DNA Methylation Profiling of Uniparental Disomy Subjects Provides a Map of Parental Epigenetic Bias in the Human Genome.

Genomic imprinting is a mechanism in which gene expression varies depending on parental origin. Imprinting occurs through differential epigenetic marks on the two parental alleles, with most imprinted loci marked by the presence of differentially methylated regions (DMRs). To identify sites of parental epigenetic bias, here we have profiled DNA methylation patterns in a cohort of 57 individuals with uniparental disomy (UPD) for 19 different chromosomes, defining imprinted DMRs as sites where the maternal and paternal methylation levels diverge significantly from the biparental mean. Using this approach we identified 77 DMRs, including nearly all those described in previous studies, in addition to 34 DMRs not previously reported. These include a DMR at TUBGCP5 within the recurrent 15q11.2 microdeletion region, suggesting potential parent-of-origin effects associated with this genomic disorder. We also observed a modest parental bias in DNA methylation levels at every CpG analyzed across ∼1.9 Mb of the 15q11-q13 Prader-Willi/Angelman syndrome region, demonstrating that the influence of imprinting is not limited to individual regulatory elements such as CpG islands, but can extend across entire chromosomal domains. Using RNA-seq data, we detected signatures consistent with imprinted expression associated with nine novel DMRs. Finally, using a population sample of 4,004 blood methylomes, we define patterns of epigenetic variation at DMRs, identifying rare individuals with global gain or loss of methylation across multiple imprinted loci. Our data provide a detailed map of parental epigenetic bias in the human genome, providing insights into potential parent-of-origin effects.

PGD for the m.14487 T>C mitochondrial DNA mutation resulted in the birth of a healthy boy.

We report on the first PGD performed for the m.14487 T>C mitochondrial DNA (mtDNA) mutation in the MT-ND6 gene, associated with Leigh syndrome. The female carrier gave birth to a healthy baby boy at age 42. This case adds to the successes of PGD for mtDNA mutations.

Preimplantation genetic diagnosis in mitochondrial DNA disorders: challenge and success.

BACKGROUND: Mitochondrial or oxidative phosphorylation diseases are relatively frequent, multisystem disorders; in about 15% of cases they are caused by maternally inherited mitochondrial DNA (mtDNA) mutations. Because of the possible severity of the phenotype, the lack of effective treatment, and the high recurrence risk for offspring of carrier females, couples wish to prevent the transmission of these mtDNA disorders to their offspring. Prenatal diagnosis is problematic for several reasons, and concern the often poor correlation between mutation percentages and disease severity and the uncertainties about the representativeness of a fetal sample. A new option for preventing transmission of mtDNA disorders is preimplantation genetic diagnosis (PGD), which circumvents these problems by transferring an embryo below the threshold of clinical expression. METHODS: We present the data on nine PGD cycles in four female carriers of mitochondrial diseases: three mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) (m.3243A>G), and one Leigh (m.8993T>G). Our threshold for transfer after PGD is 15% for the m.3243A>G mutation and 30% for the m.8993T>G mutation. RESULTS: All four female carriers produced embryos eligible for transfer. The m.8993T>G mutation in oocytes/embryos showed more skewing than the m.3243A>G. In about 80% of the embryos the mutation load in the individual blastomeres was fairly constant (interblastomere differences <10%). However, in around 11% (in embryos with the m.3243A>G mutation only), the mutation load differed substantially (>15%) between blastomeres of a single embryo, mostly as a result of one outlier. The m.8993T>G carrier became pregnant and gave birth to a healthy son. CONCLUSIONS: PGD provides carriers of mtDNA mutations the opportunity to conceive healthy offspring.

Comprehensive embryo testing. Experts' opinions regarding future directions: an expert panel study on comprehensive embryo testing.

