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Autosomal dominant polycystic kidney disease (ADPKD) is primarily characterized by renal cysts and progression to renal failure. It is a genetically heterogeneous disease, with mutations in the PKD1 gene accounting for the majority of cases. Direct mutation detection for PKD1-linked ADPKD or type 1 is complicated by the large size and complex genomic structure of PKD1. This paper describes a microsatellite marker-based assay for PGD in couples at risk of transmitting ADPKD type 1. During PGD, genetic analysis is carried out on single blastomeres biopsied from preimplantation embryos obtained after IVF, and only embryos unaffected by the disease under investigation are selected for transfer. Single-cell genetic analysis relied on a fluorescent duplex-PCR of linked polymorphic markers followed by fragment length determination on an automated sequencer. The co-amplification of the intragenic KG8 and the extragenic D16S291 marker at the single-cell level was evaluated in pre-clinical tests on lymphoblasts and research blastomeres. The developed assay proved to be efficient (96.1% amplification) and accurate (1.4% allele drop-out and 4.3% contamination), and can be applied in all informative ADPKD type 1 couples. From five clinical cycles carried out for three couples, two pregnancies ensued, resulting in the birth of two healthy children. Mol Hum Reprod

Cystic fibrosis (CF) is an autosomal recessive disease characterized by obstruction and chronic infection of the respiratory tract and pancreatic insufficiency. The first preimplantation genetic diagnosis (PGD) for CF was carried out in 1992. At our centre the first cycle was performed in 1993. However, the number of known CF mutations is >1000, so developing mutation-specific PCR protocols for PGD is unfeasible. This is why a number of marker-based duplex PCRs were developed at the single cell level. A duplex PCR of a mutation and one or two microsatellites is not only a diagnostic tool, but it can also be used as a control for allele drop-out and contamination. During PGD, embryos obtained in vitro are analysed for the presence or absence of a particular genetic disease, after which only embryos shown to be free of this disease are returned to the mother. In total, 22 PGD cycles with duplex PCR (IVS8CA/IVS17BTA, DeltaF508/IVS8CA, DeltaF508/IVS17BTA and D7S486/D7S490) were carried out in 16 couples, which resulted in four ongoing pregnancies and one miscarriage.

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.

Development of β-cells from human embryonic stem cells (hESCs) could compensate for the shortage of islet donors required for diabetes therapy. Although pancreatic progenitors have been derived from hESCs using various protocols, no fully functional β-cells could be generated in vitro. We evaluated the in vivo growth and differentiation of PDX1+ pancreatic endoderm cells obtained from hESCs. Here we show site-specific survival and differentiation when comparing cells grafted in the epididymal fat pad or the subcutaneous space of NOD/SCID mice after 12 weeks follow-up. Subcutaneous grafts persisted and expressed PDX1 at all time points analyzed, showed PDX1 and NKX6.1 coexpression after 6 weeks, and contained NGN3+ cells after 12 weeks. These findings suggest that further specification along the pancreatic lineage occured at the subcutaneous site. In sharp contrast, in the fat pad grafts only a minority of PDX1+ cells remained after 2 weeks, and no further pancreatic differentiation was observed later on. In addition, contaminating mesenchymal cells present in the implants further developed into cartilage tissue after 6 weeks implantation in the fat pad, but not in the subcutaneous space. These findings indicate that the in vivo microenvironment plays a critical role in the further differentiation of transplanted pancreatic endoderm cells.

The interface between assisted reproductive technologies (ART) and genetics comprises several sensitive and important issues that affect infertile couples, families with severe genetic diseases, potential children, professionals in ART and genetics, health care, researchers and the society in general. Genetic causes have a considerable involvement in infertility. Genetic conditions may also be transmitted to the offspring and hence create transgenerational infertility or other serious health problems. Several studies also suggest a slightly elevated risk of birth defects in children born following ART. Preimplantation genetic diagnosis (PGD) has become widely practiced throughout the world for various medical indications, but its limits are being debated. The attitudes towards ART and PGD vary substantially within Europe. The purpose of the present paper was to outline a framework for development of guidelines to be issued jointly by European Society of Human Genetics and European Society of Human Reproduction and Embryology for the interface between genetics and ART. Technical, social, ethical and legal issues of ART and genetics will be reviewed.

