. RESULT(S): The patients in group 2 had lower mean E2 levels after 5 days and 9 days of stimulation than the patients in group 1. The number of recombinant FSH ampules administered and the number of days of stimulation required were higher in group 2 than in group 1. These differences were prominent in the subgroups of older patients (> or =36 years) and patients who had no evidence of polycystic ovaries on ultrasound examination. The number of oocytes retrieved and fertilized, the cancelation rate, and the pregnancy rate were similar in the two groups. CONCLUSION(S): Prolonged administration of a GnRH agonist to achieve suppression leads to a reduced ovarian response, particularly in women > or =36 years of age, but does not affect the success rate of IVF-ET.

Adult↗
PubMed2000

Anti-apoptotic action of insulin-like growth factor-I during human preimplantation embryo development.

Insulin-like growth factor I (IGF-I) has been shown to increase the proportion of embryos forming blastocysts and the number of inner cell mass cells in human and other mammalian preimplantation embryos. Here we examined whether the increased cell number resulted from increased cell division or decreased cell death. Normally fertilized, Day 2 human embryos of good morphology were cultured to Day 6 in glucose-free Earle's balanced salt solution supplemented with 1 mM glutamine, with (n = 42) and without (n = 45) 1.7 nM IGF-I. Apoptotic cells in Day 6 blastocysts were identified using terminal deoxynucleotidyl dUTP terminal transferase (TUNEL) labeling to detect DNA fragmentation and 4'-6-diamidino-2-phenylindole (DAPI) counterstain to evaluate nuclear morphology. The number of nuclei and extent of DNA and nuclear fragmentation was assessed using laser scanning confocal microscopy. IGF-I significantly increased the proportion of embryos developing to the blastocyst stage from 49% (control) to 74% (+IGF-I) (P < 0.05). IGF-I also significantly decreased the mean proportion of apoptotic nuclei from 16.3 +/- 2.9% (-IGF-I) to 8.7 +/- 1.4% (+IGF-I) (P < 0.05). The total number of cells remained similar between both groups (61.7 +/- 4.6 with IGF-I; 54.5 +/- 5.1 without IGF-I). The increased number of blastocysts combined with reduced cell death suggests that IGF-I is rescuing embryos in vitro which would otherwise arrest and acting as a survival factor during preimplantation human development.

Apoptosis↗
PubMed2000

In vitro maturation of oocytes.

Only about 400 of the one million oocytes present at birth will be ovulated, while the rest will die by atresia. The ability to rescue oocytes destined to die and mature them in vitro would provide invaluable information about folliculogenesis and oocyte maturation, and could provide oocytes for infertile women. In vitro maturation (IVM) is challenging in the human because folliculogenesis is a lengthy process encompassing many complex cellular changes in the oocyte and its surrounding follicle cells. A few live births have resulted from the maturation and fertilization of immature human oocytes aspirated from small antral follicles. Furthermore, it is possible to grow primordial follicles to pre-antral stages in slices of ovarian tissue, and support antrum formation in isolated pre-antral follicles. However, we are still a considerable way from growing and maturing pre-antral follicles to pre-ovulatory stages in vitro. The importance of the follicular environment for producing a healthy and developmentally competent oocyte is illustrated by the oocyte's susceptibility to errors during meiosis. This counsels considerable caution in the development of IVM for clinical application.

Animals↗
PubMed1999

Successful preimplantation genetic diagnosis for sex Link Lesch--Nyhan Syndrome using specific diagnosis.