STUDY QUESTION: What do scientists in the field of preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS) consider to be the future direction of comprehensive embryo testing? SUMMARY ANSWER: Although there are many biological and technical limitations, as well as uncertainties regarding the meaning of genetic variation, comprehensive embryo testing will impact the IVF/PGD practice and a timely ethical reflection is needed. WHAT IS KNOWN ALREADY: Comprehensive testing using microarrays is currently being introduced in the context of PGD and PGS, and it is to be expected that whole-genome sequencing will also follow. Current ethical and empirical sociological research on embryo testing focuses on PGD as it is practiced now. However, empirical research and systematic reflection regarding the impact of comprehensive techniques for embryo testing is missing. STUDY DESIGN, SIZE AND DURATION: In order to understand the potential of this technology and to be able to adequately foresee its implications, we held an expert panel with seven pioneers in PGD. PARTICIPANTS/MATERIALS, SETTING, METHODS: We conducted an expert panel in October 2011 with seven PGD pioneers from Belgium, The Netherlands, Germany and the UK. MAIN RESULTS AND THE ROLE OF CHANCE: Participants expected the use of comprehensive techniques in the context of PGD. However, the introduction of these techniques in embryo testing requires timely ethical reflection as it involves a shift from choosing an embryo without a particular genetic disease (i.e. PGD) or most likely to result in a successful pregnancy (i.e. PGS) to choosing the best embryo based on a much wider set of criteria. Such ethical reflection should take account of current technical and biological limitations and also of current uncertainties with regard to the meaning of genetic variance. However, ethicists should also not be afraid to look into the future. There was a general agreement that embryo testing will be increasingly preceded by comprehensive preconception screening, thus enabling smart combinations of genetic testing. LIMITATIONS, REASONS FOR CAUTION: The group was composed of seven participants from four Western Europe countries. As willingness to participate in this study may be connected with expectations regarding the pace and direction of future developments, selection bias cannot be excluded. WIDER IMPLICATIONS OF THE FINDINGS: The introduction of comprehensive screening techniques in embryo testing calls for further ethical reflection that is grounded in empirical work. Specifically, there is a need for studies querying the opinions of infertile couples undergoing IVF/PGS regarding the desirability of embryo screening beyond aneuploidy. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the CSG, Centre for Society and Life Sciences (project number: 70.1.074). The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Further evidence that culture media affect perinatal outcome: findings after transfer of fresh and cryopreserved embryos.

BACKGROUND: We have previously shown that the medium used for culturing IVF embryos affects the birthweight of the resulting newborns. This observation with potentially far-reaching clinical consequences during later life, was made in singletons conceived during the first IVF treatment cycle after the transfer of fresh embryos. In the present study, we hypothesize that in vitro culture of embryos during the first few days of preimplantation development affects perinatal outcome, not only in singletons conceived in all rank order cycles but also in twins and in children born after transfer of frozen embryos. Furthermore, we investigated the effect of culture medium on gestational age (GA) at birth. METHODS: Oocytes and embryos from consecutive treatment cycles were alternately assigned to culture in either medium from Vitrolife or from Cook. Data on a cohort of 294 live born singletons conceived after fresh transfer during any of a patient's IVF treatment cycles, as well as data of 67 singletons conceived after frozen embryo transfer (FET) and of 88 children of 44 twin pregnancies after fresh transfer were analysed by means of multiple linear regression. RESULTS: In vitro culture in medium from Cook resulted in singletons after fresh transfer with a lower mean birthweight (adjusted mean difference, 112 g, P= 0.03), and in more singletons with low birthweight (LBW) <2500 g (P= 0.006) and LBW for GA ≥ 37 weeks (P= 0.015), when compared with singletons born after culture in medium from Vitrolife AB. GA at birth was not related to the medium used (adjusted difference, 0.05 weeks, P = 0.83). Among twins in the Cook group, higher inter-twin mean birthweight disparity and birthweight discordance were found. Z-scores after FET were -0.04 (± 0.14) in the Cook group compared with 0.18 (± 0.21) in the Vitrolife group (P> 0.05). CONCLUSIONS: Our findings support our hypothesis that culture medium influences perinatal outcome of IVF singletons and twins. A similar trend is seen in case of singletons born after FET. GA was not affected by culture medium. These results indicate that in vitro culture might be an important factor explaining the poorer perinatal outcome after assisted reproduction technology (ART). Further research is needed to confirm this culture medium-induced effect in humans and to provide more insight into whether it is caused by epigenetic disturbance of imprinted genes in fetal or placental tissues. Moreover, embryo culture media and their effects need to be investigated thoroughly to select the best embryo culture medium in order to minimize or prevent short-term risks and maybe even long-term disease susceptibility.