Preimplantation genetic diagnosis (PGD) was introduced at the beginning of the 1990s as an alternative to prenatal diagnosis, to prevent termination of pregnancy in couples with a high risk for offspring affected by a sex-linked genetic disease. At that time, embryos obtained in vitro were tested to ascertain their sex, and only female embryos were transferred. Since then, techniques for genetic analysis at the single-cell level, involving assessment of first and second polar bodies from oocytes or blastomeres from cleavage-stage embryos, have evolved. Fluorescence in-situ hybridisation (FISH) has been introduced for the analysis of chromosomes and PCR for the analysis of genes in cases of monogenic diseases. In-vitro culture of embryos has also improved through the use of sequential media. Here, we provide an overview of indications for, and techniques used in, PGD, and discuss results obtained with the technique and outcomes of pregnancies. A brief review of new technologies is also included.

Embryoid body (EB) formation is a standard approach to assess differentiation potential of human pluripotent stem cells (hPSC). However, EB size has been shown to influence the differentiation course of the cells. In order to compare functional variability between individual hPSC lines it is therefore mandatory to use equal-sized EBs, using a standardized EB protocol. Our cells are routinely cultured on laminin-521TM (LN521) in NutristemTM (NS) medium and passaged as single cells, providing feeder- and xeno-free conditions. As previously published and commercially available protocols were optimized for different culture systems, we had to develop a new single cell-based EB protocol. We aimed to obtain equal-sized EBs after spinning the single cell suspension in round bottom 96-well plates (5000 cells seeded per well) in the presence of ROCK inhibitor. In a first approach, cells were pre-differentiated for 2 days before harvesting. Results were highly variable, with cells either aggregating, partially aggregating or completely failing to form EBs. We did not observe significant differences between enzymatic vs non-enzymatic cell harvest approaches, but we noticed that differentiation in the presence of serum caused poor aggregation of EBs and early disaggregation, whereas aggregate formation and subsequent growth for 21 days was much improved in KnockoutTM Serum Replacement (KOSR)-based medium. Finally, we observed that the crucial factor influencing the aggregation of single hPSC was the growth phase of the cells. Only cells that were freshly passaged 2-3 days before EB formation were consistently forming aggregates, not only in KOSR-based medium but also in commercially available xeno-free APELTM medium and in NS medium. With this approach we even managed to obtain hanging drop EBs, which was not possible in our previous experiments. Our optimized EB formation protocol using 96-well plates has until now been successful on 6 different hESC lines and 12 hiPSC lines. We are currently quantifying the size of EBs formed by different numbers of cells during a 21-day differentiation course and investigating the effect of EB size on expression of early lineage specification genes.

no abstract available

Individual human embryonic stem cell (hESC) lines often demonstrate a differentiation bias towards one of the embryonic germ layers. It is necessary to better understand the molecular mechanisms causing this phenomenon, as it can hamper the efficiency of hESC-based biomedical applications. Moreover, knowledge of the pathways implicated may help to improve differentiation protocols. However, an accurate quantification of line-specific differentiation bias is challenging, as culture conditions also influence the differentiation outcome. In this study, we compared different standardized methods to quantify differentiation potential towards the definitive endoderm (DE) of four hESC lines (VUB01, VUB02, VUB07 and VUB14). All lines carried a balanced chromosomal content, confirmed by array comparative genome hybridization, and were cultured on laminin-521TM in NutristemTM medium. First, we used our in-house optimized embryoid body (EB) formation protocol to generate equal-sized EBs, followed by a 21-day spontaneousdifferentiation in APEL medium. Gene expression analysis by real-time PCR did not show a lineage bias between differently sized EBs from the same hESC line, but we detected consistent differences between individual lines. In contrast, when performing a 7-day spontaneous EB differentiation and evaluating gene expression levels using the TaqMan® hPSC Scorecard™ Panel, we did not obtain reproducible results for biological replicates. We therefore concluded that this set-up was not applicable for our purpose. Next, we performed a 3-day DE induction with defined seeding density (20000 cells/cm2) to normalize the method. VUB14 DE samples showed statistically significant lower expression levels of SOX17, FOXA2 and GATA4 in comparison to DE samples from the other hESC lines. These results were in concordance with the data obtained from the 21-day EB spontaneous differentiation, suggesting that these two standardized methods can serve as a tool for evaluating DE differentiation bias. Our next step will be comparing our approach with an external technique to additionally confirm its accuracy.

