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A H Handyside

Publications and source records attributed to A H Handyside.

At least 19 recordsLinked to original sources
PubMed1992

Birth of a normal girl after in vitro fertilization and preimplantation diagnostic testing for cystic fibrosis.

BACKGROUND: Cystic fibrosis is a common, severe autosomal recessive disease caused in a majority of cases by a three-nucleotide deletion (delta F508) in the cystic fibrosis transmembrane regulator gene. Current methods of prenatal diagnosis involve chorionic-villus sampling or amniocentesis. In vitro fertilization and diagnosis during embryonic development before implantation would allow only unaffected embryos to be selected for transfer to the uterus, thereby avoiding the need to terminate a pregnancy. METHODS: Preimplantation diagnosis of cystic fibrosis was attempted in the cases of three couples, both members of which carried the delta F508 deletion. In vitro fertilization techniques were used to recover oocytes from each woman and fertilize them with her husband's sperm. Three days after insemination, embryos in the cleavage stage underwent biopsy and removal of one or two cells for DNA amplification and analysis. RESULTS: Only two oocytes from one woman were fertilized normally; DNA analysis of one of the embryos failed and cystic fibrosis was diagnosed in the other (i.e., it was homozygous for delta F508), so neither was transferred. The oocytes of each of the other two women produced noncarrier, carrier, and affected embryos. Both couples chose to have one noncarrier embryo and one carrier embryo transferred. One woman became pregnant and gave birth to a girl free of the deletion in both chromosomes. CONCLUSIONS: Preimplantation diagnosis of the delta F508 deletion causing cystic fibrosis is possible through in vitro fertilization, biopsy of a cleavage-stage embryo, and amplification of DNA from single embryonic cells. This approach should be equally applicable to other single-gene diseases in which the defect has been identified. Analysis of a series of pregnancies, however, will be required to assess the method adequately.

Base Sequence↗
PubMed1992

Dual fluorescent in situ hybridisation for simultaneous detection of X and Y chromosome-specific probes for the sexing of human preimplantation embryonic nuclei.

Dual fluorescent in situ hybridisation has been used for the simultaneous detection of X and Y chromosome-specific probes in single cleavage nuclei from disaggregated 4- to 7-cell human embryos. Based on the presence of a Y signal or 2 X signals in the absence of a Y, 89% of poor quality metaphases and 72% of interphase nuclei could be classified as male or female. With further refinements, this technique will offer a credible alternative to the polymerase chain reaction for the diagnosis of sex in human preimplantation embryos in families segregating for X-linked genetic disease.

Blastomeres↗
PubMed1992

Human embryo biopsy on the 2nd day after insemination for preimplantation diagnosis: removal of a quarter of embryo retards cleavage.

OBJECTIVE: To assess any reduction in viability and development in vitro after biopsy of a quarter of the cells of human embryos on day 2 after insemination. DESIGN: A prospective study in which normally fertilized surplus embryos of good morphology with two to eight cells approximately 48 hours after insemination were randomly allocated to a control or biopsied group, respectively. SETTING: In vitro fertilization (IVF) unit and laboratories of the Hammersmith Hospital, Institute of Obstetrics and Gynaecology, London University. PATIENTS, PARTICIPANTS: One hundred twenty-nine embryos from 28 infertile IVF patients. INTERVENTIONS: Follicular aspiration by ultrasound-guided transvaginal puncture and embryo biopsy by micromanipulative procedures. MAIN OUTCOME MEASURE(S): Pyruvate uptake and cell number at the blastocyst stage. RESULTS: Embryo biopsy did not have an adverse effect on either the proportion developing to the blastocyst stage (50% [32 of 64] and 47.7% [31 of 65] for the control and biopsied groups, respectively) or embryo viability, measured indirectly through pyruvate uptake. However, the proportion of embryos that reached the morula stage after day 4 (retarded embryos) was significantly higher (44%, 11 of 25 versus 8.7%, 2 of 23) in the biopsied group. The total number of cells (29.6 +/- 3.1 versus 62.4 +/- 4.7), numbers of inner cell mass (7.7 +/- 2.2 versus 24.5 +/- 1.4) and trophectoderm (24.0 +/- 5.2 versus 45.0 +/- 6.4) cells, and the inner cell mass:trophectoderm ratio (34.7 +/- 7.9 versus 59.5 +/- 11.7) were strikingly reduced at the blastocyst stage in the biopsied group. This reduction was greater in embryos that reached the morula stage after day 4. CONCLUSIONS: More investigation is needed to assess whether the detrimental effects observed were because of the biopsy method used in this study or to a high sensitivity of human embryos at early stages to manipulation in vitro.

