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Biomedical subjects

R M Winston

Publications and source records attributed to R M Winston.

At least 55 records · Page 3Linked to original sources
PubMed1997

Right or left ovary--which one is better?

This study tests the hypothesis that there is a different response after gonadotrophin-releasing hormone and human menopausal gonadotrophins (GnRH/HMG) stimulation for in-vitro fertilization (IVF) in patients with either a right or left ovary. A total of 56 cycles in 44 women after left oophorectomy (group A) was compared with 42 cycles in 29 women following right oophorectomy (group B). Tubal disease was the sole cause of infertility in all cases. The two groups were similar in age. There was no difference in total amount or days of HMG required for ovulation induction, peak oestradiol concentrations, number of oocytes retrieved and fertilization rate. The pregnancy rate was identical in the two groups (20.0 and 20.9% respectively). Our results indicate that with GnRH/HMG ovulation induction protocol for IVF there is no predilection of one ovary over the other.

Adult↗
PubMed1996

Clinical experience with preimplantation genetic diagnosis of cystic fibrosis (delta F508).

Preimplantation genetic diagnosis (PGD) was attempted in 12 couples in whom both parents carry the common delta F508 deletion causing cystic fibrosis (CF). In vitro fertilization (IVF) was followed by cleavage stage biopsy on days 2 and 3 and removal of one or two cells for genetic analysis by nested polymerase chain reaction (PCR) and heteroduplex formation. A total of 18 cycles resulted in 137 normally fertilized embryos, of which 115 developed to cleavage stages and 114 were successfully biopsied. Genetic analysis was successful in 83 embryos (73 per cent). With the remaining embryos, either results from two or more cells were discordant or amplification failed. In 15 cycles, one or two either normal or carrier embryos were transferred and five (33 per cent) clinical pregnancies were established. Five singletons have been born and at birth all five babies have been confirmed as homozygous for the normal allele. Our experience demonstrates that IVF and cleavage stage biopsy consistently provides sufficient embryos, diagnosed as unaffected, for transfer in this autosomal recessive disease and that pregnancy rates are comparable to those following IVF.

Adult↗
PubMed1996

Pregnancies resulting from embryos biopsied for preimplantation diagnosis of genetic disease: biochemical and ultrasonic studies in the first trimester of pregnancy.

PURPOSE: Our purpose was to investigate early biochemical and ultrasonic measurements of pregnancies resulting from embryos biopsied for preimplantation diagnosis of inherited disease. RESULTS: Singleton pregnancies following biopsy had lower initial hCG levels [10 or 12 days after oocyte recovery (OR)], which rose steeply to match the controls by 16 days after OR. Twin biopsied pregnancies showed hCG levels lower than those of twin control pregnancies, which rose in parallel with the controls but remained lower for a longer period than the singletons. Progesterone levels showed a wide variation. Ultrasound measurements showed that overall the mean sac diameter and crown-rump length at 28 and 42 days after egg collection were similar in biopsied and control pregnancies. CONCLUSIONS: Pregnancies resulting from biopsied embryos behave similarly to control IVF pregnancies. However, the reduction in cell mass following embryo biopsy occasionally results in reduced levels of circulating serum hCG and smaller ultrasound measurements in early pregnancy.

Biopsy↗
PubMed1996

Reduced allele dropout in single-cell analysis for preimplantation genetic diagnosis of cystic fibrosis.

BACKGROUND: For couples at risk of transmitting a known single-gene defect, preimplantation genetic diagnosis (PGD) allows the identification and transfer of only unaffected embryos following in vitro fertilisation (IVF), single-cell biopsy at about the eight-cell stage, and genetic analysis by PCR. This technique therefore avoids the risk of terminating an affected pregnancy diagnosed later in gestation. METHODS AND RESULTS: Using nested PCR, the delta F508 mutation causing cystic fibrosis can be detected in single cells and we previously reported successful PGD in a couple in whom both partners carry the delta F508 mutation. To date we have treated 12 couples in a total of 18 cycles. This resulted in five singleton births confirmed to be homozygous normal. Single blastomeres from disaggregated embryos which had not been transferred were analysed to confirm the original diagnosis and assess reliability in clinical practice. Amplification efficiency and accuracy were high, with blastomeres from embryos diagnosed as homozygous normal or affected. In a proportion of blastomeres from presumed carrier embryos, one of the parental alleles failed to amplify, apparently at random (allele dropout, ADO). A possible explanation is the relative inaccessibility of one of the target allele early in the PCR. To test this we have used single lymphocytes from delta F508 carriers and investigated the effects of various denaturation temperatures in the early cycles of amplification. CONCLUSIONS: Increasing the denaturation temperature reduced the rate of ADO without affecting amplification efficiency.