Profiles and motives for PGD: a prospective cohort study of couples referred for PGD in the Netherlands.

BACKGROUND: PGD is nowadays a well-established alternative to prenatal diagnosis. However, information with respect to couples' motives and profiles for choosing PGD is scarce. METHODS: A prospective cohort of 264 couples referred for PGD was interviewed semi-structurally after intake, and follow-up data were collated after 6-8 years. Outcome measures were: the primary choice shortly after intake (PGD intention), and their definitive use, until maximum 8 years later (PGD use). Logistic regression analysis was performed with clinical impact of the genetic disorder, couples' experiences, obstetric history and psychosocial factors as putative predictors. RESULTS: About 53.4% of the couples showed PGD intention. The experience of one or more miscarriages, the loss of an affected child and the absence of (acceptable) alternatives for the female partner positively contributed to PGD intention. For PGD use (45.8% of couples), infertility, a history of pregnancy termination(s) and the absence of alternatives according to the female partner were positive determinants. A living affected child reduced PGD use. Mode of inheritance and clinical impact of the disorder did not contribute. CONCLUSIONS: Fewer than 50% of the referred couples actually started PGD treatment. Personal experiences and reproductive history [the presence of a living affected child, infertility or a history of termination of pregnancy (TOP)] were more important determinants of eventual PGD use than the mode of inheritance or the expected clinical impact of the disorder.

Prevalence of at-risk genotypes for genotoxic effects decreases with age in a randomly selected population in Flanders: a cross sectional study.

BACKGROUND: We hypothesized that in Flanders (Belgium), the prevalence of at-risk genotypes for genotoxic effects decreases with age due to morbidity and mortality resulting from chronic diseases. Rather than polymorphisms in single genes, the interaction of multiple genetic polymorphisms in low penetrance genes involved in genotoxic effects might be of relevance. METHODS: Genotyping was performed on 399 randomly selected adults (aged 50-65) and on 442 randomly selected adolescents. Based on their involvement in processes relevant to genotoxicity, 28 low penetrance polymorphisms affecting the phenotype in 19 genes were selected (xenobiotic metabolism, oxidative stress defense and DNA repair, respectively 13, 6 and 9 polymorphisms). Polymorphisms which, based on available literature, could not clearly be categorized a priori as leading to an 'increased risk' or a 'protective effect' were excluded. RESULTS: The mean number of risk alleles for all investigated polymorphisms was found to be lower in the 'elderly' (17.0 ± 2.9) than the 'adolescent' (17.6 ± 3.1) subpopulation (P = 0.002). These results were not affected by gender nor smoking. The prevalence of a high (> 17 = median) number of risk alleles was less frequent in the 'elderly' (40.6%) than the 'adolescent' (51.4%) subpopulation (P = 0.002). In particular for phase II enzymes, the mean number of risk alleles was lower in the 'elderly' (4.3 ± 1.6 ) than the 'adolescent' age group (4.8 ± 1.9) P < 0.001 and the prevalence of a high (> 4 = median) number of risk alleles was less frequent in the 'elderly' (41.3%) than the adolescent subpopulation (56.3%, P < 0.001). The prevalence of a high (> 8 = median) number of risk alleles for DNA repair enzyme-coding genes was lower in the 'elderly' (37,3%) than the 'adolescent' subpopulation (45.6%, P = 0.017). CONCLUSIONS: These observations are consistent with the hypothesis that, in Flanders, the prevalence of at-risk alleles in genes involved in genotoxic effects decreases with age, suggesting that persons carrying a higher number of at risk alleles (especially in phase II xenobiotic-metabolizing or DNA repair genes) are at a higher risk of morbidity and mortality from chronic diseases. Our findings also suggest that, regarding risk of disease associated with low penetrance polymorphisms, multiple polymorphisms should be taken into account, rather than single ones.

