Chromosome fragility at FRAXA in human cleavage stage embryos at risk for fragile X syndrome.

Fragile X syndrome (FXS), the most common inherited intellectual disability syndrome, is caused by expansion and hypermethylation of the CGG repeat in the 5' UTR of the FMR1 gene. This expanded repeat, also known as the rare fragile site FRAXA, causes X chromosome fragility in cultured cells from patients but only when induced by perturbing pyrimidine synthesis. We performed preimplantation genetic diagnosis (PGD) on 595 blastomeres biopsied from 442 cleavage stage embryos at risk for FXS using short tandem repeat (STR) markers. In six blastomeres, from five embryos an incomplete haplotype was observed with loss of all alleles telomeric to the CGG repeat. In all five embryos, the incomplete haplotype corresponded to the haplotype carrying the CGG repeat expansion. Subsequent analysis of additional blastomeres from three embryos by array comparative genomic hybridization (aCGH) confirmed the presence of a terminal deletion with a breakpoint close to the CGG repeat in two blastomeres from one embryo. A blastomere from another embryo showed the complementary duplication. We conclude that a CGG repeat expansion at FRAXA causes X chromosome fragility in early human IVF embryos at risk for FXS.

In vitro screening of embryos by whole-genome sequencing: now, in the future or never?

STUDY QUESTION: What are the analytical and clinical validity and the clinical utility of in vitro screening of embryos by whole-genome sequencing? SUMMARY ANSWER: At present there are still many limitations in terms of analytical and clinical validity and utility and many ethical questions remain. WHAT IS KNOWN ALREADY: Whole-genome sequencing of IVF/ICSI embryos is technically possible. Many loss-of-function mutations exist in the general population without serious effects on the phenotype of the individual. Moreover, annotations of genes and the reference genome are still not 100% correct. STUDY DESIGN, SIZE, DURATION: We used publicly available samples from the 1000 Genomes project and Complete Genomics, together with 42 samples from in-house research samples of parents from trios to investigate the presence of loss-of-function mutations in healthy individuals. PARTICIPANTS/MATERIALS, SETTING, METHODS: In the samples, we looked for mutations in genes that are associated with a selection of severe Mendelian disorders with a known molecular basis. We looked for mutations predicted to be damaging by PolyPhen and SIFT and for mutations annotated as disease causing in Human Genome Mutation Database (HGMD). MAIN RESULTS AND THE ROLE OF CHANCE: More than 40% of individuals who can be considered healthy have mutations that are predicted to be damaging in genes associated with severe Mendelian disorders or are annotated as disease causing. LIMITATIONS, REASONS FOR CAUTION: The analysis relies on current knowledge and databases are continuously updated to reflect our increasing knowledge about the genome. In the process of our analysis several updates were already made. WIDER IMPLICATIONS OF THE FINDINGS: At this moment it is not advisable to use whole-genome sequencing as a tool to set up health profiles to select embryos for transfer. We also raise some ethical questions that have to be addressed before this technology can be used for embryo selection. TRIAL REGISTRATION NUMBER: N/A.

Current issues in medically assisted reproduction and genetics in Europe: research, clinical practice, ethics, legal issues and policy.