Individual human embryonic stem cell (hESC) lines often demonstrate a differentiation bias towards a specific germ layer, which may hamper the efficiency of hESC-based biomedical applications. A better understanding of the molecular mechanisms causing this phenomenon is therefore needed. However, an accurate quantification of differentiation bias is challenging, as culture conditions also influence differentiation outcome. Here, we compared standardized methods to quantify definitive endoderm (DE) differentiation potential of four hESC lines. All lines carried a balanced chromosomal content and were cultured in identical conditions on laminin-521TM in NutristemTM medium. First, we used our in-house optimized embryoid body (EB) formation protocol to generate equal-sized EBs, followed by 21-days of spontaneous differentiation in APEL medium. Gene expression analysis did not show a lineage bias between differently sized EBs within the same line, but we detected consistent differences between individual hESC lines. Next, we performed a 3-day DE induction with a defined seeding density. DE samples from hESC line VUB14 showed a statistically significant reduction in expression levels of SOX17, FOXA2 and GATA4 in comparison to the other hESC lines. Also, immunocytochemistry analysis showed that the VUB14-derived DE population had the lowest percentage of SOX17-positive cells and the highest number of POU5F1-positive cells. These results were in concordance with the EB spontaneous differentiation experiment. Additionally, we performed a 12-day EB differentiation and evaluated gene expression levels using the TaqMan® hPSC Scorecard™ Panel. These results showed that VUB14 in general had the lowest tri-lineage differentiation potential amongst the evaluated lines. Our data shows that standardized EB and DE differentiation followed by gene expression analysis can serve as a reliable tool for evaluating DE differentiation bias. Our next step will be evaluating a potential reason for VUB14’s resistance to differentiation and establishing whether this hindrance is maintained during further differentiation towards hepatocytes.

The first clinical application of preimplantation genetic diagnosis (PGD) was reported almost a decade ago. Since then, the range of genetic defects that can be detected at single cell level has increased dramatically. At the 13th Annual Meeting of ESHRE in Edinburgh in 1997, a PGD Consortium was formed to undertake the first systematic and long-term study of the efficacy and clinical outcome of PGD. We report here the first data collection covering the period of January 1997 to September 1998. Referral data on 323 couples have been collected for a variety of monogenic and chromosomal disorders, providing information about which patients, at risk for which genetic diseases, are interested in PGD. Data were collected on 392 PGD cycles, resulting in 302 embryo transfers and 66 clinical pregnancies. Because of the importance of follow-up of the children born after PGD, participating centres were asked to contribute data on the pregnancies achieved and the children born after PGD since the start of their PGD programme. Data on 82 pregnancies and 110 fetal sacs were collected, and information was available on 79 children. Finally, biopsy, fluorescence in-situ hybridization and polymerase chain reaction protocols were collected, clearly showing that no consensus exists on technical aspects such as which culture medium to use, and emphasizing the role the PGD Consortium could play in setting up guidelines for good laboratory practice. In conclusion, it is clear that the effort of gathering data on PGD cycles is worthwhile and will be continued in the future, preferably using electronic data collection.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease which is most often caused by a deficiency in steroid 21-hydroxylase. The disease is characterized by a range of impaired adrenal cortisol and aldosterone synthesis combined with an increased androgen synthesis. These metabolic abnormalities lead to an inability to conserve sodium and virilization of females. The most common mutation causing the severe form of CAH is a conversion of an A or C at nucleotide (nt) 656 to a G in the second intron of the steroid 21-hydroxylase gene (CYP21) causing aberrant splicing of mRNA. A couple was referred to our centre for preimplantation genetic diagnosis (PGD) for 21-hydroxylase deficiency in CAH. A PGD was set up to detect the nt656 A/C-->G mutation using fluorescent polymerase chain reaction (PCR) and subsequent restriction enzyme digestion and fragment analysis on an automated sequencer. Using DNA or single cells from the father, the normal allele could not be amplified. Non-amplification of the normal allele has been previously described in asymptomatic carriers, therefore the PCR was further developed using heterozygous lymphoblasts from the mother. The PCR was shown to be highly efficient (96% amplification), accurate (0% contamination) and reliable (0% allelic drop-out). The couple started PGD treatment and the second PGD cycle resulted in a twin pregnancy. The genotype of the fetuses was determined in our laboratory using chorionic villus sampling material using the method described here. Both fetuses were shown to be heterozygous carriers of the mutation, and two healthy girls were born.