Electrical signals affect the cardiomyocyte transcriptome independently of contraction.

Cardiomyocytes in vivo are continuously subjected to electrical signals that evoke contractions and instigate drastic changes in the cells' morphology and function. Studies on how electrical stimulation affects the cardiac transcriptome have remained limited to a small number of heart-specific genes. Furthermore, these studies have ignored the interplay between the electrical excitation and the subsequent contractions. We carried out a genomewide assessment of the effects of electrical signaling on gene expression, while distinguishing between the effects deriving from the electrical pulses themselves and the effects instigated by the evoked contractions. Changes in gene expression in primary cultures of neonatal ventricular cardiomyocytes from Lewis Rattus norvegicus were investigated with microarrays and RT-quantitative PCR (QPCR). A series of experiments was included in which the culture medium was supplemented with the contraction inhibitor blebbistatin to allow for electrical stimulation in the absence of contraction. Electrical stimulation was shown to directly enhance calcium handling and induce cardiomyocyte differentiation by arresting cell division and activating key cardiac transcription factors as well as additional differentiation mechanisms such as wnt signaling. Several genes involved in metabolism were also directly activated by electrical stimulation. Furthermore, our data suggest that contraction exerts negative feedback on the transcription of various genes. Together, these observations indicate that intercellular electric currents between adjacent cardiomyocytes have an important role in cardiomyocyte development. They act at least partially through a pulse-specific gene expression program that is activated independently from the evoked contractions.

Effect of in vitro culture of human embryos on birthweight of newborns.

BACKGROUND: In animal models, in vitro culture of preimplantation embryos has been shown to be a risk factor for abnormal fetal outcome, including high and low birthweight. In the human, mean birthweight of singletons after in vitro fertilization (IVF) is considerably lower than after natural conception, but it is not known whether culture conditions play a role in this. METHODS: We compared pregnancy rates and perinatal outcomes from singleton pregnancies resulting from a total of 826 first IVF treatment cycles in which oocytes and embryos were randomly allocated to culture in either of two commercially available sequential media systems. RESULTS: When the 110 live born singletons in the Vitrolife group were compared with the 78 singletons in the Cook group, birthweight +/- SEM (3453 +/- 53 versus 3208 +/- 61 g, P = 0.003), and birthweight adjusted for gestational age and gender (mean z-score +/- SEM: 0.13 +/- 0.09 versus -0.31 +/- 0.10, P = 0.001) were both significantly higher in the Vitrolife group. When analyzed by multiple linear regression together with several other variables that could possibly affect birthweight as covariates, the type of culture medium was significantly (P = 0.01) associated with birthweight. CONCLUSIONS: In vitro culture of human embryos can affect birthweight of live born singletons.

Differential gene expression in cumulus cells as a prognostic indicator of embryo viability: a microarray analysis.

Besides the established selection criteria based on embryo morphology and blastomere number, new parameters for embryo viability are needed to improve the clinical outcome of IVF and more particular of elective single-embryo transfer. Genome-wide gene expression in cumulus cells was studied, since these cells surround the oocyte inside the follicle and therefore possibly reflect oocyte developmental potential. Early cleavage (EC) was chosen as a parameter for embryo viability. Gene expression in cumulus cells from eight oocytes resulting in an EC embryo (EC-CC; n = 8) and from eight oocytes resulting in a non-EC (NEC) embryo (NEC-CC; n = 8) was analysed using microarrays (n = 16). A total of 611 genes were differentially expressed (P < 0.01), mainly involved in cell cycle, angiogenesis, apoptosis, epidermal growth factor, fibroblast growth factor and platelet-derived growth factor signalling, general vesicle transport and chemokine and cytokine signalling. Of the 25 selected differentially expressed genes analysed by quantitative real-time PCR 15 (60%) genes could be validated in the original samples. Of these 8 (53%) could also be validated in 24 (12-EC-CC and 12 NEC-CC) extra independent samples. The most differentially expressed genes among these were CCND2, CXCR4, GPX3, CTNND1 DHCR7, DVL3, HSPB1 and TRIM28, which probably point to hypoxic conditions or a delayed oocyte maturation in NEC-CC samples. This opens up perspectives for new molecular embryo or oocyte selection parameters which might also be useful in countries where the selection has to be made at the oocyte stage before fertilization instead of at the embryonic stage.