STUDY QUESTION: How has the interface between genetics and assisted reproduction technology (ART) evolved since 2005? SUMMARY ANSWER: The interface between ART and genetics has become more entwined as we increase our understanding about the genetics of infertility and we are able to perform more comprehensive genetic testing. WHAT IS KNOWN ALREADY: In March 2005, a group of experts from the European Society of Human Genetics and European Society of Human Reproduction and Embryology met to discuss the interface between genetics and ART and published an extended background paper, recommendations and two Editorials. STUDY DESIGN, SIZE, DURATION: An interdisciplinary workshop was held, involving representatives of both professional societies and experts from the European Union Eurogentest2 Coordination Action Project. PARTICIPANTS/MATERIALS, SETTING, METHODS: In March 2012, a group of experts from the European Society of Human Genetics, the European Society of Human Reproduction and Embryology and the EuroGentest2 Coordination Action Project met to discuss developments at the interface between clinical genetics and ART. MAIN RESULTS AND THE ROLE OF CHANCE: As more genetic causes of reproductive failure are now recognized and an increasing number of patients undergo testing of their genome prior to conception, either in regular health care or in the context of direct-to-consumer testing, the need for genetic counselling and PGD may increase. Preimplantation genetic screening (PGS) thus far does not have evidence from RCTs to substantiate that the technique is both effective and efficient. Whole genome sequencing may create greater challenges both in the technological and interpretational domains, and requires further reflection about the ethics of genetic testing in ART and PGD/PGS. Diagnostic laboratories should be reporting their results according to internationally accepted accreditation standards (ISO 15189). Further studies are needed in order to address issues related to the impact of ART on epigenetic reprogramming of the early embryo. LIMITATIONS, REASONS FOR CAUTION: The legal landscape regarding assisted reproduction is evolving, but still remains very heterogeneous and often contradictory. The lack of legal harmonization and uneven access to infertility treatment and PGD/PGS fosters considerable cross-border reproductive care in Europe, and beyond. WIDER IMPLICATIONS OF THE FINDINGS: This continually evolving field requires communication between the clinical genetics and IVF teams and patients to ensure that they are fully informed and can make well-considered choices. STUDY FUNDING/COMPETING INTERESTS: Funding was received from ESHRE, ESHG and EuroGentest2 European Union Coordination Action project (FP7 - HEALTH-F4-2010-26146) to support attendance at this meeting.

Human trophectoderm cells are not yet committed.

STUDY QUESTION: Are human trophectoderm (TE) cells committed or still able to develop into inner cell mass (ICM) cells? SUMMARY ANSWER: Human full blastocyst TE cells still have the capacity to develop into ICM cells expressing the pluripotency marker NANOG, thus they are not yet committed. WHAT IS KNOWN ALREADY: Human Day 5 full blastocyst TE cells express the pluripotency markers POU5F1, SOX2 and SALL4 as well as the TE markers HLA-G and KRT18 but not yet CDX2, therefore their developmental direction may not yet be definite. STUDY DESIGN, SIZE, DURATION: The potency of human blastocyst TE cells was investigated by determining their in vitro capacity to develop into a blastocyst with ICM cells expressing NANOG; TE cells were isolated either by aspiration under visual control or after labeling with fluorescent 594-wheat germ agglutinin. Further on, aspirated TE cells were also labeled with fluorescent PKH67 and repositioned in the center of the original embryo. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human preimplantation embryos were used for research after obtaining informed consent from IVF patients. The experiments were approved by the Local Ethical Committee and the 'Belgian Federal Committee on medical and scientific research on embryos in vitro'. Outer cells were isolated and reaggregated by micromanipulation. Reconstituted embryos were analyzed by immunocytochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: Isolated and reaggregated TE cells from full human blastocysts are able to develop into blastocysts with ICM cells expressing the pluripotency marker NANOG. Moreover, the majority of the isolated TE cells which were repositioned in the center of the embryo do not sort back to their original position but integrate within the ICM and start to express NANOG. LIMITATIONS, REASONS FOR CAUTION: Owing to legal and ethical restrictions, manipulated human embryos cannot be transferred into the uterus to determine their totipotent capacity. The definitive demonstration that embryos reconstructed with TE cells are a source of pluripotent cells is to obtain human embryonic stem cell 'like' line(s), which will allow full characterization of the cells. WIDER IMPLICATIONS OF THE FINDINGS: Our finding has important implications in reproductive medicine and stem cell biology because TE cells have a greater developmental potential than assumed previously. STUDY FUNDING/COMPETING INTEREST(S): Scientific Research Foundation-Flanders (FWO-Vlaanderen) and Research Council (OZR) of the Vrije Universiteit Brussel. None of the authors declared a conflict of interest.

Pregnancy outcome in carriers of Robertsonian translocations.