Myotonic dystrophy (DM) or Steinert's disease is a progressive autosomal dominant disease characterized by increasing muscle weakness, myotonia, cataracts, and endocrine abnormalities such as diabetes and testicular atrophy. The gene for DM was cloned in 1992 and the mutation was shown to be an expanded trinucleotide (CTG) repeat. A polymerase chain reaction (PCR)-based assay was described soon after that would allow (prenatal) diagnosis of the disease. Based on these PCR assays, we have developed a method for carrying out single-cell PCR for DM. In preimplantation diagnosis, embryos obtained in vitro are checked for the presence or absence of a disease, after which only embryos shown to be free of the disease under consideration are returned to the mother. A single-cell assay was developed for preimplantation diagnosis in couples where one of the parents is afflicted with DM. Twenty intracytoplasmic sperm injection (ICSI) cycles were carried out in eight patients and between one and four embryos were replaced in 17 out of 20 cycles. Two of the patients became pregnant and have had prenatal diagnosis which has confirmed that they are unaffected.

This paper describes the 5 years' experience of preimplantation genetic diagnosis (PGD) at the Brussels Free University. Our first PGD was carried out in February 1993. Up to October 1998, we carried out 183 PGD cycles on fresh cleavage embryos of 92 couples for 25 different conditions. Patients were treated for autosomal recessive (n = 39), autosomal dominant (n = 65) and X-linked recessive (n = 47) monogenic disorders as well as for autosomal structural aberrations (n = 10), sex chromosome numerical and structural aberrations (n = 21) and a combination of the two latter (n = 1). Specific diagnosis was carried out by polymerase chain reaction (n = 108). Fluorescence in-situ hybridization was used for sexing (n = 64) and structural aberrations (n = 11). We transferred 1.6 +/- 1.1 embryos per cycle, resulting in an implantation rate of 12.0% per replaced embryo. Ongoing pregnancies were achieved in 29 cycles, i.e. 23 singletons, five twins and one dichorionic triplet with an acardius acranius. The ongoing pregnancy rates per cycle, per transfer and per couple were 16.4, 19.9 and 31.5% respectively. While 28 ongoing pregnancies resulted in the births of 34 infants, one pregnancy was terminated after misdiagnosis. The results of 24 PGD were confirmed by prenatal diagnosis or after birth while no information was available in four pregnancies. Our series demonstrates that PGD is a feasible technique by which to avoid the birth of genetically affected children to couples at risk.

OK, The scarcity of genomic DNA can be a limiting factor in some fields of genetic research. One of the methods developed to overcome this difficulty is whole genome amplification (WGA). Recently, multiple displacement amplification (MDA) has proved very efficient in the WGA of small DNA samples and pools of cells, the reaction being catalyzed by the phi29 or the Bst DNA polymerases. The aim of the present study was to develop a reliable, efficient, and fast protocol for MDA at the single-cell level. We first compared the efficiency of phi29 and Bst polymerases on DNA samples and single cells. The phi29 polymerase generated accurately, in a short time and from a single cell, sufficient DNA for a large set of tests, whereas the Bst enzyme showed a low efficiency and a high error rate. A single-cell protocol was optimized using the phi29 polymerase and was evaluated on 60 single cells; the DNA obtained DNA was assessed by 22 locus-specific PCRs. This new protocol can be useful for many applications involving minutequantities of starting material, such as forensic DNA analysis, prenatal and preimplantation genetic diagnosis, or cancer research.

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.

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 co-culture 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.

Charcot-Marie-Tooth (CMT) disease type 1A is an autosomal dominant peripheral neuropathy characterized by slow progressive distal muscle wasting and weakness, and decreased nerve conduction velocities. Most CMT1A cases (>98%) are caused by a duplication of a 1.5 Mb region on the short arm of chromosome 17 containing the PMP22 gene. A couple with a previous history of CMT followed by termination of pregnancy was referred to our centre for preimplantation genetic diagnosis (PGD). The husband carries the CMT1A duplication which can be detected by polymerase chain reaction (PCR) analysis using polymorphic (CA)n markers localized within the duplication. PCR amplification of genomic DNA of the parents-to-be with one of the two primers labelled with fluorescein, followed by automated laser fluorescence (ALF) gel electrophoresis of the amplified fragments allows the distinction between both genotypes. Embryos obtained after intracytoplasmic sperm injection (ICSI) were evaluated for the presence of the normal allele of the father. PCR with single Epstein-Barr virus-transformed lymphoblasts and blastomeres resulted in 91.4 and 93.5% amplification efficiency respectively, whereas none of the blank controls gave a positive signal. Allele drop-out (ADO) was observed in eight out of 32 lymphoblasts (25%) or in five out of 21 blastomeres (23.8%). However, within this set-up ADO will never lead to transfer of an affected embryo. A first ICSI-PGD cycle did not result in embryo transfer for the patient. A second cycle involved 10 mature oocytes of which eight were fertilized, resulting in five embryos for biopsy. Two unaffected embryos were available for transfer and resulted in a singleton pregnancy. The genotype of the fetus has been confirmed healthy by chorionic villus sampling.