Variable clinical manifestation of a novel missense mutation in the alpha-tropomyosin (TPM1) gene in familial hypertrophic cardiomyopathy.

OBJECTIVES: This study was initiated to identify the disease-causing genetic defect in a family with hypertrophic cardiomyopathy (HCM) and high incidence of sudden death. BACKGROUND: Familial hypertropic cardiomyopathy (FHC) is an autosomal dominant transmitted disorder that is genetically and clinically heterogeneous. Mutations in 11 genes have been associated with the pathogenesis of the disease. METHODS: We studied a large FHC family, first by linkage analysis, to identify the gene involved, and subsequently screened the gene, encoding alpha-tropomyosin (TPM1), for mutations by using single-strand conformation polymorphism and sequencing analysis. RESULTS: Twelve family members presented clinical features of HCM, five of whom died at young age, while others had only mild clinical features. Marker analysis showed linkage for the TPM1 gene on chromosome 15q22 (maximal logarithm of the odds score is 5.16, theta = 0); subsequently, a novel missense mutation (Glu62Gln) was identified. CONCLUSIONS: The novel mutation identified in TPM1 is associated with the clinical features of cardiac hypertrophy in all but one genetically affected member of this large family. The clinical data suggest a malignant phenotype at young age with a variable clinical manifestation and penetrance at older age. The Glu62Gln mutation is the sixth TPM1 mutation identified as the cause of FHC, indicating that mutations in this gene are very rare. This is the first reported amino acid substitution at the f-position within the coiled-coil structure of the tropomyosin protein.

PGD for hereditary breast and ovarian cancer: the route to universal tests for BRCA1 and BRCA2 mutation carriers.

Preimplantation Genetic Diagnosis (PGD) is a method of testing in vitro embryos as an alternative to prenatal diagnosis with possible termination of pregnancy in case of an affected child. Recently, PGD for hereditary breast and ovarian cancer caused by BRCA1 and BRCA2 mutations has found its way in specialized labs. We describe the route to universal single-cell PGD tests for carriers of BRCA1/2 mutations. Originally, mutation-specific protocols with one or two markers were set up and changed when new couples were not informative. This route of changing protocols was finalized after 2 years with universal tests for both BRCA1 and BRCA2 mutation carriers based on haplotyping of, respectively, 6 (BRCA1) and 8 (BRCA2) microsatellite markers in a multiplex PCR. Using all protocols, 30 couples had a total of 47 PGD cycles performed. Eight cycles were cancelled upon IVF treatment due to hypostimulation. Of the remaining 39 cycles, a total of 261 embryos were biopsied and a genetic diagnosis was obtained in 244 (93%). In 34 of the 39 cycles (84.6%), an embryo transfer was possible and resulted in 8 pregnancies leading to a fetal heart beat per oocyte retrieval of 20.5% and a fetal heart beat per embryonic transfer of 23.5%. The preparation time and costs for set-up and validation of tests are minimized. The informativity of microsatellite markers used in the universal PGD-PCR tests is based on CEPH and deCODE pedigrees, making the tests applicable in 90% of couples coming from these populations.

Fertility preservation in female classic galactosemia patients.