Robertsonian translocation carriers are at increased risk for infertility, spontaneous abortions, or chromosomally unbalanced offspring. Reproductive counseling of these carriers is challenging. We performed a retrospective analysis of all prenatal diagnoses from Robertsonian translocation carriers during the time period January 1, 1992 through December 31, 2007. Data on the carriers and the results of their prenatal analyses were retrieved as well as data on their previous pregnancies. We identified 28 female and 20 male carriers of Robertsonian translocations and results on 79 prenatal samples were obtained. Among female carriers, 10.3% of chorionic villus sampling and 5.9% of amniocentesis results were unbalanced, whereas for male carriers, this was 3.6% and 0%, respectively. When considering all pregnancies involving carriers, 52.7% of those to female carriers and 61.8% of those to male carriers led to the birth of a healthy child. Male carriers in whom the translocation was ascertained because of infertility or recurrent miscarriages appear to be at higher risk, whereas carriers in whom ascertainment was because of a family history are at lower risk. We conclude that pregnancies of Robertsonian translocation carriers are at increased risk for chromosomal imbalance, and prenatal chromosomal testing should be discussed. More than half of the pregnancies led to the birth of a healthy child, but prediction of which couples will be successful in obtaining a pregnancy with or without assisted reproductive technologies and/or embryo selection remains difficult. The reason for ascertainment of the translocation should be taken into account when counseling these couples. The possibility of preimplantation genetic diagnosis should also be discussed with the couples.

Two-year auxological and medical outcome of singletons born after embryo biopsy applied in preimplantation genetic diagnosis or preimplantation genetic screening.

BACKGROUND: Embryo biopsy is an essential but invasive procedure to perform preimplantation genetic diagnosis (PGD) or preimplantation genetic screening (PGS). The major objective of this study was to determine whether embryo biopsy might cause post-natal growth restriction. METHODS: We compared growth data and physical findings at birth and 2 years for singletons born either after PGD/PGS (n = 70), ICSI (n = 70) or natural conception (NC) (n = 70). Children were matched for gender, maternal educational level, mother tongue and birth order. RESULTS: No significant differences were found between the three groups regarding weight, height and head circumference standard deviation scores (SDS) at birth and at age 2 years, although the PGD/PGS children tended to have a lower birthweight compared with the NC children. At 2 years, the mean BMI SDS in PGD/PGS children was significantly lower compared with NC children (P = 0.005). PGD/PGS children were more frequently born after Caesarian section than ICSI children, but had no more congenital malformations, hospital admissions and surgical interventions compared with ICSI and NC children. CONCLUSIONS: Singleton children at age 2 years born after embryo biopsy applied in PGD/PGS present a similar post-natal linear growth compared with ICSI and NC children. PGD/PGS singletons appear not to be at higher risk for congenital malformations and surgical interventions during the first 2 years of life. To date, there have been no observable detrimental effects of the PGD/PGS procedure on children.

A proposal for reproductive counselling in carriers of Robertsonian translocations: 10 years of experience with preimplantation genetic diagnosis.

BACKGROUND: Carriers of Robertsonian translocations are at increased risk for infertility, repeated miscarriage and aneuploid offspring. In the present study, 10 years of experience with preimplantation genetic diagnosis (PGD) for Robertsonian translocations is reviewed and these data are used to improve the reproductive counselling in the carriers. METHODS: A retrospective analysis was performed of all requests and cycles for PGD for Robertsonian translocations at our centre between January 1997 and December 2006. Data on the characteristics of the couples and on the PGD cycles were retrieved from the medical records. These data were recorded for the whole group and according to the sex of the carrier. RESULTS: A total of 111 couples made a request for PGD in our centre, of which 76 had at least one PGD cycle. In the PGD cycles embryo transfer could take place in 66.1% of the cycles with oocyte pick-up and positive hCG was found in 42.7% of the cycles with embryo transfer. The live born delivery rate was 20.2% per cycle with oocyte retrieval and 30.5% per cycle with embryo transfer. CONCLUSIONS: With a live birth delivery rate of 32.9% per couple, PGD is considered a good option for these couples, especially when there is a coexisting fertility problem. PGD reduces the risk of miscarriage and allows couples to have a healthy child within a relatively short time span compared with spontaneous pregnancies. However, for young, fertile couples, the chances of having a healthy child after a number of spontaneous pregnancies, should not be ignored.

The experience of two European preimplantation genetic diagnosis centres on human leukocyte antigen typing.