Lesch-Nyhan syndrome (LN) is a severe X-linked recessive disorder caused by a deficiency of the enzyme hypoxanthine phosphoribosyl transferase (HRT). Clinical features displayed by affected boys are particularly severe and disturbing and include hyperuricaemia, Characteristic neurological features including self-mutilation, choreothetosis, spasticity and mental retardation. A couple with a boy diagnosed with LN and a history of pregnancy termination was referred to the Hammersmith Hospital. Their affected son was born in 1982 after an uncomplicated pregnancy and vaginal delivery. Eight subsequent pregnancies had been unsuccessful. There were five therapeutic terminations and three spontaneous abortions, one at least directly caused by the sampling procedure during amniocentesis. From 1989 to 1991 two unsuccessful preimplantation genetic diagnosis (PGD) cycles by sexing were performed by DNA amplification. The mutation was characterized and a nested PCR protocol was designed which allowed the efficient amplification of the affected loci followed by the detection of the mutant allele by restriction digestion. Three PGD cycles were performed using this specific diagnostic test before a successful pregnancy was achieved resulting in the birth of a healthy unaffected baby girl.

Embryo Transfer↗
PubMed1999

A trisomic germ cell line and precocious chromatid segregation leads to recurrent trisomy 21 conception.

A chromosomally normal 37-year-old woman was referred for preimplantation genetic diagnosis after having several conceptuses with trisomy 21. Segregation of chromosome 21 was assessed in unfertilised meiosis II oocytes and preimplantation embryos from PGD cycles using fluorescent in situ hybridisation (FISH). Of 7 preimplantation embryos, 5 were chromosomally abnormal with 4 having trisomy 21 and one being tetraploid. Of 4 oocytes, 3 had an abnormal chromosomal constitution with either an extra chromosome 21 or an extra chromatid 21. In one oocyte an extra chromatid 21 was detected in both the metaphase II complement and the first polar body providing the first direct evidence of a maternal trisomic germ cell line. Moreover, this result shows that the extra chromosome 21 can precociously divide into its two chromatids at the first meiotic division.

Adult↗
PubMed1999

Human primordial, primary and secondary ovarian follicles in long-term culture: effect of partial isolation.

Ovarian cortical tissue, donated by 20 women aged 25-43 years during gynaecological laparoscopies or laparotomies, was first cultured for 7-9 days as tissue slices, 0.1-0.3 mm in thickness, in extracellular matrix, to initiate the growth of the primordial and primary follicles. It was then divided into two parts, one of which was cultured further as slices, and the other one used for enzymatic (collagenase at 1, 0.5 or 0.25 mg/ml; 17 patients) or mechanical (four patients) partial isolation of the follicles. The tissue slices and the partially isolated follicles were cultured for a further 1-3 weeks in the matrix. After approximately 2 weeks in culture, some oocytes began to extrude from the follicles, which were usually at the secondary stage. They were small, 20-80 micrometer in diameter, and had a thin or absent zona. Polar bodies and meiotic chromosomes could be seen in these naked oocytes. This premature extrusion probably resulted from sub-optimal culture conditions. It occurred sooner in follicles that had been partially isolated using collagenase. Histologically, larger numbers of oocytes were observed in non-isolated slice cultures than in the partially isolated cultures. Initiation of growth of the follicles occurred during the first 7-9 days in culture within slices. In non-isolated slices and following mechanical partial isolation there were significantly more secondary follicles after 11-18 days in culture than following isolation with collagenase. The proportion of atretic follicles increased during all cultures, and it was significantly higher after partial isolation. Because partial isolation did not improve the survival or development of the follicles the optimal method for human ovarian follicles could be to culture them non-isolated within small tissue slices.

Adult↗
PubMed1999

Effects of follicle-stimulating hormone and serum substitution on the in-vitro growth of human ovarian follicles.

