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Biomedical subjects

A H Handyside

Publications and source records attributed to A H Handyside.

At least 37 records · Page 2Linked to original sources
PubMed1997

XIST expression from the maternal X chromosome in human male preimplantation embryos at the blastocyst stage.

In the somatic cells of female mammals, either the maternally or paternally derived X chromosome (X(M) or X(P)) is randomly inactivated to achieve dosage compensation for X-linked genes. In early mouse development, however, selective inactivation of X(P) occurs first in extraembryonic lineages at the blastocyst stage around the time of implantation before later random inactivation in the embryonic ectoderm from which the fetus is derived. Xist, a gene mapping to the X-inactivation centre (Xic), is exclusively expressed from the inactive X-chromosome and is thought to be involved in the initiation of X-inactivation. Consistent with this, Xist is first expressed at the 4-to 8-cell stages, prior to functional inactivation at the blastocyst stage, exclusively from X(P) in female embryos. This also suggests that genomic imprinting may influence the earliest expression of Xist resulting in selective inactivation of X(P) and a candidate methylation site in the promoter region has recently been described. Here we report the expression of the human homologue, XIST, in human preimplantation embryos from the 5- to 10-cell stage onwards consistent with its role in the initiation of inactivation. In contrast to the mouse, however, transcripts were detected in both male and female embryos demonstrating XIST expression from the X(M) in male embryos (X(M)Y).

Animals↗
PubMed1996

Metabolism and cell allocation during parthenogenetic preimplantation mouse development.

Diploid parthenogenetic postimplantation mouse embryos, containing two maternal genomes, are characterized by poor development of extraembryonic membranes derived from the trophectoderm and primitive endoderm of the blastocyst. This is thought to be caused by a deficiency of expression of paternally derived imprinted genes. Here we have compared the inner cell mass, from which the primitive endoderm and fetal lineages are derived, and the trophectoderm, which forms a major component of the placenta, in parthenogenetic and fertilized preimplantation embryos. We have also studied the metabolism from the 1-cell to the blastocyst stage. Cell numbers were reduced in the ICM and TE of parthenogenetic blastocysts compared to fertilized blastocysts. This was thought to be due to the increased levels of cell death observed in these lineages. Pyruvate and glucose uptake by parthenogenetic embryos was similar to that by fertilized embryos throughout preimplantation development. However, at the expanded blastocyst stage glucose uptake by parthenogenetic embryos was significantly higher than by fertilized embryos. The implications of the actions of imprinted genes and of X-inactivation is discussed.

Animals↗
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

Identifying the sex of human preimplantation embryos in X-linked disease: amplification efficiency of a Y-specific alphoid repeat from single blastomeres with two lysis protocols.

INTRODUCTION: Preimplantation diagnosis involves detecting genetic defects in one or two blastomeres biopsied from cleavage stage embryos following a vitro fertilization (IVF). For X-linked recessive disease, identification of the sex of embryos allows transfer of only unaffected females. To examine how critical the preparation of the single blastomere is for amplification of a Y chromosome specific repeat sequence using the polymerase chain reaction (PCR), the incidence of amplification failure has been examined following two lysis protocols. MATERIALS AND METHODS: Amplification of a Y alphoid repeat sequence from single blastomeres disaggregated from cleavage stage embryos was examined after either (1) lysis in distilled water and freeze-thawing twice or (2) a two-step lysis protocol involving an initial treatment in potassium hydroxide and dithiothreitol. Some of the embryos had been previously sexed by cleavage-stage biopsy and fluorescent in situ hybridization with X- and Y-specific probes. RESULTS: Amplification failure occurred in 6 of 50 (12%) and 4 of 60 (7%) single blastomeres from male embryos following lysis in distilled water or using the two-step protocol, respectively. Conversely, amplification from contaminating DNA occurred in 5 of 63 (8%) single blastomeres from female embryos and 6 of 94 (6%) of control medium-blanks. CONCLUSIONS: The incidence of amplification failure was improved but not eliminated using the two-step lysis protocol. At least two cells, therefore, would be necessary for accurate identification of males by amplification of Y-specific repeat sequences alone. Nevertheless, this protocol for preparing cleavage-stage blastomeres is likely to give more consistent amplification of any unique or repeat sequences.