BACKGROUND: Two European centres report on human leukocyte antigen (HLA) typing of preimplantation embryos for haematopoietic stem cell (HSC) transplantation: 'UZ Brussel' in Brussels and 'Genoma' in Rome. Both centres have 6 years' experience with technical and clinical aspects of this type of genetic analysis on single blastomeres. METHODS: Both centres apply a similar technique for preimplantation HLA typing using short tandem repeats linked to the HLA locus in multiplex PCR for haplotyping. RESULTS: At present, a conclusive HLA diagnosis could be assured in 92.8% and 90.3% of the embryos at UZ Brussel and at Genoma, respectively. The implantation rates were 32.4% and 28.2%, respectively, and the birth rates per cycle were 9.4% and 18.6%, respectively. The HLA programme at UZ Brussel and at Genoma resulted in the birth of 9 babies and 3 successful HSC transplantations, and 42 babies and 7 successful HSC transplantations, respectively, so far. CONCLUSIONS: Drastic embryo selection for preimplantation HLA typing (in theory 1/4 for HLA, 1/8 for HLA in combination with sexing for X-linked recessive diseases, 3/16 for HLA in combination with autosomal recessive disorders) resulted overall in the birth of 51 babies (15.9% live birth rate per started cycle) in two European centres.

Human embryonic stem cell lines derived from single blastomeres of two 4-cell stage embryos.

BACKGROUND: Recently, we demonstrated that single blastomeres of a 4-cell stage human embryo are able to develop into blastocysts with inner cell mass and trophectoderm. To further investigate potency at the 4-cell stage, we aimed to derive pluripotent human embryonic stem cells (hESC) from single blastomeres. METHODS: Four 4-cell stage embryos were split on Day 2 of preimplantation development and the 16 blastomeres were individually cultured in sequential medium. On Day 3 or 4, the blastomere-derived embryos were plated on inactivated mouse embryonic fibroblasts (MEFs). RESULTS: Ten out of sixteen blastomere-derived morulae attached to the MEFs, and two produced an outgrowth. They were mechanically passaged onto fresh MEFs as described for blastocyst ICM-derived hESC, and shown to express the typical stemness markers by immunocytochemistry and/or RT-PCR. In vivo pluripotency was confirmed by the presence of all three germ layers in the teratoma obtained after injection in immunodeficient mice. The first hESC line displays a mosaic normal/abnormal 46, XX, dup(7)(q33qter), del(18)(q23qter) karyotype. The second hESC line displays a normal 46, XY karyotype. CONCLUSION: We report the successful derivation and characterization of two hESC lines from single blastomeres of four split 4-cell stage human embryos. These two hESC lines were derived from distinct embryos, proving that at least one of the 4-cell stage blastomeres is pluripotent.

What next for preimplantation genetic screening? High mitotic chromosome instability rate provides the biological basis for the low success rate.

Preimplantation genetic screening is being scrutinized, as recent randomized clinical trials failed to observe the expected significant increase in live birth rates following fluorescence in situ hybridization (FISH)-based screening. Although these randomized clinical trials are criticized on their design, skills or premature stop, it is generally believed that well-designed and well-executed randomized clinical trials would resolve the debate about the potential benefit of preimplantation genetic screening. Since FISH can analyze only a limited number of chromosomal loci, some of the embryos transferred might be diagnosed as 'normal' but in fact be aneuploid for one or more chromosomes not tested. Hence, genome-wide array comparative genome hybridization screening enabling aneuploidy detection of all chromosomes was thought to be a first step toward a better design. We recently showed array screening indeed enables accurate determination of the copy number state of all chromosomes in a single cell. Surprisingly, however, this genome-wide array screening revealed a much higher frequency and complexity of chromosomal aberrations in early embryos than anticipated, with imbalances in a staggering 90% of all embryos. The mitotic error rate in cleavage stage embryos was proven to be higher than the meiotic aneuploidy rate and as a consequence, the genome of a single blastomere is not representative for the genome of the other cells of the embryo. Hence, potentially viable embryos will be discarded upon screening a single blastomere. This observation provides a biological basis for the failure of the randomized clinical trials to increase baby-take-home rates using FISH on cleavage stage embryos.

Generation of lung epithelial-like tissue from human embryonic stem cells.