Alleles↗
PubMed1996

Preimplantation genetic diagnosis of cystic fibrosis (delta F508).

Cystic fibrosis is a common autosomal recessive condition caused by mutations in the cystic fibrosis transmembrane regulator gene. The major mutation is a three base pair deletion (delta F508). If both partners carry this deletion, the chance of having an affected child is 1 in 4. In vitro fertilization (IVF) with preimplantation genetic diagnosis allows the selection of the unaffected embryos only to be returned to the uterus. Preimplantation genetic diagnosis was attempted in 14 couples in which both partners carry the delta F508 deletion. A total of 22 cycles resulted in 170 normally fertilized embryos of which, 145 embryos were successfully biopsied and in 18 cycles, one or two unaffected embryos were transferred. A total of five clinical pregnancies established and at birth all five singletons have been confirmed as homozygous for the normal allele. From our experience, cleavage stage biopsy after in vitro fertilization provides sufficient embryos diagnosed as unaffected for transfer in this autosomal recessive disease. Also, pregnancy rates after the preimplantation diagnosis are similar to those with infertile couples. Prospects for applying preimplantation genetic diagnosis to autosomal dominant conditions, where incidences of having affected embryos would be higher, therefore, appear good.

Cystic Fibrosis↗
PubMed1996

Expression of intercellular junctions during preimplantation development of the human embryo.

A total of 74 human embryos were stained with gap junction protein specific anti-peptide antibodies an antibodies to the desmosomal protein desmoplakin to reveal the expression pattern of intercellular junctions during preimplantation development. Prior to implantation, the human embryo expresses predominantly connexin (Cx43)-containing gap junctions. Gap junctions were first detected in apposing cell membranes at the 4-cell stage and became increasingly organized as development proceeded. In normal blastocysts, trophectoderm (TE) cells were linked by dense arrays of gap junctions while inner cell mass (ICM) cells were linked by small, punctate gap junctions. Gap junctions containing Cx32 or Cx26 were observed occasionally in the TE of late blastocysts. Desmosomes appeared between outer cells prior to cavitation and were retained in the TE, but not in the ICM. Levels of gap junction protein expression were variable in morphologically normal embryos at the same stage, suggesting that a normal appearance may not be a reliable indicator of future viability. Morphologically normal embryos often possessed multinucleate, apoptotic and decompacting cells. They could show either extensive, disorganized over-expression or reduced expression of gap junction protein. The results fit the view that only embryos destined to survive display an organized pattern of intercellular junctions.

Blastocyst↗
PubMed1996

Cryopreservation of human ovarian tissue using dimethylsulphoxide and propanediol-sucrose as cryoprotectants.

Pieces of ovarian cortical tissue (0.3-2 mm in diameter) were obtained during gynaecological operations by biopsy or as a result of oophorectomy from 19 women aged 19-44 years. The tissue was frozen in a programmable freezer using one of two different cryoprotectants, either 1.5 M dimethylsulphoxide (DMSO), or a combination of 1,2-propanediol (1.5 M) and sucrose (0.1 M). After cryopreservation lasting from 24 h to 5 weeks, the ovarian pieces were thawed and studied histologically. Specimens taken before and after cryopreservation with either protectant showed no signs of tissue necrosis. Follicles at similar developmental stages were found before and after freezing. The proportions of follicles showing signs of atresia, 27% in the non-frozen tissue and 19% in the frozen-thawed tissue, were not significantly different. Oocytes, too, had the same appearance after freezing and thawing with both cryoprotectants as was seen in the specimens taken before freezing. These results suggest that cryopreservation of human ovarian tissue is feasible. However, the normality of the oocytes taken from tissue which has been frozen still needs to be established. Cryopreservation of ovarian tissue would be potentially an excellent method for storage of human oocytes once methods for their maturation in vitro have been developed.