Blastomeres↗
PubMed1996

Increasing the denaturation temperature during the first cycles of amplification reduces allele dropout from single cells for preimplantation genetic diagnosis.

Single cell polymerase chain reaction (PCR) for preimplantation genetic diagnosis (PGD) needs to be highly efficient and accurate. In some single cells from human embryos presumed to be heterozygous for the delta F508 deletion causing cystic fibrosis (CF), we recently observed random amplification failure of one of the two parental alleles following nested PCR. To investigate allele dropout (ADO), we have examined two different lysis protocols and the effect of altering the denaturation temperature in the primary PCR using single lymphocytes heterozygous for delta F508 or for two beta-thalassaemia mutations IVS 1 nt 1 (G/T) and 5 (G/C) using a nested PCR protocol to amplify the 5' region of the beta-globin gene. Amplification rates were high after lysis in either water or lysis buffer and at all denaturation temperatures studied (> or = 92%). With a typical denaturation temperature (93 degrees C), ADO was detected at both loci. When the denaturation temperature was lowered to 90 degrees C, however, ADO increased substantially and conversely by raising the denaturation temperature to 96 degrees C during the first 10 cycles ADO was reduced but not eliminated. ADO was also reduced with cells in lysis buffer. We suggest that ADO may be caused by a combination of inefficient denaturation and degradation of one of the genomic alleles in the first cycles of PCR. For autosomal recessive conditions in which both parents are carrying the same mutation, ADO would not cause serious misdiagnosis. For compound heterozygotes or autosomal dominant conditions, however, extensive testing of the amplification protocol with single heterozygous cells and individual calibration of each thermocycler for the effect of denaturation temperature on ADO is essential before clinical application.

Alleles↗
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↗
PubMed1996

Moisaicism in the human preimplantation embryo.

Recent studies have revealed widespread mosaicism in the human preimplantation embryo at the nuclear and chromosomal level arising at fertilization and preimplantation development. Molecular cytogenetic analysis by fluorescent in situ hybridization (FISH), in particular, has for the first time made it possible to analyse all or almost all nuclei in cleavage stage embryos. The genotype of an individual is therefore not strictly defined at conception by the genotype of the germ cells. Rather it seems (at least in vitro) that the processes of fertilization, syngamy and the postzygotic mitotic divisions can be irregular. In some cases, therefore, the eventual genotype only emerges by a process of natural selection.

Cell Nucleus↗
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

Single-cell analysis of the RhD blood type for use in preimplantation diagnosis in the prevention of severe hemolytic disease of the newborn.

OBJECTIVE: Our purpose was to develop a molecular assay to determine the fetal RhD blood type on single diploid cells, including blastomeres. STUDY DESIGN: Polymerase chain reaction amplification of a 99 bp deoxyribonucleic acid fragment of the RhD gene or a 113 bp fragment from the RhCE gene was performed from 20 venous blood samples and 20 amniotic fluid samples and from 60 single-cultured lymphoblasts and 12 media blanks mixed in a blinded fashion. This reaction was similarly tested after whole-genome amplification on 10 lymphoblasts and seven human blastomeres. RESULTS: Deoxyribonucleic acid amplification was successful and correct from all genomic deoxyribonucleic acid samples. Ninety-seven percent of single cells amplified; correct diagnosis was made in 96%. Five blastomeres successfully amplified. No media blanks produced amplified, contaminating deoxyribonucleic acid. CONCLUSIONS: The RhD blood type can be determined reliably from single cells and can be used for preimplantation genetic diagnosis for the prevention of rhesus hemolytic disease.

Amniotic Fluid↗
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

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

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↗