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 assisted reproductive technology (ART), and published an extended background paper, recommendations and two Editorials. Seven years later, in March 2012, a follow-up interdisciplinary workshop was held, involving representatives of both professional societies, including experts from the European Union Eurogentest2 Coordination Action Project. The main goal of this meeting was to discuss developments at the interface between clinical genetics and ARTs. As more genetic causes of reproductive failure are now recognised and an increasing number of patients undergo testing of their genome before conception, either in regular health care or in the context of direct-to-consumer testing, the need for genetic counselling and preimplantation genetic diagnosis (PGD) may increase. Preimplantation genetic screening (PGS) thus far does not have evidence from randomised clinical trials 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 (International Standards Organisation - ISO 15189). Further studies are needed in order to address issues related to the impact of ART on epigenetic reprogramming of the early embryo. The legal landscape regarding assisted reproduction is evolving but still remains very heterogeneous and often contradictory. The lack of legal harmonisation and uneven access to infertility treatment and PGD/PGS fosters considerable cross-border reproductive care in Europe and beyond. The aim of this paper is to complement previous publications and provide an update of selected topics that have evolved since 2005.

It is well known that human embryonic stem cells (hESCs) frequently acquire recurrent chromosomal abnormalities, very rem- iniscent of those found in cancerous cells. Given the parallels between cancer and stem cell biology, we set out to investigate the occurrence of a common form of genome instability in tumors, namely microsatellite instability (MSI), in hESCs. MSI is caused by a deficiency in mismatch repair (MMR) genes, which leads to the accumulation of mutations during DNA replication. In this study, we analyzed up to 122 microsatellites in a total of 10 hESC lines, for 1-11 different passages, ranging from passage 7 to passage 334. In two lines, this revealed that two microsatellites had altered allelic patterns. Small-pool PCR for several microsatellites and testing of the Bethesda panel microsatellites (commonly used in cancer studies) revealed that, whilst MSI is common in all tested lines, it occurs at a very low and variable frequency, ranging from � 1 to 20% of the total number of alleles. In cancerous cells, MSI leads to multiple large shifts in allele sizes within the majority of the cells, while hESCs show small changes in a mi- nority of the cells. Since these genetic alterations do not consistently take over the culture, we assume that they are not concurrent with a selective advantage as it is in tumors. Finally, the MMR genes showed a very variable gene expression that could not be correlated with the variable (low) levels of MSI in the different hESC lines.

BACKGROUND: Preimplantation genetic diagnosis (PGD) and subsequently preimplantation genetic screening (PGS) have been introduced since I990. The difference from the already existing in vitro fertilization (IVF) technology, using intracytoplasmic sperm injection (ICSI), was the embryo biopsy at day 3 after fertilization. Although healthy children post-PGD/PGS have been born, the question of whether embryo biopsy could have any harmful effects has to be studied on large series in a prospective manner. METHODS: A prospective cohort study was undertaken from I992 until 2005, using the same approach as for the follow-up of IVF and ICSI children conceived in the same centre. Questionnaires were sent to physicians and parents at conception and at delivery. Children were examined at 2 months of age by trained clinical geneticists whenever possible. RESULTS: Data collected on 58I post-PGD/PGS children showed that term, birthweight and major malformation rates were not statistically different from that of 2889 ICSI children, with overall rates of major malformation among these post-PGD/PGS and ICSI children being 2.I3 and 3.38%, respectively (odds ratio [OR]: 0.62; exact 95% confidence limits [95% CL]: 0.31 - 1.15). However, the overall perinatal death rate was significantly higher among post-PGD/PGS children compared with ICSI children (4.64 versus I.87%; OR: 2.56; 95% CL: I.54―4.I8). When stratified for multiple births, perinatal death rates among PGD/PGS singleton and ICSI singleton children were similar (I.03 versus I.30%; OR: 0.83; 95% CL: 0.28―2.44), but significantly more perinatal deaths were seen in post-PGD/PGS multiple pregnancies compared with ICSI multiple pregnancies (II.73 versus 2.54%; OR: 5.09; 95% CL: 2.80-9.90). The overall misdiagnosis rate was below I%. CONCLUSIONS: Embryo biopsy does not add risk factors to the health of singleton children born after PGD or PGS. The perinatal death rate in multiple pregnancies is such that both caution and long-term follow-up are required.