Almost every female classic galactosemia patient develops primary ovarian insufficiency (POI) as a diet-independent complication of the disease. This is a major concern for patients and their parents, and physicians are often asked about possible options to preserve fertility. Unfortunately, there are no recommendations on fertility preservation in this group. The unique pathophysiology of classic galactosemia with a severely reduced follicle pool at an early age requires an adjusted approach. In this article recommendations for physicians based on current knowledge concerning galactosemia and fertility preservation are made. Fertility preservation is only likely to be successful in very young prepubertal patients. In this group, cryopreservation of ovarian tissue is currently the only available technique. However, this technique is not ready for clinical application, it is considered experimental and reduces the ovarian reserve. Fertility preservation at an early age also raises ethical questions that should be taken into account. In addition, spontaneous conception despite POI is well described in classic galactosemia. The uncertainty surrounding fertility preservation and the significant chance of spontaneous pregnancy warrant counseling towards conservative application of these techniques. We propose that fertility preservation should only be offered with appropriate institutional research ethics approval to classic galactosemia girls at a young prepubertal age.

SNP array-based copy number and genotype analyses for preimplantation genetic diagnosis of human unbalanced translocations.

Preimplantation genetic diagnosis (PGD) for chromosomal rearrangements (CR) is mainly based on fluorescence in situ hybridisation (FISH). Application of this technique is limited by the number of available fluorochromes, the extensive preclinical work-up and technical and interpretative artefacts. We aimed to develop a universal, off-the-shelf protocol for PGD by combining single-nucleotide polymorphism (SNP) array-derived copy number (CN) determination and genotyping for detection of unbalanced translocations in cleavage-stage embryos. A total of 36 cleavage-stage embryos that were diagnosed as unbalanced by initial PGD FISH analysis were dissociated (n=146) and amplified by multiple displacement amplification (MDA). SNP CNs and genotypes were determined using SNP array. Epstein-Barr Virus-transformed cell lines with known CR were used for optimising the genomic smoothing (GS) length setting to increase signal to noise ratio. SNP CN analysis showed 23 embryos (64%) that were unbalanced in all blastomeres for the chromosomes involved in the translocation, 5 embryos (14%) that were normal or balanced in all blastomeres and 8 embryos (22%) that were mosaic. SNP genotyping, based on analysis of informative SNP loci with opposing homozygous parental genotypes, confirmed partial monosomies associated with inheritance of unbalanced translocation in surplus embryos. We have developed a universal MDA-SNP array technique for chromosome CN analysis in single blastomeres. SNP genotyping could confirm partial monosomies. This combination of techniques showed improved diagnostic specificity compared with FISH and may provide more reliable PGD analysis associated with higher embryo transfer rate.

Preimplantation genetic diagnosis (PGD) for Huntington's disease: the experience of three European centres.

This study provides an overview of 13 years of experience of preimplantation genetic diagnosis (PGD) for Huntington's disease (HD) at three European PGD centres in Brussels, Maastricht and Strasbourg. Information on all 331 PGD intakes for HD, couples' reproductive history, PGD approach, treatment cycles and outcomes between 1995 and 2008 were collected prospectively. Of 331 couples for intake, 68% requested direct testing and 32% exclusion testing (with a preponderance of French couples). At the time of PGD intake, 39% of women had experienced one or more pregnancies. A history of pregnancy termination after prenatal diagnosis was observed more frequently in the direct testing group (25%) than in the exclusion group (10%; P=0.0027). PGD workup was based on two approaches: (1) direct testing of the CAG-triplet repeat and (2) linkage analysis using intragenic or flanking microsatellite markers of the HTT gene. In total, 257 couples had started workup and 174 couples (70% direct testing, 30% exclusion testing) completed at least one PGD cycle. In total, 389 cycles continued to oocyte retrieval (OR). The delivery rates per OR were 19.8%, and per embryo transfer 24.8%, resulting in 77 deliveries and the birth of 90 children. We conclude that PGD is a valuable and safe reproductive option for HD carriers and couples at risk of transmitting HD.

eSET irrespective of the availability of a good-quality embryo in the first cycle only is not effective in reducing overall twin pregnancy rates.

