Biomedical subjects
A H Handyside
Publications and source records attributed to A H Handyside.
Sex determination of preimplantation embryos.
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Presence of chromosomal mosaicism in abnormal preimplantation embryos detected by fluorescence in situ hybridisation.
The extent of chromosomal mosaicism in human preimplantation embryos was examined using an improved procedure for the preparation and spreading of interphase nuclei for use in fluorescence in situ hybridisation, allowing the analysis of every nucleus within an embryo. One cell showed no hybridisation signals in only three of the 38 embryos that were included in this study, i.e. the hybridisation efficiency per successfully spread nucleus was 99% (197/200). Double-target in situ hybridisation analyses with X- and Y-chromosome-specific probes was performed to analyse nine embryos resulting from normal fertilisation, 22 polypronucleate embryos and seven cleavage-stage embryos where no (apronucleate) or only one pronucleus (monopronucleate) was observed. We also analysed autosomes 1 and 7 by double-target in situ hybridisation in the nuclei of two apronucleate, one monopronucleate and four polypronucleate embryos. All nine embryos that resulted from normal fertilisation were uniformly XY or XX. None of the apronucleate or monopronucleate embryos was haploid: three were diploid, one was triploid and three were mosaic. Fertilisation was detected by the presence of a Y-specific signal in four of these embryos. Of the polypronucleate embryos, two were diploid, two were triploid and 18 were mosaic for the sex chromosomes and/or autosomes 1 and 7. These results demonstrate that fertilisation sometimes occurs in monopronucleate embryos and that chromosomal mosaicism can be detected with high efficiency in apronucleate, monopronucleate and polypronucleate human embryos using fluorescence in situ hybridisation.
Clinical experience with preimplantation diagnosis of sex by dual fluorescent in situ hybridization.
PURPOSE: Our purpose was to assess the clinical application of dual fluorescent in situ hybridization (FISH) for the diagnosis of sex in the human preimplantation embryo. RESULTS: Over a 2-year period, 18 couples at risk of transmitting X-linked recessive disorders underwent preimplantation diagnosis of embryo sex by dual FISH with X and Y chromosome-specific DNA probes. A total of 27 in vitro fertilization (IVF) treatment cycles led to nine pregnancies; 7 reached the stage of clinical recognition, of which 2 spontaneously aborted. There were five live births, three singleton and two twin: none in disagreement with the diagnosed sex. The diagnosis was corroborated in 51 of the 74 nontransferred embryos. The efficiency of the procedure improved throughout the four treatment cycles. This was reflected in the increased proportion of double embryo transfers (from 50% in series 1 and 2 to 100% in series 3 and 4), with a consequent improvement in pregnancy rate (from 28 to 71% per embryo transfer). The excess of male embryos (male:female, 60:40 overall) and the high proportion of biopsied embryos with abnormal numbers of X and Y chromosome signals (14.5%) effectively reduced the number of normal female embryos available for transfer. CONCLUSION: Dual FISH is an efficient technique for determination of the sex of human preimplantation embryos and the additional ability to detect abnormal chromosome copy numbers, which is not possible via the polymerase chain reaction, (PCR), makes FISH the preferred technique.
Pre-implantation diagnosis of inherited predisposition to cancer.
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Transcription of paternal Y-linked genes in the human zygote as early as the pronucleate stage.
Global activation of the embryonic genome occurs at the 4- to 8-cell stage in human embryos and is marked by continuation of early cleavage divisions in the presence of transcriptional inhibitors. Here we demonstrate, using reverse transcriptase-polymerase chain reaction (RT-PCR), the presence of transcripts for two paternal Y chromosomal genes, ZFY and SRY in human preimplantation embryos. ZFY transcripts were detected as early as the pronucleate stage, 20-24 h post-insemination in vitro and at intermediate stages up to the blastocyst stage. SRY transcripts were also detected at 2-cell to blastocyst stages. The expression of SRY and ZFY at these early stages and the faster cleavage rate of male embryos observed in many mammalian species focuses attention on the role of events in sex determination prior to gonad differentiation.
The zona reaction in human oocytes as seen with scanning electron microscopy.