Study question: What are the dynamics of X chromosome inactivation (XCI) in a large cohort of female human embryonic stem cell (hESC) lines during long-term culture and differentiation? Summary answer: We found a stable, though aberrant XCI pattern during long-term undifferentiated culture and after differentiation to somatic and tro- phoblast lineages: all lines displayed stage III XCI, with major loss of XCI marks, and a completely non-random XCI pattern in lines informative for the microsatellite markers under study. What is known already: Female hESC cultures have previously been shown to display variable XCI patterns. Unlike mouse embryonic stem cells, most hESC already display XCI at the undifferentiated state. Moreover, a predominant oc- currence of non-random XCI patterns has been reported; but the extent and origin of this non-random pattern in hESC remains unresolved. Study design, size, duration: XCI patterns of 22 female hESC lines were ana- lyzed at different passages during long-term culture,and after differentiation into somatic osteoprogenitor-like cells and the trophoblast lineage. The parental origin of the inactivated X chromosome was identi ed if DNA from the embryo donors was available. Participants/materials, setting, methods: We used methylation-sensitive DNA restriction and PCR, and massive parallel bisulphite sequencing for mic- rosatellite markers to study DNA methylation. Histone modi cations were ana- lysed by immunostaining. Expression of XIST was investigated using real time PCR, while cDNA mini sequencing was used to determine mono- or biallelic gene expression. Main results and the role of chance: Methylation analysis revealed XCI in all investigated lines. One line was non-informative for the studied markers; all other lines displayed a completely skewed, non-random inactivation pattern, from early passage on and in undifferentiated cells as well as after differentiation. We ob- served a transition from a random to a non-random pattern in only one line. From ten lines for which donor DNA was available, six lines displayed inactivation of the male donor allele; four of the female donor allele. Massive parallel bisulphite sequencing revealed partially methylated pro les, suggesting erosion of methyla- tion. While in some early passage lines we could still observe repressing histone marks and XIST expression, these were very rapidly lost after limited culture time. Strikingly, loss of histone marks occurred in a colony-speci c manner. Limitations, reason for caution: We investigated the methylation status of the X chromosome using three microsatellite markers. Further re nement of the analysis at multiple loci spread over the whole chromosome could offer a more in-depth view of the XCI patterns in hESC. Wider implications of the ndings: The extent of the observed non-random XCI patterns is striking. We excluded a parent-of-origin-related mechanism and also its occurrence as passenger aberration is unlikely because of the number of cell lines involved and the presence at early passages. The transition found in one line rules out an embryonic origin of the skewed XCI, therefore we hypothesize that this XCI skewing originates from a culture advantage conferred by a speci c XCI pattern. Study funding/competing interest(s): Funding by University(ies) – Funding by national/international organization(s) – This work was supported by grants from the Research Foundation - Flanders (FWO-Vlaanderen) [grant number 1502512N] and the Methusalem grant for K. Sermon of the Research Council of the Vrije Universiteit Brussel. Trial registration number: NA. Keywords: hESC, X chromosome inactivation

no abstract available

In this report, we present the derivation and characterization of 15 hESC lines established at the Vrije Universiteit Brussel, Belgium in collaboration with the Universitair Ziekenhuis Brussel, Belgium, using surplus in vitro fertilization embryos and embryos carrying monogenic disorders donated for research. Four lines were derived from blastocyst-stage embryos presumed to be genetically normal, and 11 hESC lines were obtained from embryos shown to carry genetic mutations by preimplantation genetic diagnosis. All the lines express markers of pluripotency as determined by immunocytochemistry and RT-PCR, and formed teratomas when injected into SCID mice. All VUB hESC lines, except for VUB17, are reported in the European hESC registry and are available upon request after signing a Material Transfer Agreement from the VUB (contact person: Prof. Dr. Karen Sermon; Karen.Sermon@uzbrussel.be).