Biopsy↗
PubMed1992

Biopsy of cleavage stage human embryos and diagnosis of single gene defects by DNA amplification.

Preimplantation diagnosis offers an alternative to current prenatal diagnostic techniques for patients known to be at risk of transmitting an inherited disease. One or two cells can be removed from cleavage stage embryos from patients undergoing in vitro fertilization. Affected embryos can be identified by analyzing the cells for the defect using the polymerase chain reaction. Following analysis, embryos identified as unaffected are replaced in the patient. The identification and transfer of female embryos was attempted in eight couples known to be at risk of transmitting various X-linked diseases. Five of the eight women became pregnant, two with twin and three with singleton pregnancies, after a total of 13 treatment cycles. Six of the seven fetuses were confirmed as being female after chorionic villus sampling. The prospects for the specific diagnosis of single gene defects are described.

Biopsy↗
PubMed1991

Fluorescent in-situ hybridization to interphase nuclei of human preimplantation embryos with X and Y chromosome specific probes.

Fluorescent in-situ hybridization (ISH) to interphase nuclei of human preimplantation embryos has been demonstrated with the X and Y chromosome-specific DNA probes, pBamX7 and pHY2.1, respectively. Assigning the sex on the basis of the number of hybridization signals in the majority of nuclei, the efficiencies with both probes to nuclei from male embryos were considerably higher than those previously reported for pHY2.1 detected by isotopic or conventional non-isotopic methods. Only approximately 15% of nuclei from male embryos failed to hybridize with these probes. With pBamX7, a high incidence (18%) of nuclei with two (or more) signals in embryos classified as males and four signals in a female embryo was observed. In some cases, the double spot nuclei were larger than those with single spots, providing evidence of tetraploidy. The feasibility of using fluorescent ISH for sexing biopsied embryos in couples at risk of X-linked disease and for the preimplantation diagnosis of chromosome abnormalities is discussed.

Blastocyst↗
PubMed1991

Hypoxanthine phosphoribosyl transferase deficiency, haematopoiesis and fertility in the mouse.

We have looked for effects of deficiency in hypoxanthine phosphoribosyl transferase (HPRT) in the mouse comparable to non-behavioural consequences of HPRT-deficiency in humans. HPRT-deficient humans show abnormalities in haematopoiesis and, in heterozygotes, there is strong selection in haematopoietic tissues against HPRT-deficient cells arising as a result of X-chromosome inactivation. We have examined two situations in mice in which HPRT- and HPRT+ cells occur in the same individual. First, in chimaeras resulting from the injection of HPRT- embryonal stem cells into HPRT+ blastocysts the fate of HPRT- and HPRT+ cell populations was monitored by their expression of different isozymes of glucose phosphate isomerase and also, in those chimaeras that resulted from injecting the male ES cells into female blastocysts, by in situ hybridisation using a Y-chromosome-specific repetitive DNA probe. There was a small statistically significant selection against the HPRT- population in haematopoietic tissues in both XX in equilibrium with XY and XY in equilibrium with XY chimaeras. Second, in female mice doubly heterozygous for HPRT-deficiency and for an electrophoretic variant of the X-linked enzyme phosphoglycerate kinase, there was a similar small statistically significant selection against the HPRT- population in haematopoietic tissues. While further work is required to establish whether this selection is a consequence of the HPRT mutation, it is clear that any selection against cells in the haematopoietic system as a consequence of HPRT-deficiency is at most small compared with the effect seen in humans. In HPRT-deficient human males surviving beyond the normal age of puberty, there is testicular atrophy. However, we find no effect of HPRT-deficiency on the fertility of either male or female mice. Thus, as with effects on behaviour, the consequences of HPRT-deficiency for haematopoiesis and testis development in the mouse are at most small compared with those in the human. We conclude that the reason for the difference in effects between the two species lies in a difference in purine-related intermediary metabolism per se, rather than in its interaction with brain amine biochemistry.