Using scanning electron microscopy we found differences in the fine structure of the zona pellucida between unfertilized and fertilized human pronuclear stage oocytes in an in-vitro fertilization programme. In unfertilized oocytes, the zona pellucida appeared porous, comprising a large number of ring-shaped structures, called hoops, randomly superimposed in several layers. Superficial pores had a mean diameter of 4 microns, with the diameter decreasing in more inner lying pores. In fertilized oocytes, the zona pellucida was compact; the hoops appeared to melt and the pores to be obliterated by an amorphous material emerging from the inner zona. The micrographs provide ultrastructural evidence of the zona reaction in human oocytes and give insights into the morphological and mechanical aspects of the polyspermy-blocking mechanism in humans.
Identification of the sex of human preimplantation embryos in two hours using an improved spreading method and fluorescent in-situ hybridization (FISH) using directly labelled probes.
Dual fluorescent in-situ hybridization (FISH) using X and Y chromosome specific probes has been used to identify the sex of human embryos for preimplantation diagnosis of X-linked disease. With a modified spreading method and directly labelled fluorescent DNA probes, we have examined the possibility of reducing the time of the FISH procedure from 7 to 2 h. A total of 17 normally fertilized human embryos were disaggregated and 98 intact blastomeres obtained. The spreading efficiency was 96% and FISH signals were obtained from 97% of nuclei. In all cases, sibling blastomeres from the same embryo were the same sex. Mosaicism was observed in some embryos. Five cells which lysed during the disaggregation process were spread to determine whether FISH was possible in these cells, but in all cases the morphology of the nuclei was poor and multiple signals were observed so that reliable diagnosis of sex was not possible. The data reported here confirm that by using an improved spreading method in combination with directly labelled DNA probes, we have increased the efficiency and reduced the time required for sexing embryos for preimplantation diagnosis of X-linked disease.
Detection of fertilization in embryos with accelerated cleavage by fluorescent in-situ hybridization (FISH).
Embryos from a couple undergoing routine in-vitro fertilization for unexplained infertility had shown cleavage by day 1 in two consecutive cycles. The response of the woman to fertility drugs had been normal, and there were no known sperm abnormalities. In a subsequent cycle, accelerated cleavage occurred again and we used a modified method of spreading whole embryos and dual fluorescent in-situ hybridization (FISH) with directly labelled probes for chromosomes X and Y to determine if fertilization had occurred in these embryos. Nine oocytes were collected, five of which had cleaved when examined for pronuclei early on day 1 following late insemination. Pronuclei were observed in one of the remaining oocytes, but in this case, three were present. On day 2, all of the oocytes/embryos had cleaved and two were transferred to the patient. The remaining seven were spread for FISH analysis but nuclei were only obtained from five. In three, a Y signal was detected, indicating that fertilization had occurred. In all five embryos, a wide range of X chromosome signals were observed. These data suggest that the embryos had undergone abnormal fertilization and accelerated cleavage.
Diagnosis of sex in preimplantation embryos by fluorescent in situ hybridisation.
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New challenges in human in vitro fertilization.
This review assesses some scientific and ethical problems with human in vitro fertilization. Improved selection of viable embryos, better culture conditions, and greater understanding of the uterine environment will increase success and prevent multiple pregnancy. Further advances will also improve oocyte cryopreservation, in vitro maturation of oocytes, knowledge of sperm function, and sperm microinjection. Preimplantation diagnosis will help avoid genetic diseases and increase understanding of embryonic defects and the viability of zygotes. The greatest ethical problem with all these developments seems to be delivery of these complex treatments when health-care resources are increasingly limited.
Cell allocation in twin half mouse embryos bisected at the 8-cell stage: implications for preimplantation diagnosis.
Previous work in the mouse and human has shown that removal of cells from 2- and 4-cell embryos disproportionately reduces the inner cell mass (ICM), from which the fetus is derived, suggesting that preimplantation biopsy at these stages would be inadvisable. Here we have examined allocation in twin half mouse embryos produced by bisection at the 8-cell stage. Half embryos and intact zona-free control embryos were differentially labeled at the time of cavitation using polynucleotide-specific fluorochromes, and the numbers of cells in the ICM and trophectoderm (TE) counted. Adding the numbers of cells in the twin halves together, the numbers of ICM cells are not significantly different from those in intact controls. In contrast, numbers of TE cells were significantly higher (P < 0.01), producing a small increase in the total cell number of half embryos. There is no concomitant decrease in ICM cell number. We propose that as the surface area of a half embryo is greater than half an intact control, TE cells in half embryos divide faster in order to reach an optimal packing density similar to that in intact control embryos. Furthermore, we suggest that preimplantation biopsy only be performed at the 8-cell stage or later.