Adult↗
PubMed1996

Compaction and surface polarity in the human embryo in vitro.

The surface morphology of the human ovum fertilized and cultured in vitro to the morula stage was studied by scanning electron microscopy with the specific aim of investigating embryo compaction and polarity. Unfertilized oocytes examined one day after attempted insemination (Day 0) were evenly and densely covered by long microvilli. The length and density of microvilli appeared to decrease in fertilized polypronuclear oocytes; a further decrease was observed in Day 2 and Day 3 embryos with 2-12 cells. No evidence of compaction or surface polarity was observed in any of these stages. On Day 4, compaction was evident in the majority of embryos with 10 or more cells, and the microvilli appeared dense again with a polarized distribution over the free surface of the compacted blastomeres. This study provides ultrastructural evidence that the human conceptus undergoes a relatively marked compaction at the morula stage during Day 4 postinsemination development in vitro.

Blastomeres↗
PubMed1996

Obstetric outcome of pregnancies resulting from embryos biopsied for pre-implantation diagnosis of inherited disease.

OBJECTIVE: Pre-implantation diagnosis of inherited disease is now a viable option for some couples at risk of transmitting inherited disorders to their children. Since the pregnancy begins knowing that the embryo is not at risk, the need for repeated terminations is eliminated. Up to 25% of the embryo is removed during the procedure, and so it is important to study the resulting pregnancies. Here we report on the obstetric outcome of our first 16 pregnancies resulting from embryo biopsy and preimplantation diagnosis of inherited disease. SETTING: Teaching hospital. SAMPLE: The first 16 pregnancies (12 singletons and 4 twins) following pre-implantation diagnosis. RESULTS: Three singleton pregnancies were lost in the first trimester. Of the remaining pregnancies, two had no prenatal diagnosis, six cases of X-linked disease had the sex confirmed by ultrasound and chorionic villus sampling was performed in the remaining five. All the singleton pregnancies had an uneventful antenatal course and the birthweights and Apgar scores of the babies were normal. The twin pregnancies presented obstetric complications but these were not unusual. CONCLUSIONS: Fifteen healthy infants were born, but for the foreseeable future pre-implantation diagnosis pregnancies should be closely followed up.

Biopsy↗
PubMed1995

Mosaicism of autosomes and sex chromosomes in morphologically normal, monospermic preimplantation human embryos.

We have previously detected chromosome abnormalities in human embryos whilst identifying the sex for preimplantation diagnosis of X-linked disease. In this study we assess the incidence of these abnormalities, both for sex chromosomes and autosomes 1 and 17, using dual fluorescent in situ hybridization (FISH). Sixty-nine normally fertilized embryos of good morphology at the 6-10 cell stage (day 3 post-insemination) were examined. The embryos were spread whole using HCl and Tween 20 to dissolve the cytoplasm. Thirty-four embryos were analyzed for the sex chromosomes and 35 for autosomes 1 and 17. All probes were directly labelled with fluorochromes allowing analysis in 2 h. Control lymphocytes demonstrated that the probes were of high specificity. For the sex chromosomes, five embryos were mosaic (15 per cent) with the remaining 29 being uniformly XX or XY. In no case was an XX nucleus found in an otherwise XY embryo, indicating that even though mosaicism for the sex chromosomes is present, such abnormalities would not lead to a misdiagnosis of sex. For the autosomes, 16 embryos were abnormal (46 per cent); one embryo was triploid, one was monosomic for chromosome 1, and ten others were diploid mosaics (three diploid/aneuploid, three diploid/polyploid, and four diploid/haploid). A further four embryos had variable chromosome numbers in the majority of nuclei which appeared to be the result of uncontrolled mitotic division. The presence of haploidy or double monosomy, which occurred in 15 per cent of nuclei, has important implications for the diagnosis of trisomies and dominant disorders.

Blastomeres↗
PubMed1995

The potential value of magnetic resonance imaging in infertility.