Animals↗
PubMed1991

Sex chimaerism, fertility and sex determination in the mouse.

Adult intraspecific mouse chimaeras, derived by introducing male embryonal stem cells into unsexed host blastocysts, were examined to determine whether gonadal sex was correlated with the sex chromosome composition of particular cell lineages. The fertility of XX in equilibrium XY and XY in equilibrium XY male chimaeras was also compared. The distribution of XX and XY cells in 34 XX in equilibrium XY ovaries, testes and ovotestes was determined by in situ hybridisation using a Y-chromosome-specific probe. Both XX and XY cells were found in all gonadal somatic tissues but Sertoli cells were predominantly XY and granulosa cells predominantly XX. The sex chromosome composition of the tunica albuginea and testicular surface epithelium could not, in general, be fully resolved, owing to diminished hybridisation efficiency in these tissues, but the ovarian surface epithelium (which like the testicular surface epithelium derives from the coelomic epithelium) was predominantly XX. These findings show that the claim that Sertoli cells were exclusively XY, on which some previous models of gonadal sex determination were based, was incorrect, and indicate instead that in the mechanism of Sertoli cell determination there is a step in which XX cells can be recruited. However, it remains to be established whether the sex chromosome constitution of the coelomic epithelium lineage plays a causal role in gonadal sex determination. Male chimaeras with XX in equilibrium XY testes were either sterile or less fertile than chimaeras with testes composed entirely of XY cells. This impaired fertility was associated with the loss of XY germ cells in atrophic seminiferous tubules. Since this progressive lesion was correlated with a high proportion of XX Leydig cells, we suggest that XX Leydig cells are functionally defective, and unable to support spermatogenesis.

Animals↗
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

Extragonadal teratocarcinoma derived from embryonal stem cells in chimaeric mice.

Three tumours which arose in two (one male and one hermaphrodite) out of 63 chimaeric mice resulting from injection of E14TG2a embryo stem (ES) cells into host blastocysts have been investigated. All of the tumours appeared within the first 3 weeks after birth. The tumour in the male chimaera and one of the tumours in the hermaphrodite were in the perigenital region but were extragonadal. The third, smaller tumour in the hermaphrodite was on the caecum. The perigenital tumour in the male chimaera was a teratocarcinoma with a wide variety of differentiated tissues, including non-pigmented retina, as well as nests of undifferentiated embryonal carcinoma (EC) cells with high levels of alkaline phosphatase activity. The perigenital tumour in the hermaphrodite was a teratoma, less differentiated and with no evidence of EC cells. Glucose phosphate isomerase isozyme analysis indicated that both perigenital tumours were predominantly of the injected ES cell rather than the host blastocyst type. The possible origins of these tumours, which are the first reported to have arisen from ES cells in chimaeric mice, are discussed.

Abdominal Neoplasms↗
PubMed1990

Future developments in IVF.

Among the many potential developments in IVF, four topics are chosen for discussion. New concepts in the regulation of follicle growth should permit the introduction of improved methods of ovarian stimulation, eg through the use of LHRH antagonists or growth factors. Detailed analyses on human capacitation, the nature of sperm-egg binding and sperm maturation in the epididymis will assist the treatment of male infertility. Research on embryos will clarify details of the growth and metabolism of embryos and their cryopreservation, and indicate the preferred methods to diagnose genetic disease in preimplantation embryos. Analyses on embryonic growth to day 10 and beyond, on interactions between uterine stroma and epithelium, and between these tissues and the trophoblast, should clarify the physiology and biochemistry of human implantation.

Embryo Implantation↗
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↗
PubMed1989

Embryo biopsy.

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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↗