Selection criteria for human embryo transfer: a comparison of pyruvate uptake and morphology.
PURPOSE: Pyruvate uptake is higher in human embryos developing to the blastocyst stage than those arresting at cleavage stages. To investigate whether pyruvate uptake provides an improved criterion for selecting embryos for transfer, we have measured uptakes by individual embryos noninvasively over 24-hr periods between the first day (day 1) postinsemination and embryo transfer on day 2 to 3 and correlated the levels with implantation and pregnancy outcome. RESULTS: The mean uptake was significantly lower for embryos that implanted than for those which failed to implant: 22.9 +/- 1.0 and 27.1 +/- 0.6 pmol/embryo/hr, respectively on day 2, and 22.4 +/- 1.5 and 26.9 +/- 0.8 pmol/embryo/hr, respectively, on day 3, but the wide range of uptakes by individual embryos was overlapping. CONCLUSION: We conclude that pyruvate uptake as the sole criterion for embryo selection cannot predict which embryos will implant after transfer. Assessment of embryos using morphological and developmental criteria, therefore, remains the most consistent, though inefficient, indicator of pregnancy potential.
Embryo biopsy strategies for preimplantation diagnosis.
OBJECTIVE: To analyze different biopsy methods, embryo stages, and cellular masses that can be removed for preimplantation diagnosis of genetic diseases to find optimal biopsy conditions compatible with the subsequent development of the conceptus, the acquisition of intact viable blastomeres, and the reliability of the genetic analysis. DATA IDENTIFICATION: The most important published studies have been identified through a computerized bibliographical search (MEDLINE; Dialog, Palo Alto, CA). STUDY SELECTION: Studies reporting different embryo biopsy methods practiced at different stages have been selected. RESULTS: The analysis carried out in the current review shows, at the present time, the following: [1] the displacement and push methods may be more suitable than the stitch and pull and aspiration (puncturing the zona pellucida) approaches at cleavages stages; [2] the aspiration and stitch and pull procedures may assure higher success rates than the herniation procedure at the blastocyst stage; [3] the mechanical division method and the use of acid Tyrode's solution would not be advisable before the eight-cell stage; [4] human embryos at the two-cell and blastocyst stages may not be suitable for preimplantation diagnosis because of an excessive reduction of cellular mass at the two-cell stage and a low or zero pregnancy rate after transfer at the blastocyst stage; and [5] biopsy of a quarter of the embryonic cellular mass on day 2 after insemination may increase biochemical pregnancies if the cleavage rate is not preserved. CONCLUSIONS: At the present time, biopsy of a quarter of the embryo on day 3 after insemination may be the most feasible approach for preimplantation diagnosis.
Detection of aneuploidy and chromosomal mosaicism in human embryos during preimplantation sex determination by fluorescent in situ hybridisation, (FISH).
Five couples at risk of producing offspring with X-linked recessive disease underwent in vitro fertilisation with a view to preimplantation determination of embryo sex and selective transfer of females. On day three postinsemination, one or two blastomeres were removed by embryo biopsy, and used for dual fluorescent in situ hybridisation with X and Y chromosome-specific DNA probes. In two cases, two female embryos were transferred and one pregnancy, (sex confirmed), is ongoing at 19 weeks. All eight embryos from one couple were of such poor quality that diagnosis was possible in one only. In the remaining two cases no embryos were transferred due to the detection of an abnormal number of X chromosome signals. Investigation of the biopsied embryos that were not transferred revealed evidence of mitotic non-disjunction in one and of complete X monosomy in a second. A surviving fetus with this latter constitution would have developed Turner syndrome and would also have been at high risk of X-linked disease. The use of fluorescent in situ hybridisation rather than the polymerase chain reaction allowed the detection of abnormal copy numbers of X chromosomes thus preventing the transfer of potentially abnormal zygotes.
Preimplantation prevention of X-linked disease: reliable and rapid sex determination of single human cells by restriction analysis of simultaneously amplified ZFX and ZFY sequences.