In-vitro maturation (IVM) of human ovarian follicles and oocytes could benefit infertile women, and allow the development of in-vitro systems for the study of human follicular development. Little is known about the initiation of growth of primordial follicles and the regulation of early folliculogenesis. An ovarian tissue-slice culture system was used to examine the effects of media composition, follicle stimulating hormone (FSH) and serum substitution on the development of small human follicles in vitro. Human ovarian cortex biopsies were cut into small pieces and cultured for 5, 10 or 15 days. Control (non-cultured) and cultured tissue was fixed, serially sectioned, and stained. The follicles contained within the tissue pieces were counted, measured, and assessed for stage of development and viability. Comparison of the ability of alpha-minimum essential medium (alpha-MEM), Waymouth's, or Earle's balanced salt solution (EBSS) culture media (all with 10% human serum) to support follicle growth demonstrated significantly increased initiation and growth of follicles in alpha-MEM during the first 10 days of culture. The supplementation of alpha-MEM with 300 mIU/ml FSH significantly reduced levels of atresia and increased the mean diameter of healthy follicles. Follicles in tissue cultured for 10 days with human serum albumin and ITS (insulin/transferrin/selenium mix) were significantly larger, more developed and showed significantly less atresia than those cultured with serum alone. Primordial to small preantral follicles can be grown under serum-substituted conditions in tissue-slice culture, and are responsive to FSH, which is thought to be acting mainly as a survival factor at these early stages.

Adult↗
PubMed1999

An analysis of the demand for and cost of preimplantation genetic diagnosis in the United Kingdom.

Research in the field of preimplantation genetic diagnosis (PGD) has concentrated on increasing the number of diseases diagnosed, different strategies for single cell analysis and improving efficiency and reliability. Of equal importance are clinical issues such as the demand for and cost of PGD. This study assesses patient awareness of PGD and its potential benefits; additionally the awareness, attitudes and referral patterns of Assisted Conception Units, Regional Genetics Centres and Health Authorities (funding bodies) have been analysed to establish the demand for PGD within the United Kingdom. The licensed units are able to perform 128 cycles of PGD annually, however 256 cases were referred within the last year. It is clear that the currently licensed units operating at their present capacity are unable to meet the demand for PGD in the U.K. Concerns raised by this study include the unequal geographical distribution of PGD services and the lack of a uniform funding policy by Health Authorities. The investment in personnel and technology to establish a PGD service is considered and a costing provided. We estimate an initial investment in the region of 139,000 Pounds with annual running costs of 55,000 Pounds. This information should contribute towards an appropriate allocation of resources at a national level in the U.K.

Awareness↗
PubMed1999

Preimplantation genetic diagnosis for couples at high risk of Down syndrome pregnancy owing to parental translocation or mosaicism.

The population risk for trisomy 21 is 1 in 700 births but some couples are at a much higher risk owing to parental translocation or mosaicism. We report on the first attempt to carry out preimplantation genetic diagnosis for two such couples using cleavage stage embryo biopsy and dual colour FISH analysis. Each couple underwent two treatment cycles. Couple 1 (suspected gonadal mosaicism for trisomy 21) had two embryos normal for chromosome 21 transferred, but no pregnancy resulted; 64% (7/11) unfertilised oocytes/embryos showed chromosome 21 aneuploidy. Couple 2 (46,XX,t(6;21)(q13;q22.3)) had a single embryo transferred resulting in a biochemical pregnancy; 91% (10/11) oocytes/embryos showed chromosome 21 imbalance, most resulting from 3:1 segregation of this translocation at gametogenesis. The opportunity to test embryos before implantation enables the outcome of female meiosis to be studied for the first time and the recurrence risk for a Down syndrome pregnancy to be assessed.

Adult↗
PubMed1998

Assessment of the reliability of single blastomere analysis for preimplantation diagnosis of the delta F508 deletion causing cystic fibrosis in clinical practice.