BACKGROUND: Human embryonic stem cells (hESC) have the capacity to differentiate in vivo and in vitro into cells from all three germ lineages. The aim of the present study was to investigate the effect of specific culture conditions on the differentiation of hESC into lung epithelial cells. METHODS: Undifferentiated hESC, grown on a porous membrane in hESC medium for four days, were switched to a differentiation medium for four days; this was followed by culture in air-liquid interface conditions during another 20 days. Expression of several lung markers was measured by immunohistochemistry and by quantitative real-time RT-PCR at four different time points throughout the differentiation and compared to appropriate controls. RESULTS: Expression of CC16 and NKX2.1 showed a 1,000- and 10,000- fold increase at day 10 of differentiation. Other lung markers such as SP-C and Aquaporin 5 had the highest expression after twenty days of culture, as well as two markers for ciliated cells, FOXJ1 and beta-tubulin IV. The results from qRT-PCR were confirmed by immunohistochemistry on paraffin-embedded samples. Antibodies against CC16, SP-A and SP-C were chosen as specific markers for Clara Cells and alveolar type II cells. The functionality was tested by measuring the secretion of CC16 in the medium using an enzyme immunoassay. CONCLUSION: These results suggest that by using our novel culture protocol hESC can be differentiated into the major cell types of lung epithelial tissue.

Blurring boundaries. Interviews with PGT couples about comprehensive chromosome screening.

OBJECTIVE: Comprehensive chromosome examination is a promising approach to Preimplantation Genetic Testing (PGT). Next to testing of specific chromosomes, such as in the case of reduced fertility due to chromosomal translocations, it allows testing of all chromosomes. Hence it potentially reduces the time to pregnancy and the risk of miscarriage. But comprehensive testing also introduces some ethical issues. For example, what is the role of the professional in the decision making regarding embryos with chromosomal abnormalities that are potentially viable? Which chromosomal abnormalities should be communicated to people undergoing fertility treatment? With this paper we wanted to explore the ethical issues related to comprehensive chromosome screening in Preimplantation Genetic Testing. DESIGN: In order to explore these issues, we interviewed seven couples undergoing PGT for chromosomal translocations at the VUB University Hospital, Belgium. We presented them with three fictional cases: the transfer of an embryo with trisomy 21, of an embryo with a sex chromosome aneuploidy and of an embryo with a chromosomal microdeletion. RESULTS: We found that opinions regarding the role of fertility professionals in deciding which embryos to transfer were mixed. Moreover, where to draw the line between healthy and unhealthy embryos was unclear. We also found that couples, although they thought that comprehensive chromosome testing had certain benefits, also considered the increased waiting time for transfer a heavy burden. CONCLUSIONS: In the light of comprehensive chromosome screening of embryos, persons undergoing fertility treatment may have views on the burdens and benefits of the techniques that are not analogous to the views of professionals.

The four blastomeres of a 4-cell stage human embryo are able to develop individually into blastocysts with inner cell mass and trophectoderm.

BACKGROUND: Early mammalian blastomeres are thought to be flexible and totipotent allowing the embryo to overcome perturbations in its organization during preimplantation development. In the past, experiments using single blastomeres from 2-, 4- and 8-cell stage mammalian embryos have provided evidence that at least some of the isolated cells can develop into healthy fertile animals and therefore are totipotent. We investigated whether isolated blastomeres of human 4-cell stage embryos could develop in vitro into blastocysts with trophectoderm (TE) and inner cell mass (ICM). METHODS: Six 4-cell stage human embryos were split and the four blastomeres were cultured individually. The expression of NANOG, a marker for ICM cells, was analysed by immunocytochemistry. RESULTS: The majority of the blastomere-derived embryos followed the normal pattern of development with compaction on Day 4 and cavitation on Day 5 and developed into small blastocysts with TE and ICM on Day 6 (n = 12). The four cells of one embryo were individually capable of developing into blastocysts with TE and ICM, and NANOG was expressed in the ICM. CONCLUSIONS: Although based on a small number of embryos, we conclude that the blastomeres of a 4-cell stage human embryo are flexible and able to develop into blastocysts with ICM and TE.

Do we need to search for gr/gr deletions in infertile men in a clinical setting?