PURPOSE: This study aims to analyze the relationship between trinucleotide repeat length and reproductive outcome in a large cohort of DM1 patients undergoing ICSI and PGD. METHODS: Prospective cohort study. The effect of trinucleotide repeat length on reproductive outcome per patient was analyzed using bivariate analysis (T-test) and multivariate analysis using Kaplan-Meier and Cox regression analysis. RESULTS: Between 1995 and 2005, 205 cycles of ICSI and PGD were carried out for DM1 in 78 couples. The number of trinucleotide repeats does not have an influence on reproductive outcome when adjusted for age, BMI, basal FSH values, parity, infertility status and male or female affected. Cox regression analysis indicates that cumulative live birth rate is not influenced by the number of trinucleotide repeats. The only factor with a significant effect is age (p

OBJECTIVE. The purpose of this study is to investigate the contributing effect of contrast media (CM) iodine dose on radiation-induced DNA damage in blood lymphocytes during a cardiac CT scan. MATERIALS AND METHODS. The minipigs were exposed 12 times in total to a fixed cardiac CT scan protocol. An unenhanced and two CM injection protocols were considered, the latter with 50% saline diluted (160 mg I/mL) and standard iodixanol. Blood samples were collected before and after CT, and radiation-induced DNA double-strand breaks were assessed using γ-H2AX (H2A histone family member X) immunofluorescent staining of the blood lymphocytes. Significant differences in foci numbers were investigated with an independent sample t test. In addition, a numeric dosimetry model was applied that simulates the cardiac CT scan, with the heart represented by a blood volume containing a mixture of six iodine concentrations (0, 10, 20, 30, 40, and 50 mg I/mL). RESULTS. Compared with the unenhanced (0 mg I/mL) protocol, the number of γ-H2AX foci per cell increased significantly (p < 0.038), by 56.1% for the reduced iodine dose (160 mg I/mL) and by 141.1% for the standard iodine dose (320 mg I/mL) protocols. These in vivo results are confirmed by the dosimetry simulation model, in which 78.8% and 133.7% increases in locally absorbed blood dose in the left ventricle were observed for the reduced and standard iodine dose protocols, respectively. CONCLUSION. Administration of CM during a cardiac CT examination significantly increases radiation-induced DNA damage in blood lymphocytes. Moreover, a lower CM iodine dose results in a reduced level of DNA damage, at constant radiation exposure.

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. ispartof: Nucleic Acids Research vol:34 issue:9 ispartof: location:England status: published

Charcot-Marie-Tooth (CMT) disease is the 'common' name for a range of hereditary peripheral neuropathies. CMT1 is the most common form and is transmitted in an autosomal dominant manner. CMT1A maps to chromosome 17p11.2 and is caused, in the majority of cases, by a 1.5 Mb DNA duplication, that includes the peripheral myelin protein 22 (PMP) gene. This paper reports on preimplantation genetic diagnosis (PGD) for CMT1A in five couples. The CMT1A duplication was detected by fluorescent PCR analysis using polymorphic (CA)n markers localized within the duplication. Single-cell PCR on blastomeres allowed genetic analysis of embryos obtained after ICSI. Only healthy unaffected embryos were transferred to the uterus. PCR experiments with single EBV-transformed lymphoblasts or with research blastomeres allowed the evaluation of amplification efficiencies, as well as contamination and allele drop-out (ADO) rates for each PCR protocol. Three simplex PCR protocols (using one primer pair) and two duplex PCR protocols (using two primer pairs) were developed for CMT1A. Additionally, a protocol using all three primer pairs in triplex was also established. Thirteen clinical ICSI-PGD cycles were performed for five couples (12 simplex PCR cycles and one duplex PCR cycle), resulting in seven embryo transfers. Three singleton pregnancies ensued in two couples and three healthy babies were delivered. This report describes different fluorescent PCR-based tests which allow efficient and accurate single-cell level detection of the CMT1A duplication. On the basis of the presence of the healthy allele of the affected parent-to-be (and/or absence of the affected one), healthy embryos can be selected for transfer. The assays are suitable for PGD for other couples who present with the same CMT1A duplication [depending on their informativity for the (CA)n markers available] as described here.

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

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common defect in fatty acid oxidation. The disease is inherited in an autosomal recessive fashion (carrier frequency around 1 in 70) and probably affects as many as 1 in 10000 new-borns. Affected children usually present within the two first years of life with recurrent episodes of hypoketotic hypoglycaemia and lethargy leading to death in approximately 25% of the cases. One mutation (c985A-->G) accounts for approximately 90% of the carrier chromosomes. We developed a preimplantation genetic diagnosis (PGD) strategy for MCAD for a couple who had already lost two affected children. When tested on heterozygous lymphoblasts, the amplification efficiency was 67 out of 71 (94%) and the allele drop-out rate was 0 out of 67. The patient became pregnant after one PGD cycle during which two embryos were replaced. The twin pregnancy was checked by chorionic villus sampling (CVS) and was shown to be unaffected. The twins have been born and are healthy.