INTRODUCTION: In several clinics, elective single-embryo transfer (eSET) is applied in a selected group of patients based on age and the availability of a good-quality embryo. Whether or not eSET can be applied irrespective of the presence of a good-quality embryo in the first cycle, to further reduce the twin pregnancy rate, remains to be elucidated. METHODS: In patients <38 years two transfer strategies were compared, which differed in the first cycle only: group A (n = 141) received eSET irrespective of the availability of a good-quality embryo, and group B (n = 174) received eSET when a good-quality embryo was available while otherwise they received double embryo transfer (DET; referred to as eSET/DET transfer policy). In any subsequent cycle, in both groups the eSET/DET transfer policy was applied. RESULTS: After completion of their IVF treatment (including a maximum of three fresh cycles and the transfer of frozen-thawed embryos), comparable cumulative live birth rates (62.4% in group A and 62.6% in group B) and twin pregnancy rates (10.1 versus 13.4%) were found. However, patients in group A required significantly more fresh (2.0 versus 1.8) and frozen (0.8 versus 0.5) cycles. CONCLUSIONS: The transfer of one embryo in the first cycle, irrespective of the availability of a good-quality embryo, in all patients <38 years, is not an effective transfer policy for reducing the overall twin pregnancy rate.

In unselected patients, elective single embryo transfer prevents all multiples, but results in significantly lower pregnancy rates compared with double embryo transfer: a randomized controlled trial.

BACKGROUND: Elective single embryo transfer (eSET) in a selected group of patients (i.e. young patients with at least one good quality embryo) reduces the number of multiple pregnancies in an IVF programme. However, the reduced overall multiple pregnancy rate (PR) is still unacceptably high. Therefore, a randomized controlled trial (RCT) was conducted comparing eSET and double embryo transfer (DET) in an unselected group of patients (i.e. irrespective of the woman's age or embryo quality). METHODS: Consenting unselected patients were randomized between eSET (RCT-eSET) (n = 154) or DET (RCT-DET) (n = 154). Randomization was performed just prior to the first embryo transfer, provided that at least two 2PN zygotes were available. Non-participants received our standard transfer policy [SP-eSET in a selected group of patients (n = 100), otherwise SP-DET (n = 122)]. RESULTS: The ongoing PR after RCT-eSET was significantly lower as compared with RCT-DET (21.4 versus 40.3%) and the twin PR was reduced from 21.0% after RCT-DET to 0% after RCT-eSET. The ongoing PRs after SP-eSET and SP-DET did not differ significantly (33.0 versus 30.3%), with an overall twin PR of 12.9%. CONCLUSION: To avoid twin pregnancies resulting from an IVF treatment, eSET should be applied in all patients. The consequence would be a halving of the ongoing PR as compared with applying a DET policy in all patients. The transfer of one embryo in a selected group of good prognosis patients leads to a less drastic reduction in PR but maintains a twin PR of 12.9%.

Toward positional cloning of the curly tail gene.

BACKGROUND: The curly tail (ct) mutant mouse is one of the best-studied mouse models of spina bifida. The ct mutation has been localized to distal chromosome 4 in two independent studies and was recently postulated to be in the Grhl-3 gene. METHODS: A recombinant BALB/c-ct strain was generated and used to precisely map the ct gene. RESULTS: We report the absence of gross chromosomal abnormalities and the precise mapping of the ct gene to a 3-Mb region at 135 Mb (66 cM) from the centromere, closely linked to the polymorphic microsatellite marker D4Mit148. Candidate genes, Idb3, Wnt4, Cdc42, and perlecan, all localized in the critical region, were studied by sequence and expression analyses. Our data indicate that these genes in all probability do not account for the ct phenotype. In addition, our expression data do not provide strong evidence that Grhl-3 is indeed the ct gene. CONCLUSIONS: The ct gene has not yet been identified. A total of 29 candidate genes remain present in the critical region. Refined mapping studies need to be performed to further narrow the region and additional candidate genes need to be examined. Supplementary material for this article can be found on the Birth Defects Research (Part A) website (http://www.mrw.interscience.wiley.com/suppmat/1542-0752/suppmat/2005/73/tables_S3-S6.doc).