BACKGROUND: Partial deletions of the AZFc region of the Y chromosome such as gr/gr deletions have been detected in infertile patients as well as in control groups. The impact of these gr/gr deletions on the etiology of male infertility remains unknown. In the present study, we investigated the presence of gr/gr deletions in Caucasian men. METHODS: gr/gr deletions were analyzed by using markers sY1291, sY1191 and sY1197 and by investigating the presence of single nucleotide variants (SNV) in DAZ and CDY1 genes in patients with azoospermia (n = 44), cryptozoospermia (n = 51) or severe oligozoospermia (n = 92). Control groups consisted of men with normal spermatogenesis on testicular biopsy (n = 33), normozoospermia (n = 278) or proven fertility (n = 83). RESULTS: We observed 20 gr/gr deletions, with eight in infertile patients (4.3%) and 12 in the control groups (3.0%), which was not significantly different. DAZ SNV analysis revealed eight different deletion patterns in patients and controls. CONCLUSIONS: In the present study, no significant differences in the frequency of gr/gr deletions between different patient and control groups were observed. We concluded that the relationship between gr/gr deletions and male infertility remains unclear and that it is too early to systematically test for gr/gr deletions for infertile couples seeking assisted reproduction treatment.

Diagnostic efficiency, embryonic development and clinical outcome after the biopsy of one or two blastomeres for preimplantation genetic diagnosis.

BACKGROUND: Preimplantation genetic diagnosis or screening (PGD, PGS) involves embryo biopsy on Day 3. Opting for one- or two-cell biopsy is a balance between the lowest risk for misdiagnosis on the one hand and the highest chance for a pregnancy on the other hand. METHODS: A prospective controlled trial was designed and 592 ICSI cycles were randomly assigned to the one-cell (group I) or the two-cell group (group II). Primary outcomes were diagnostic efficiency and embryonic development to delivery with live birth (analysed by cycle). The false-positive rate for the PCR cycles is presented as a secondary outcome (analysed by embryo). RESULTS: A strong significant correlation was observed between embryonic developmental stage on Day 3 and post-biopsy in vitro development on Day 5 (P < 0.0001). The influence of the intervention on Day 3 was less significant (P = 0.007): the biopsy of one cell is less invasive than the biopsy of two cells. PCR diagnostic efficiency was 88.6% in group I and 96.4% in group II (P = 0.008). For the fluorescence in situ hybridization (FISH) PGD cycles no significant difference in efficiency was obtained (98.2 and 97.5% in group I and II, respectively). Similar delivery rates with live birth per started cycle were obtained [58/287 or 20.2% in group I versus 52/303 or 17.2% in group II, P = 0.358; the absolute risk reduction = 3.05%; 95% confidence interval (CI): -3.24, 9.34]. Post-PGD PCR reanalysis showed six false positives in 97 embryos (6.2%) in group II and none in group I (91 embryos reanalysed). No false negatives were found. CONCLUSIONS: While removal of two blastomeres decreases the likelihood of blastocyst formation, compared with removal of one blastomere, Day 3 in vitro developmental stage is a stronger predictor for Day 5 developmental potential than the removal of one or two cells. The biopsy of only one cell significantly lowers the efficiency of a PCR-based diagnosis, whereas the efficiency of the FISH PGD procedure remains similar whether one or two cells are removed. Delivery rates with live birth per started cycle were not significantly different.

The future (r)evolution of preimplantation genetic diagnosis/human leukocyte antigen testing: ethical reflections.

There has been increasing support for combining preimplantation genetic diagnosis (PGD) for specific diseases with a test for human leukocyte antigens (HLA) because the generation of HLA-matched umbilical cord blood cells may save the life of a diseased sibling. To date, this procedure has taken place in the context of conceiving another child--PGD/HLA testing type 1. However, it may well become possible to perform PGD/HLA testing outside this context, that is, to select matched embryos from which embryonic stem cells could be derived and used in cell therapy--PGD/HLA testing type 2. A proactive ethical analysis is needed and is presented in this article. Although PGD/HLA testing type 1 can be morally justified, the risks, pitfalls, and practical limitations of this procedure make it necessary to develop alternative strategies. PGD/HLA testing type 2 may provide an alternative strategy. From an ethical point of view, the controversial issue is that this procedure creates embryos purely for instrumental use. However, given the dominant view that the preimplantation embryo has only limited moral value, this alternative may be as morally justified as PGD/HLA testing type 1.