The potential value of magnetic resonance imaging as a diagnostic tool in infertility was investigated. Twenty-six women with primary or secondary infertility and symptoms of dysmenorrhoea or menorrhagia were studied prospectively using conventional T1- and T2-weighted spin-echo techniques. Positive diagnoses were obtained in 20 of 26 (76.9%) patients. Of these, 18 (69.2%) had lesions likely to be significantly contributing to infertility. Adenomyosis was detected in 14 patients (53.8%) with 11 showing the diffuse pattern while three had discrete adenomyomas. Cystic lesions typical of endometriosis were detected in seven patients (26.9%), four of these also had evidence of adenomyosis. The endometriotic lesions were also seen at laparoscopy in each case. Five patients (19.2%) had leiomyomas, one in a patient with adenomyosis and endometriosis and one in a patient with endometriosis alone. Only one patient had submucous leiomyomas causing significant distortion of the endometrial mucosa likely to affect fertility. Magnetic resonance imaging is valuable in the investigation of unexplained infertility where it provides a high diagnostic yield particularly if uterine pathology is suspected.

Adult↗
PubMed1995

Delaying transfer to the third day post-insemination, to select non-arrested embryos, increases development to the fetal heart stage.

The purpose of this study was to determine whether delaying embryo transfer by 24 h, until day 3 post-insemination, allowed improved selection of non-arrested embryos for transfer. We have retrospectively analysed pregnancy rates in a large series of patients who had embryo transfer either on day 2 or on day 3 post-insemination over a 27 month period. From January 1990 to March 1992, 567 patients received embryo transfer on day 2, and 661 patients had transfer on day 3 post-insemination, but these transfers were not contemporary. Pregnancy rates were slightly higher in patients who had embryo transfer on day 3 (37%) than in those patients who had their embryos transferred on day 2 (35%), but this difference was not significant. The implantation rate, as measured by the proportion of embryos developing to the fetal heart stage, was significantly higher following transfer on day 3 (23%) than after transfer on day 2 (19%) (P < 0.05), suggesting that selection of viable embryos is improved on day 3. Furthermore, of the embryos which gave rise to a fetal sac, significantly fewer miscarried before the fetal heart stage (P < 0.05) following transfer on day 3 (6%) than after transfer on day 2 (12%). Delaying transfer until day 3 provides a further 24 h to observe embryo development. During this period 16% of embryos arrested or became developmentally retarded; thus waiting until day 3 allowed these embryos to be identified and avoided for consideration for transfer. Embryo transfer may be safely delayed until day 3, and this may help in selecting embryos most likely to implant and develop after transfer.

Adult↗
PubMed1995

Proliferation of blastomeres from biopsied cleavage stage human embryos in vitro: an alternative to blastocyst biopsy for preimplantation diagnosis.

Normally fertilized human embryos were biopsied at cleavage stages on the third day after in-vitro fertilization (IVF). One or two blastomeres at the 8-cell stage were removed and co-cultured with the biopsied embryos. Embryos and blastomeres were assessed daily for morphological development until day 6, when the number of cells were counted by labelling the nuclei. In all, 53% of the biopsied embryos (25 out of 47) reached the blastocyst stage between day 5 and 6 and the proportion was the same irrespective of the number of cells removed. There was no significant difference between biopsied embryos from which one or two blastomeres respectively had been removed with regard to total cell numbers at the blastocyst stage (56.2 +/- 3.0 and 64.7 +/- 5.5), number of trophectoderm (45.4 +/- 3.5 and 44.0 +/- 5.7) and inner cell mass cells (14.0 +/- 1.2 and 16.6 +/- 1.8). Overall, 72% of the isolated blastomers divided at least once over 3 days in culture and 50% divided more than once. The mean overall cell number after 3 days in culture was 3.7 +/- 0.48 per blastomere (range 1-8 cells) if one cell was removed and 6.9 +/- 1.0 if two cells were removed. If the undivided blastomeres are excluded, the mean cell number was 4.8 +/- 0.51 and 8.3 +/- 1.0 respectively. Over this period, 55% of the blastomeres cavitated. Of the blastomeres taken from embryos that developed to the blastocyst stage, 92% divided and 76% cavitated. In those from arrested embryos, 50% divided (P < 0.002) and 32% cavitated (P < 0.003).(ABSTRACT TRUNCATED AT 250 WORDS)

Biopsy, Needle↗
PubMed1995

Results of IVF in patients with endometriosis: the severity of the disease does not affect outcome, or the incidence of miscarriage.