In vitro fertilization (IVF), blastomere biopsy of the 6-8 cell embryo, and single cell DNA diagnosis allows couples at risk of transmitting an X-linked or autosomal disease to start a pregnancy knowing their child will not be affected. We present a quick and reliable nested PCR strategy for sex determination at the single cell level by simultaneous amplification and subsequent restriction fragment analysis of the homologous but non-allelic ZFX and ZFY genes present on the X and Y chromosomes respectively. Amplified ZFX and ZFY sequences are of equal size and produce distinguishable HaeIII digestion products. In a randomized, blinded study of 194 individually isolated lymphoblasts, amniocytes, chorion villus cells, and blastomeres, 191 amplified successfully (98.4% sensitivity). None of the sample blanks showed any PCR product, all 90 of the karyotypically XY cells were correctly genotyped as ZFX/ZFY, all 83 of the 84 XX cells that amplified were correctly genotyped as ZFX only, and analyses of all same-embryo blastomeres were completely concordant (100% specificity). This strategy avoids a source of misdiagnosis observed in methods which detect only Y-specific sequences, where amplification failure in an XY cell results in an erroneous XX diagnosis. This rapid (6 hr) and simple method of analysis, when applied to preimplantation embryo diagnosis, allows the avoidance of offspring affected with an X-linked recessive disorder by transferring only female embryos for implantation and ensuing pregnancy.
Preimplantation diagnosis of aneuploidy using fluorescent in-situ hybridization: evaluation using a chromosome 18-specific probe.
Fluorescent detection of in-situ hybridization (FISH) with a chromosome 18-specific probe (P5041 B.5 D18Z1) has been used to assess the use of this method for preimplantation diagnosis of aneuploidy. Interphase nuclei (n = 802) have been analysed from 59 normally fertilized embryos developing in vitro at the normal rate between days 2 and 7 postinsemination. The efficiency of hybridization in control cells, as assessed by the proportion with two signals in normal female lymphocytes was 88.9% (n = 353) and with three signals in a trisomic (48,XXX+18) fibroblast cell line 74.0% (n = 290). Fifty-four of the human embryos were considered to be diploid on the basis that the majority of nuclei had two signals. Some nuclei in these embryos had one or no signal, especially on day 2, and tetraploid nuclei were also widespread. Among the remaining five embryos, one 5-cell embryo on day 2 had three hybridization signals in 4/5 nuclei and was trisomic for chromosome 18, one 4-cell embryo on day 2 had only one signal in 4/4 nuclei and was monosomic, and the three other embryos were aneuploid mosaics and/or had multi-nucleated blastomeres. Analysis of the incidence of interphase nuclei with more or less than the diploid number of hybridization signals indicates that more than a single nucleus will be necessary for accurate preimplantation diagnosis of aneuploidy.
Binucleate blastomeres in preimplantation human embryos in vitro: failure of cytokinesis during early cleavage.
The nuclei of disaggregated blastomeres from two hundred preimplantation human embryos were examined between days 2 and 4 after insemination in vitro by vital labelling with a polynucleotide-specific fluorochrome. Although the majority of blastomeres had a single nucleus, binucleate blastomeres containing two nuclei of equal size were common and other blastomeres had fragmented nuclei or were anucleate. Seventeen per cent of normally fertilized embryos at two- to four-cell stage had at least one binucleate blastomere, and this increased to 65% at the nine- to 16-cell stage when individual embryos had between one and six binucleate blastomeres. The proportion of binucleate blastomeres in normally fertilized embryos increased from 5 to 10% over this period, whereas in abnormally fertilized, polyspermic or parthenogenetic, embryos the proportion was significantly higher during early cleavage stages but decreased at the nine- to 16-cell stage when the majority of these embryos arrest (25 and 6%, respectively). The incidence of anucleate blastomeres in normally fertilized embryos was also high, especially in those of poor morphology. In contrast, blastomeres with fragmented nuclei were relatively uncommon and the incidence was variable among classes and stages of development. Estimates of the volume of binucleate blastomeres based on measurement of their diameters and comparison with mononucleate blastomeres at various cleavage stages indicated that these blastomeres arise from a failure of cytokinesis between the second and fourth cleavage divisions. On this basis, assignment of binucleate blastomeres to particular cleavage stages in normally fertilized day 4 embryos suggests that at least some of these blastomeres arising during early cleavage persist without further cell division for up to 48 h. At the cellular level, therefore, blastomeres with either binucleate or abnormal nuclei contribute to cleavage stage arrest in vitro.