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.

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.

ESHRE PGD Consortium data collection VIII: cycles from January to December 2005 with pregnancy follow-up to October 2006.

The eighth report of the European Society of Human Reproduction and Embryology PGD Consortium is presented documenting cycles collected for the calendar year 2005 and follow-up of the pregnancies and babies born until October 2006 which resulted from these cycles. For the first time, the delivery rates for each indication are presented and also the pregnancy rates for each centre are reported anonymously. Since the first data collections, there has been a steady increase in the number of cycles, pregnancies and babies reported annually. For data collection VIII, 39 centres have participated, reporting on 3488 cycles to oocyte retrieval (OR), along with details of the follow-up on 845 pregnancies and 670 babies born. Five hundred and twenty OR were reported for chromosomal abnormalities, 108 OR for sexing for X-linked diseases, 500 OR for monogenic diseases, 2275 OR for preimplantation genetic screening and 85 OR for social sexing. Data VIII is compared with the cumulative data for data collections I-VII.

ESHRE PGD consortium data collection VII: cycles from January to December 2004 with pregnancy follow-up to October 2005.

The seventh report of the ESHRE PGD Consortium is presented documenting cycles collected for the calendar year 2004 and follow-up of the pregnancies and babies born subsequent to these cycles up to October 2005. Since the beginning of the data collections, there has been a steady increase in the number of cycles, pregnancies and babies reported. For data collection VII, 45 centres have participated, reporting on 3358 cycles to oocyte retrieval (OR), 679 pregnancies and 528 babies born. Five hundred and fifty nine OR were reported for chromosomal abnormalities, 113 OR for sexing for X-linked diseases, 520 OR for monogenic diseases, 2087 OR for PGS, and 79 OR for social sexing. Data VII is compared with the cumulative data for data collections I-VI.

Communicating chromosome rearrangements and their outcomes using simple computer-generated color ideograms.

Simple computer-generated color ideograms (CGCIs) conforming to ISCN (1995) are presented, not only to represent partial karyotypes, but also to depict reproductive outcomes displaying potential imbalances in a form that is easy to understand. This approach has been found to facilitate communication greatly between cytogeneticist, clinician, and patient, and could be easily adopted by most centers.

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.

ESHRE PGD Consortium data collection VI: cycles from January to December 2003 with pregnancy follow-up to October 2004.

The sixth report of the ESHRE PGD Consortium is presented, relating to cycles collected for the calendar year 2003 and follow-up of the pregnancies and babies born up to October 2004. Since the beginning of the data collections, there has been a steady rise in the number of cycles, pregnancies and babies reported. For this report, 50 centres participated, reporting on 2984 cycles, 501 pregnancies and 373 babies born. Five hundred and twenty-nine cycles were reported for chromosomal abnormalities, 516 cycles were reported for monogenic diseases, 137 cycles were reported for sexing for X-linked diseases, 1722 cycles were reported for preimplantation genetic screening (PGS) and 80 cycles were reported for social sexing. Data VI is compared to the cumulative data for data collections I-V.

ESHRE PGD Consortium data collection V: cycles from January to December 2002 with pregnancy follow-up to October 2003.

The fifth report of the ESHRE PGD Consortium is presented (data collection V). For the first time, the cycle data were collected for one calendar year (2002) in the following October, so that data collection was complete for pregnancies and babies. The data were collected using a Filemaker Pro database and divided into referrals, cycles, pregnancies and babies. There are currently 66 active centres registered with the consortium; however, the data presented here were obtained from 43 centres and included 1603 referrals, 2219 cycles, 485 pregnancies and 382 babies born. The cycle data were divided into preimplantation genetic diagnosis (PGD) for inherited disorders (including chromosome abnormalities, sexing for X-linked disease and monogenic disorders), aneuploidy screening (PGS) and the use of PGD for social sexing. Data collection V is compared with the previous cumulative data collection (I-IV), which comprised 4058 PGD/PGS cycles that reached oocyte retrieval.

ESHRE PGD Consortium 'Best practice guidelines for clinical preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS)'.

