Paul N. Scriven

Chromosome translocations: segregation modes and strategies for preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) offers polymerase chain reaction tests for an increasing range of single gene defects, and fluorescence in situ hybridization tests for sex determination (for X‐linked conditions) and for aneuploidy detection. Patients carrying chromosome translocations with a high reproductive risk are increasingly seeking to increase their chances of a normal pregnancy with the help of PGD, for which they present a special challenge. This paper describes the behaviour of reciprocal translocations at meiosis, discusses current methods of detecting meiotic outcomes at the preimplantation stage and outlines ways forward for preimplantation diagnosis of these common rearrangements. We also propose a more general strategy using recently developed chromosome‐specific sub‐telomeric probes, combined, if possible, with proximal probes, to form a strong diagnostic tool. Copyright

Lack of cell cycle checkpoints in human cleavage stage embryos revealed by a clonal pattern of chromosomal mosaicism analysed by sequential multicolour FISH

Multicolour fluorescence in situ hybridisation (FISH) analysis of interphase nuclei in cleavage stage human embryos has highlighted a high incidence of postzygotic chromosomal mosaicism, including both aneuploid and ploidy mosaicism. Indeed, some embryos appear to have a chaotic chromosomal complement in a majority of nuclei, suggesting that cell cycle checkpoints may not operate in early cleavage. Most of these studies, however, have only analysed a limited number of chromosomes (3-5), making it difficult to distinguish FISH artefacts from true aneuploidy. We now report analysis of 11 chromosomes in five sequential hybridisations with standard combinations of two or three probes and minimal loss of hybridisation efficiency. Analysis of a series of arrested human embryos revealed a generally consistent pattern of hybridisation on which was superimposed frequent deletion of one or both chromosomes of a specific pair in two or more nuclei indicating a clonal origin and continued cleavage following chromosome loss. With a binucleate cell in a predominantly triploid XXX embryo, the two nuclei remained attached during preparation and the chaotic diploid/triphoid status of every chromosome analysed was the same for each nucleus. Furthermore, in each hybridisation the signals were distributed as a mirror-image about the plane of attachment, indicating premature decondensation during anaphase consistent with a lack of checkpoint control.

Preimplantation Genetic Diagnosis—An Overview

Since the early 1990s, preimplantation genetic diagnosis (PGD) has been expanding in scope and applications. Selection of female embryos to avoid X-linked disease was carried out first by polymerase chain reaction, then by fluorescence in situ hybridization (FISH), and an ever-increasing number of tests for monogenic diseases have been developed. Couples with chromosome rearrangements such as Robertsonian and reciprocal translocations form a large referral group for most PGD centers and present a special challenge, due to the large number of genetically unbalanced embryos generated by meiotic segregation. Early protocols used blastomeres biopsied from cleavage-stage embryos; testing of first and second polar bodies is now a routine alternative, and blastocyst biopsy can also be used. More recently, the technology has been harnessed to provide PGD-AS, or aneuploidy screening. FISH probes specific for chromosomes commonly found to be aneuploid in early pregnancy loss are used to test blastomeres for aneuploidy, with the aim of replacing euploid embryos and increasing pregnancy rates in groups of women who have poor IVF success rates. More recent application of PGD to areas such as HLA typing and social sex selection have stoked public controversy and concern, while provoking interesting ethical debates and keeping PGD firmly in the public eye.

The future of prenatal diagnosis: rapid testing or full karyotype? An audit of chromosome abnormalities and pregnancy outcomes for women referred for Down's Syndrome testing

Objective  To assess the implications of a change in prenatal diagnosis policy from full karyotype analysis to rapid trisomy testing for women referred primarily for increased risk of Downs Syndrome.

Strategies and outcomes of the first 100 cycles of preimplantation genetic diagnosis at the Guy's and St. Thomas' Center

OBJECTIVE To establish strategies for the implementation of a successful preimplantation genetic diagnosis (PGD) service. DESIGN Retrospective review of data from a single center. SETTING A United Kingdom National Health Service hospital. PATIENT(S) Patients (60 couples) were referred for PGD from UK genetic centers. INTERVENTION(S) We followed the protocol of ovarian stimulation, oocyte retrieval, fertilization, single cell biopsy on day 3, and embryo transfer on day 4. Pregnancies unaffected by the familial genetic condition. RESULT(S) A total of 60 couples was treated for 20 different conditions. Early cycles using nonsequential embryo culture media were less successful (13% pregnancy rate/embryo transfer) than later cycles using sequential media (33.5%). Ninety-four percent of embryos (n = 473) had a single cell removed at biopsy. The overall pregnancy rate was 24% per cycle started, 29% per egg collection, 38% per transfer, and 40% per couple treated. In one cycle, an affected pregnancy followed PGD for spinal muscular atrophy (SMA). CONCLUSION(S) The use of sequential media and single cell biopsy results in a successful PGD program with encouraging pregnancy rates.

Three hundred and thirty cycles of preimplantation genetic diagnosis for serious genetic disease : clinical considerations affecting outcome

Objective  To report on our experience with preimplantation genetic diagnosis (PGD) cycles performed for serious genetic disease in relation to the clinical factors affecting outcome.