The literature suggests that the results of in-vitro fertilization (IVF) for patients with endometriosis depend on the stage of the disease, and that patients with severe endometriosis have a higher failure rate. Miscarriage is said to be more prevalent in women treated for endometriosis. In the study reported here, 140 patients with endometriosis underwent 182 cycles of IVF using gonadotrophin-releasing hormone analogues (GnRHa). Patients with endometriosis only were allocated to one group (group 4). The results were compared with those of three other groups of patients undergoing the same treatment within the same period. Group 1 consisted of couples with male factor only (45 cycles), group 2, couples with unexplained infertility (196 cycles) and group 3, couples with a tubal factor only (1139 cycles). The mean age of the patients, mean number of human menopausal gonadotrophin (HMG) ampoules administered, oestradiol concentration on the day of human chorionic gonadotrophin administration, number of days of HMG, mean number of oocytes retrieved and retrieval rate were not significantly different. The fertilization rate was significantly lower in group 1; no difference was observed in the other three groups. The mean number of normally fertilized embryos was not significantly different. The number of transferred embryos in each cycle and the implantation rates were similar in the four groups. The overall pregnancy rate per transfer was 39% in group 1, 48% in group 2, 45% in group 3 and 40% in group 4.(ABSTRACT TRUNCATED AT 250 WORDS)

Abortion, Spontaneous↗
PubMed1995

The use of fluorescent in-situ hybridization (FISH) for the analysis of in-vitro fertilization embryos: a diagnostic tool for the infertile couple.

We use triple colour fluorescent in-situ hybridization (FISH) to sex human embryos for preimplantation diagnosis of X-linked disease, to analyse chromosome numbers in embryos donated for research purposes and as a diagnostic tool for patients undergoing infertility treatment, especially in cases where abnormal embryo development occurs. We have reported on the use of FISH in a case where all embryos showed accelerated cleavage. Here we report on the use of triple colour FISH in a case where five out of seven oocytes were multi-nucleated when examined for pronuclei. The embryos were spread whole using HCl/Tween 20 and triple colour FISH performed with probes for chromosomes X, Y and 1 in a 2 h procedure. Two embryos were normal for the probes used, and three showed abnormalities, including one 4-cell embryo where all nuclei were X,X,X,Y,1,1,1,1. FISH indicated that fertilization had occurred, but that the majority of embryos were abnormal confirming that such embryos should not be considered for transfer. In these cases, or where there is recurrent in-vitro fertilization failure or spontaneous abortions, embryos in future cycles can be examined using FISH to ascertain the level of chromosome abnormality which may aid future infertility treatment.

Blastocyst↗
PubMed1995

The variable effects of 2'-deoxyadenosine on human sperm motility and hyperactivation in vitro.

The response of human sperm motility and hyperactivation to the stimulant 2'-deoxyadenosine (2'-DEA) was studied in vitro using computer-assisted sperm motion analysis. A total of 20 randomly selected individuals with normal sperm counts as defined by the World Health Organization were chosen and their migration-separated spermatozoa exposed to a range (0.1-10.0 mM) of concentrations of 2'-DEA. The straight line velocity (VSL) was increased above control values only at 0.1 mM, while the curvilinear velocity (VCL) and lateral head displacement (ALH) were increased significantly at all concentrations. Linearity of progression (LIN), on the other hand, declined with increasing concentration of 2'-DEA. These changes were related to a significant increase in the number of spermatozoa exhibiting hyperactive-like motion. There was, however, considerable intra-individual variability in the response to 2'-DEA. In some individuals VCL and ALH exhibited little or no response to 2'-DEA, whilst in others an increase above the control of 50-55% occurred. The maximum response for VCL and ALH occurred at 2.5 mM 2'-DEA. Individuals showed greater variability in the percentage of spermatozoa exhibiting hyperactivity in response to 2'-DEA, with increases ranging from 76 to 948% of the control value, although the maximum response was also most commonly seen at 2.5 mM 2'-DEA. The diversity of response to 2'-DEA emphasizes the importance of tailoring doses to the individual rather than employing one concentration for all. Further tests on a subgroup of the individuals examined the longevity of spermatozoa in response to 24 h of continued exposure to 2'-DEA.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