Among the many educational materials produced by the European Society of Human Reproduction and Embryology (ESHRE) are guidelines. ESHRE guidelines may be developed for many reasons but their intent is always to promote best quality practices in reproductive medicine. In an era in which preimplantation genetic diagnosis (PGD) has become a reality, we must strive to maintain its efficacy and credibility by offering the safest and most effective treatment available. The dominant motivators for the development of current comprehensive guidelines for best PGD practice were (i) the absence of guidelines and/or regulation for PGD in many countries and (ii) the observation that no consensus exists on many of the clinical and technical aspects of PGD. As a consequence, the ESHRE PGD Consortium undertook to draw up guidelines aimed at giving information, support and guidance to potential, fledgling and established PGD centres. The success of a PGD treatment cycle is the result of great attention to detail. We have strived to provide a similar level of detail in this document and hope that it will assist staff in achieving the best clinical outcome for their patients.

Caithness revisited: Robertsonian chromosome polymorphism in Caithness house mice.

The purpose of this communication is to revise and clarify the position regarding house mouse populations with Robertsonian chromosomes in the northern Scottish county of Caithness.

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.

The future of preimplantation genetic diagnosis.

This paper reviews the current status of preimplantation genetic diagnosis (PGD), outlining the methods currently in use for the diagnosis of sex and single-gene defects. New approaches under development are described, e.g. fluorescent polymerase chain reaction (PCR), the use of sub-telomeric probes for patients with balanced reciprocal translocations, the analysis of first and second polar bodies, the use of lasers to facilitate the biopsy of embryos, and ways forward for infertile patients.

Simultaneous partial monosomy 10p and trisomy 5q in a case of hypoparathyroidism.

We report a case of monosomy for the distal region of the short arm of chromosome 10 (p13----ter) associated with trisomy for the terminal region of the long arm of chromosome 5 (q35.2----ter) that had originated from adjacent 1 segregation of a maternal reciprocal balanced translocation (5;10)(q35.2;p13). We review the clinical findings of previously reported cases of both partial monosomy for 10p and of partial trisomy for 5q, but to our knowledge there are no previous reports of the effects of these two chromosome anomalies together. Clinically our patient showed features typical of partial monosomy for 10p (including hypothyroidism) rather than partial trisomy 5q.

A new approach to the elucidation of complex chromosome rearrangements illustrated by a case of Rieger syndrome.

A patient with a complex chromosome rearrangement and unilateral Rieger syndrome is presented. This rearrangement involves four chromosomes and six breakpoints, one of which is at 4q25, the candidate region for Rieger syndrome. We discuss a novel approach to the elucidation of this case using a multiprobe fluorescence in situ hybridisation method to show rearrangements unpredictable from G banded analysis, and the clear and unambiguous presentation of the karyotype using computer generated colour ideograms.

Robertsonian translocations--reproductive risks and indications for preimplantation genetic diagnosis.

BACKGROUND: Robertsonian translocations carry reproductive risks that are dependent on the chromosomes involved and the sex of the carrier. We describe five couples that presented for preimplantation genetic diagnosis (PGD). METHODS: PGD was carried out using cleavage-stage (day 3) embryo biopsy, fluorescence in-situ hybridization (FISH) with locus-specific probes, and day 4 embryo transfer. RESULTS: Couple A (45,XX,der(14;21)(q10;q10)) had two previous pregnancies, one with translocation trisomy 21. A successful singleton pregnancy followed two cycles of PGD. Couple B (45,XX,der(13;14)(q10;q10)) had four miscarriages, two with translocation trisomy 14. One cycle of PGD resulted in triplets. Couple C (45,XX,der(13;14)(q10;q10)) had four years of infertility; two cycles were unsuccessful. Couple D (45,XY,der(13;14)(q10;q10)) presented with oligozoospermia. A singleton pregnancy followed two cycles of PGD. Couple E (45,XY,der(13;14)(q10;q10)) had a sperm count within the normal range and low levels of aneuploid spermatozoa. PGD was therefore not recommended. No evidence for a high incidence of embryos with chaotic or mosaic chromosome complements was found. CONCLUSIONS: For fertile couples, careful risk assessment and genetic counselling should precede consideration for PGD. Where translocation couples need assisted conception for subfertility, PGD is a valuable screen for imbalance, even when the risk of viable chromosome abnormality is low.