Efficient differentiation of human embryonic stem cells into a homogeneous population of osteoprogenitor-like cells.

The use of human embryonic stem cells (hESC) in both research and therapeutic applications requires relatively large homogeneous populations of differentiated cells. The differentiation of three hESC lines into highly homogeneous populations of osteoprogenitor-like (hESC-OPL) cells is reported here. These cells could be expanded in a defined culture system for more than 18 passages, and showed a fibroblast-like morphology and a normal stable karyotype. The cells were strongly positive for the same antigenic markers as mesenchymal stem cells but negative for markers of haematopoetic stem cells. The hESC-OPL cells were able to differentiate into the osteogenic, but not into the chondrogenic or adipogenic, lineage and were positive for markers of early stages of osteogenic differentiation. When cultured in the presence of osteogenic supplements, the cells indicated the capacity to achieve, under inductive conditions, a mature osteoblast phenotype. The differentiation protocol is based on a monolayer approach, and does not require any exogenous factors other than fetal calf serum, or coculture systems of animal or human origin. This method is likely to be amenable to large-scale production of homogeneous osteoprogenitor-like cells and thus overcomes one of the major problems of differentiation of hESC, with important relevance for further cell therapy studies.

Methylation analysis of KvDMR1 in human oocytes.

Recently, several reports have been published that showed a higher incidence of assisted reproductive technologies (ART) in patients with Beckwith-Wiedemann syndrome compared with the general population, and in most of these patients, aberrant methylation imprints of KvDMR1 have been found. This has led to the concern that ART might increase the incidence of imprinting syndromes such as Beckwith-Wiedemann syndrome. Not much is known on environmental or genetic factors that may interfere with the processes of imprint maintenance or resetting. A methylation analysis of KvDMR1 was performed in human oocytes at different stages of nuclear maturity and in sperm cells. The results indicate that the maternal methylation imprints were already established at the germinal vesicle stage, whereas all sperm cells were unmethylated, thereby showing that the KvDMR1 carries a germline methylation imprint. For one of the oocytes analysed, an unmethylated pattern was found, which highlights the need for further molecular studies that consider the safety of ART.

Single-cell chromosomal imbalances detection by array CGH.

Genomic imbalances are a major cause of constitutional and acquired disorders. Therefore, aneuploidy screening has become the cornerstone of preimplantation, prenatal and postnatal genetic diagnosis, as well as a routine aspect of the diagnostic workup of many acquired disorders. Recently, array comparative genomic hybridization (array CGH) has been introduced as a rapid and high-resolution method for the detection of both benign and disease-causing genomic copy-number variations. Until now, array CGH has been performed using a significant quantity of DNA derived from a pool of cells. Here, we present an array CGH method that accurately detects chromosomal imbalances from a single lymphoblast, fibroblast and blastomere within a single day. Trisomy 13, 18, 21 and monosomy X, as well as normal ploidy levels of all other chromosomes, were accurately determined from single fibroblasts. Moreover, we showed that a segmental deletion as small as 34 Mb could be detected. Finally, we demonstrated the possibility to detect aneuploidies in single blastomeres derived from preimplantation embryos. This technique offers new possibilities for genetic analysis of single cells in general and opens the route towards aneuploidy screening and detection of unbalanced translocations in preimplantation embryos in particular.

A single mutation in the GALC gene is responsible for the majority of late onset Krabbe disease patients in the Catania (Sicily, Italy) region.

A high proportion of patients with late onset forms of Krabbe disease is observed in a region north of Catania in Sicily. Molecular analysis in five families from this region shows that this condition is mainly due to a not previously described p.Gly41Ser substitution in the GALC gene that abolishes catalytic activity of the galactocerebrosidase enzyme, as shown by expression studies. Three patients were homozygous for this mutation, the other two were heterozygous, one with a frameshift mutation and one with a missense mutation on the second allele. Therefore, the mutation must be a mild one since it leads to late onset disease in all patients. In addition, it is on a unique haplotype indicating that it represents a founder mutation. This is also supported by the fact that the mutation was not found in three late onset patients from other regions in Sicily, in whom four novel mutations were identified.