Alan H. Handyside

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.

Multicolour FISH detects frequent chromosomal mosaicism and chaotic division in normal preimplantation embryos from fertile patients

Abstract We have used multicolour fluorescent in situ hybridisation (FISH) with DNA probes for chromosomes X, Y and 1 to analyse spare untransferred cleavage-stage embryos after preimplantation diagnosis to avoid X-linked disease. In total, 93 morphologically normal embryos were available from seven patients (six of proven fertility) who had undergone fourteen in vitro fertilisation (IVF) cycles. The chromosome patterns observed were classified into four groups; normal, abnormal (non-mosaic), mosaic and chaotic (uncontrolled division). Approximately half of the embryos were normal for the chromosomes tested. Two embryos only were aneuploid (non-mosaic) throughout but, after excluding those showing chaotic division, 30% were considered to be chromosomal mosaics. Of these, a minority had arisen because of mitotic non-disjunction or chromosome loss or gain, whereas the majority were ploidy mosaics, with haploidy being the most common. The occurrence of chaotically dividing embryos was strongly patient-related, i.e. some patients had ‘chaotic’ embryos in repeated cycles, whereas other patients were completely free of this type of anomaly. ‘Chaotic’ embryos are unlikely to progress beyond implantation. These findings have important implications both for routine IVF and preimplantation genetic diagnosis.

Karyomapping: a universal method for genome wide analysis of genetic disease based on mapping crossovers between parental haplotypes

The use of genome wide single nucleotide polymorphism (SNP) arrays for high resolution molecular cytogenetic analysis using a combination of quantitative and genotype analysis is well established. This study demonstrates that by Mendelian analysis of the SNP genotypes of the parents and a sibling or other appropriate family member to establish phase, it is possible to identify informative loci for each of the four parental haplotypes across each chromosome and map the inheritance of these haplotypes and the position of any crossovers in the proband. The resulting ‘karyomap’, unlike a karyotype, identifies the parental and grandparental origin of each chromosome and chromosome segment and is unique for every individual being defined by the independent segregation of parental chromosomes and the pattern of non-recombinant and recombinant chromosomes. Karyomapping, therefore, enables both genome wide linkage based analysis of inheritance and detection of chromosome imbalance where either both haplotypes from one parent are present (trisomy) or neither are present (monosomy/deletion). The study also demonstrates that karyomapping is possible at the single cell level following whole genome amplification and, without any prior patient or disease specific test development, provides a universal linkage based methodology for preimplantation genetic diagnosis readily available worldwide.

Polar body array CGH for prediction of the status of the corresponding oocyte. Part I: clinical results

BACKGROUND Several randomized controlled trials have not shown a benefit from preimplantation genetic screening (PGS) biopsy of cleavage-stage embryos and assessment of up to 10 chromosomes for aneuploidy. Therefore, a proof-of-principle study was planned to determine the reliability of alternative form of PGS, i.e. PGS by polar body (PB) biopsy, with whole genome amplification and microarray-based comparative genomic hybridization (array CGH) analysis. METHODS In two centres, all mature metaphase II oocytes from patients who consented to the study were fertilized by ICSI. The first and second PBs (PB1and PB2) were biopsied and analysed separately for chromosome copy number by array CGH. If either or both of the PBs were found to be aneuploid, the corresponding zygote was then also processed by array CGH for concordance analysis. RESULTS Both PBs were biopsied from a total of 226 zygotes from 42 cycles (average 5.5 per cycle; range 1–15) in 41 couples with an average maternal age of 40.0 years. Of these, the ploidy status of the zygote could be predicted in 195 (86%): 55 were euploid (28%) and 140 were aneuploid (72%). With only one exception, there was at least one predicted aneuploid zygote in each cycle and in 19 out of 42 cycles (45%), all zygotes were predicted to be aneuploid. Fresh embryos were transferred in the remaining 23 cycles (55%), and one frozen transfer was done. Eight patients had a clinical pregnancy of which seven were evolutive (ongoing pregnancy rates: 17% per cycle and 30% per transfer). The ploidy status of 156 zygotes was successfully analysed by array CGH: 38 (24%) were euploid and 118 (76%) were aneuploid. In 138 cases complete information was available on both PBs and the corresponding zygotes. In 130 (94%), the ploidy status of the zygote was concordant with the ploidy status of the PBs and in 8 (6%), the results were discordant. CONCLUSIONS This proof-of-principle study indicates that the ploidy of the zygote can be predicted with acceptable accuracy by array CGH analysis of both PBs.

Selection criteria for human embryo transfer: A comparison of pyruvate uptake and morphology

PurposePyruvate 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 or 3 and correlated the levels with implantation and pregnancy outcome.ResultsThe 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.ConclusionWe 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.

Clinical experience with preimplantation diagnosis of sex by dual fluorescent in situ hybridization

PurposeOur purpose was to assess the clinical application of dual fluorescent in situhybridization (FISH) for the diagnosis of sex in the human preimplantation embryo.ResultsOver 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.ConclusionDual 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.

What next for preimplantation genetic screening? A polar body approach!

Screening of human preimplantation embryos for numerical chromosome abnormalities has been conducted mostly at the preimplantation stage using fluorescence in situ hybridization. However, it is clear that preimplantation genetic screening (PGS) as it is currently practiced does not improve live birth rates. Therefore the ESHRE PGS Task Force has decided to start a proof of principle study with the aim of determining whether biopsy of the first and second polar body followed by subsequent analysis of the complete chromosome complement of these polar bodies using an array based technique enables a timely identification of the chromosomal status of an oocyte. If the principle of this approach can be proven, it is obvious that a multicentre randomized controlled trial should then be started to determine the clinical value of this technique. In this way the ESHRE PGS Task Force hopes to redirect preimplantation screening from the blind alley to the main road of assisted reproduction.

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.

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.

Towards the isolation of embryonal stem cell lines from the sheep

SummaryImmunosurgical isolation of inner cell masses (ICMs) from sheep embryos was most efficient at the expanded, zona-intact blastocyst stage (day 7 to 8 post oestrus) before migration of endoderm cells beyond the boundary of the ICM across the blastocoelic surface of the trophectoderm. When cultured under conditions which allow the isolation of embryonal stem (ES) cell lines from mouse ICMs, sheep ICMs attached, spread and developed areas of both ES cell-like and endoderm-like cells. After prolonged culture only endoderm-like cells were evident. The implications for the isolation of ES cell lines from sheep embryos and possible species-specific requirements are discussed.