Following the birth of a baby girl confirmed to be homozygous normal for the delta F508 deletion causing cystic fibrosis (CF), many single-gene defects have been diagnosed by polymerase chain reaction (PCR) for preimplantation genetic diagnosis (PGD). A few misdiagnoses have been reported but no large-scale studies have been performed to assess the accuracy of diagnosis in a clinical setting. Here we focus on a series of 15 delta F508 PGD cycles performed at the Hammersmith hospital in an 18 month period. All the spare embryos that had not been selected for transfer after clinical diagnosis were disaggregated and the blastomeres were analysed individually to confirm the clinical results and assess the reliability of single blastomere analysis by the nested PCR method. A total of 484 blastomeres from 112 embryos of different delta F508 genotypes were analysed. The amplification rate for nucleated blastomeres was 95 per cent and the overall accuracy of diagnosis was 89 per cent. Using these figures, we calculate that the chance of selecting an affected embryo instead of a homozygous unaffected or heterozygous carrier is 1.3 per cent, and 0.3 per cent of selecting an affected embryo as unaffected when heterozygotes were not considered for transfer. Misdiagnoses risks were negligible when embryos were considered for transfer after obtaining two concordant results from the same embryo. This study highlights the fact that heterozygous carrier embryos are more often associated with misdiagnoses, due to the failure of amplification of one of the two alleles in heterozygous cells (allele dropout (ADO)) and undetected contamination. In a recessive condition such as CF, ADO cannot result in a serious error. Misdiagnoses due to contamination are potentially more dangerous, they, however, can be limited by only selecting homozygous unaffected embryos for transfer as the risks are quadrupled when heterozygotes are also considered for transfer. For diagnoses of dominant conditions we strongly recommend the systematic analysis of two blastomeres per embryo and the transfer of only embryos with two independent concordant results.

Biopsy↗
PubMed1998

Infertile couples with Robertsonian translocations: preimplantation genetic analysis of embryos reveals chaotic cleavage divisions.

Preimplantation genetic diagnosis (PGD) may provide a feasible option for some Robertsonian translocation carriers who experience severe difficulty in achieving a normal pregnancy. We report on five PGD cycles for two such couples, 45,XY,der(13;14)(q10:q10) and 45,XX,der(13;21)(q10;q10), carried out by biopsy of two cells from day 3 post-insemination embryos generated by in vitro fertilisation. Locus-specific YAC probes for chromosomes 13, 14 and 21 were used to detect the chromosomes involved in the translocation using multicolour FISH. Three embryos transfers were carried out (two single embryo transfers and one double transfer) but no clinical pregnancies were established. In two cycles no embryos were transferred as all those biopsied were chromosomally abnormal. Combined results from both couples show 13% (6/45) of embryos analysed were normal for the translocation chromosomes and 87% (39/45) were chromosomally abnormal; these were categorised as 36% aneuploid or aneuploid mosaic and 51% chaotic where the chromosome constitution varied randomly from cell to cell. This suggests two factors may be acting to reduce fertility in these couples; the aneuploid segregation of the parental Robertsonian translocation and also a post-zygotic factor leading to uncontrolled chromosome distribution in early cleavage stages in an exceptionally high proportion of embryos.

Blastomeres↗
PubMed1998

Glutamine improves human preimplantation development in vitro.

OBJECTIVE: To study the effect of glutamine on human preimplantation embryo development in vitro. DESIGN: A randomized, controlled, prospective study of 138 normally fertilized and nontransferred human embryos. On day 2 after fertilization, the embryos were allocated randomly to glucose-free medium with or without 1 mM of glutamine. SETTING: A university hospital-based IVF-ET program. PATIENT(S): Couples undergoing IVF treatment. INTERVENTION(S): Embryo culture. MAIN OUTCOME MEASURE(S): Blastocyst formation, trophectoderm and inner cell mass numbers, and pyruvate uptake and lactate production by individual embryos. RESULT(S): A higher proportion of embryos reached the morula (89% versus 68%, respectively) and blastocyst (71% versus 54%, respectively) stages when cultured with glutamine compared with embryos cultured without glutamine. Blastocyst cell numbers were not statistically significantly different for embryos grown in the presence or absence of glutamine. Pyruvate uptake and lactate production were higher in the presence of glutamine throughout development; these increases were statistically significant at the blastocyst stage. CONCLUSION(S): This study demonstrates that supplementation of the culture medium with glutamine is beneficial for human preimplantation embryo development in vitro, increasing the proportion of embryos that develop to the morula and blastocyst stages.

Blastocyst↗