Successful pregnancy outcomes after preimplantation genetic diagnosis (PGD) for carriers of chromosome translocations.

Reciprocal translocations are found in about 1 in 500 people, whereas Robertsonian translocations occur with a prevalence of 1 in 1000. Balanced carriers of these rearrangements, although phenotypically normal, may present with infertility, recurrent miscarriage, or offspring with an abnormal phenotype after segregation of the translocation at meiosis. Once the translocation has been identified, prenatal diagnosis can be offered, followed by termination of pregnancies with chromosome imbalance. Couples who have suffered repeated miscarriage or those who have undergone termination of pregnancy as a result of the translocation carrier status of one partner are looking increasingly to preimplantation genetic diagnosis (PGD) as a way of achieving a normal pregnancy. Similarly, infertile couples in which one partner is a translocation carrier may request PGD to ensure transfer of normal embryos after in vitro fertilization. Translocation PGD has been applied successfully in several centres worldwide and should now be considered as a realistic treatment option for translocation carriers who do not wish to trust to luck for a successful natural outcome.

Diagnostic accuracy: theoretical models for preimplantation genetic testing of a single nucleus using the fluorescence in situ hybridization technique

BACKGROUND The aim of this study was to develop and use theoretical models to investigate the accuracy of the fluorescence in situ hybridization (FISH) technique in testing a single nucleus from a preimplantation embryo without the complicating effect of mosaicism. METHODS Mathematical models were constructed for three different applications of FISH in preimplantation genetic testing (sex determination for sex-linked diseases, two-way reciprocal translocations and sporadic chromosome aneuploidy). The input values were the degree of aneuploidy (initially set at 3% per chromosome for sporadic aneuploidy) and the accuracy per probe (initially set at 95%), defined as the proportion of normal diploid nuclei with a normal signal pattern. The primary statistic was the predictive value of the test result. RESULTS Testing two chromosome pairs to determine sex chromosome status or detect unbalanced translocation products had high predictive value: at least 99.5% for a normal test result (95% CI: 99-100%), and 90% for an abnormal test result (95% CI: 88-92%). However, the predictive value of an abnormal test result testing five chromosomes for sporadic chromosome aneuploidy was 41% (95% CI: 36-46%); 90% would be achieved with an aneuploidy rate per chromosome of 20.3% (equivalent to 99.5% prevalence for 23 chromosomes) rather than 3%, or with an accuracy per probe of 99.6% rather than 95%, or when testing 23 chromosome pairs, rather than 5 pairs, with either 8.3% aneuploidy (86.4% prevalence) or 99.5% accuracy. CONCLUSIONS Testing a single cell using the FISH technique has the potential to achieve acceptable analytical performance for sex determination and two-way reciprocal translocations, but is unlikely to achieve adequate performance testing for sporadic chromosome aneuploidy. New techniques for detecting the copy number of every chromosome are emerging, but it remains to be seen if the high accuracy required will be achieved.

Meiotic segregation of Robertsonian translocations ascertained in cleavage-stage embryos—implications for preimplantation genetic diagnosis

BACKGROUND The aim of this study was to ascertain the prevalence of meiotic segregation products in embryos from carriers of 13/14 and 14/21 Robertsonian translocations and to estimate the predictive value of testing single cells using the fluorescence in situ hybridization (FISH) technique, to provide more information for decision-making about PGD. METHODS In this prospective cohort study, the copy number of translocation chromosomes in nuclei from lysed blastomeres of cleavage-stage embryos was ascertained using locus-specific FISH probes. Logistic regression analysis, controlling for translocation type, female age and fertility status, was used to calculate the odds ratio (OR) of unbalanced segregation products for female and male heterozygotes. The primary diagnostic measure was the predictive value of the test result. The primary outcome measure was the live birth rate per couple. RESULTS Female carriers were four times more likely than male carriers to produce embryos with an unbalanced translocation product (OR 3.8, 95% confidence interval 2.0-7.2, P < 0.001). The prevalence of abnormality for the chromosomes tested in embryos from female or male heterozygotes was estimated to be 43 or 28%, respectively, while estimates of the predictive value were 93-100 or 96-100% for a normal test result and 79 or 57% for an abnormal test result. The live birth rate per couple was 58% for female carriers and 50% for male carriers. CONCLUSIONS For female carriers, PGD using FISH could reduce the risk of miscarriage from either translocation or the risk of Down syndrome from the 14/21 Robertsonian translocation. PGD using FISH for male carriers is unlikely to be indicated given the relatively low prevalence of chromosome imbalance and low predictive value.

Fluorescence In Situ Hybridization on Single Cells

Fluorescence in situ hybridization (FISH) is the technique of choice for preimplantation genetic diagnosis (PGD) selection of female embryos in families with X-linked disease, for which there is no mutation-specific test. FISH with target-specific DNA probes is also the primary technique used for PGD detection of chromosome imbalance associated with Robertsonian translocations, reciprocal translocations, inversions, and other chromosome rearrangements, because the DNA probes, labeled with different fluorochromes or haptens, detect the copy number of their target loci. The methods described outline strategies for PGD for sex determination and chromosome rearrangements. These methods are assessment of reproductive risks, the selection of suitable probes for interphase FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring using directly labeled and indirectly labeled probes.