Clinical pregnancy following blastomere biopsy and PGD for a reciprocal translocation carrier: analysis of meiotic outcomes and embryo quality in two IVF cycles.

A couple were referred for preimplantation genetic diagnosis (PGD) following diagnosis of a reciprocal translocation in the female partner: 46,XX,t(14;22)(q11.2;q13.3). PGD was carried out using fluorescence in situ hybridization (FISH) with probes specific for the translocated and centric segments of chromosome 22. An initial cycle was unsuccessful, producing 11 embryos for biopsy, only one of which, when followed up on day 4, yielded more than 10 nuclei (median 7.5, n=10). In addition, five of the embryos showed mosaic or chaotic chromosome constitutions; some of these embryos had fragmented or multilobed abnormal nuclei, hindering interpretation of the FISH signals. The single embryo transferred did not result in a pregnancy. A second cycle, using a revised protocol, produced 10 embryos, three of which were transferred, resulting in an ongoing singleton pregnancy. All the remaining embryos yielded 12 to 23 nuclei by day 4 (median 17, n=7). Apart from some tetraploid nuclei, only one embryo showed mosaicism. The significance of the changes in protocol leading to the successful outcome is discussed, and the pattern of meiotic segregation products is analysed and compared with other previous reports of reciprocal translocations.

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.

Two cases of 5q deletions in patients with familial adenomatous polyposis: possible link with Caroli's disease.

Two cases are reported of patients with deletions of chromosome 5q. Both have familial adenomatous polyposis (FAP) and mild mental retardation. In both, macroscopic polyposis was confined to the proximal colon in adult life (in their thirties) although microscopic adenomatosis was shown in the more distal colon with occasional single polyps. Both subjects had dermoid cysts, and congenital hypertrophy of the retinal pigment epithelium (CHRPE) was seen in case 2. Case 1 has gastroduodenal polyps and desmoid tumours; case 2 has a marfanoid habitus with an abnormal pectus, wasted calf muscles and clawing of the toes, and Caroli's syndrome. His deletion is cytogenetically more extensive than that in case 1. The paucity of adenomas in the left side of the colon suggests that FAP cannot always confidently be excluded by sigmoidoscopy alone. The expression of the disease in the colon in these cases could be milder than in the more usual autosomal dominant cases where nonsense mutations resulting from single base changes of small deletions rather than deletion of the whole gene are the usual finding.

Development and implementation of a new rapid aneuploidy diagnostic service within the UK National Health Service and implications for the future of prenatal diagnosis.

BACKGROUND: Prenatal diagnosis for chromosome abnormality is routinely undertaken by full karyotype analysis of chromosomes from cultured cells; pregnant women must wait on average 13-14 days for their results. Autosomal trisomies, which account for around 80% of significant abnormalities, can be detected by quantitative fluorescence (QF) PCR. We report on the development and implementation of this technique as the first such routine service within a diagnostic department of the UK National Health Service (NHS). METHODS: We designed a "one-tube test" comprising four primer pairs for polymorphic tetranucleotide repeat sequences on chromosome 21, four primer pairs for sequences on chromosome 18, three primer pairs for sequences on chromosome 13, and one primer pair to identify the sex chromosomes. All prenatal samples received by our NHS diagnostic department between April, 2000, and April, 2001, were tested. After DNA extraction, PCR amplification was done and the products separated on a capillary-based genetic analyser; the results were interpreted with dedicated software. Follow-up karyotype analysis was done on all samples. FINDINGS: 1148 amniotic fluid samples, 188 chorionic villus samples, and 37 fetal tissue samples were tested; the amplification failure rate was zero with our current protocol. QF-PCR results were obtained and reported on 1314 (98%) of the prenatal samples; the remaining 22 (2%) were uninformative because of maternal-cell contamination. One case of mosaicism in a chorionic villus sample, and two cases indicating somatic expansion of a tetranucleotide repeat were found. No false positive or false negative results were obtained. The mean reporting time for the last 4 months of data collection was 1.25 working days. INTERPRETATION: QF-PCR aneuploidy testing is an efficient and accurate technique for the detection of autosomal trisomies in prenatal samples. Implementation of this service has led to the rapid diagnosis of abnormalities and early reassurance for women with normal results.