This brief reminder chapter aims to freshen up what professionals in reproduction may have learned a while ago at university, and will also serve the reader as a source of information to comprehend the following, more complex chapters. At the end of this chapter, basic study books recommended for further reading are given rather than regular scientific references, to help the reader in the further understanding of this textbook. Human reproduction and genetics are intimately intertwined and indeed often confused and rolled into one. Understanding reproduction is impossible without a firm basis in genetics, and the readiness to acquire more knowledge when needed. However, human genetics is much broader than just reproduction - think of for instance oncogenetics - so in this chapter I will summarize what basic genetics is indispensable for the specialists in reproduction. In this chapter, I will introduce the general organization of our genome, how this genome behaves when it goes through a reproductive cycle (meiosis), how our genome is used as a template for making proteins, and how this is broadly regulated, major genetic and hereditary abnormalities both at the chromosome and at the monogenic level, and a brief overview of current genetic diagnostic techniques.

Multiple displacement amplification (MDA) is a recently described method of whole-genome amplification (WGA) that has proven efficient in the amplification of small amounts of DNA, including DNA from single cells. Compared with PCR-based WGA methods, MDA generates DNA with a higher molecular weight and shows better genome coverage. This protocol was developed for preimplantation genetic diagnosis, and details a method for performing single-cell MDA using the phi29 DNA polymerase. It can also be useful for the amplification of other minute quantities of DNA, such as from forensic material or microdissected tissue. The protocol includes the collection and lysis of single cells, and all materials and steps involved in the MDA reaction. The whole procedure takes 3 h and generates 1-2 microg of DNA from a single cell, which is suitable for multiple downstream applications, such as sequencing, short tandem repeat analysis or array comparative genomic hybridization.

Introduction: NANOG, SOX2, and SALL4 are transcription factors that play a key role in controlling stemness in embryonic stem cells (ESC) and are therefore candidate markers for developmental triggers in early embryos. KRT18, a trophoblast determining gene, may mark early differentiation in embryos. The expression pattern of NANOG, SOX2, SALL4 and KRT18 may identify the totipotent cells during early human development and determine when early embryonic cells lose their totipotent competence. Material and Methods: Thirtheen human oocytes and 121 human preimplantation embryos were examined for the presence of NANOG, SOX2, SALL4 or KRT18 proteins using immunostaining and confocal microscopy. Results: None of the stemness markers seemed to direct the dividing blastomeres towards the inner cell mass (ICM) or trophectoderm (TE) lineage, nor could they identify all and none other than the totipotent cells during human preimplantation development. NANOG proteins could at the earliest be detected at the blastocyststage and were consistently present in expanded blastocysts, when the two cell lineages were already visibly defined. Within the expanded blastocyst, we found NANOG proteins to be present in the nuclei of a subpopulation of the ICM. A sporadic NANOG expression was also detected in a minority of TE cells located near the ICM. A cytoplasmic SOX2 expression was detected in oocytes and throughout the whole preimplantation development. A nuclear expression appeared at day three of development and became prominent in all cells. After the ICM and the TE could clearly be distinguished, the nuclear staining increased in all cells of the ICM and disappeared in nearly all TE cells. SALL4 proteins were detected in the nucleus and the cytoplasm in oocytes and early cleavage stages until day 3 of development. During further development, the cytoplasmic staining was lost whereas the nuclear staining increased. In blastocysts, a strong expression was found in the nuclei of the ICM and TE. A joint expression of NANOG, SOX2 and SALL4 was only detected in the nuclei of a subpopulation of ICM cells. Expression of KRT18 was seen for the first time during compaction in some outer cells. During blastocyst expansion, KRT18 proteins were found in the cytoskeleton of the TE and the outer cells of the ICM adjacent to the blastocoel cavity, which will contribute to the yolk sac. Conclusions: The expression pattern of markers that define stemness in ESC could not tell us when and in which cells totipotency is lost during early human development. However, because stemness markers act in concert to keep the properties of ESC, these markers may maintain totipotency in a subpopulation of the ICM. Assessing the presence of KRT18 proteins implied that the outer cells of compacting embryos have lost their totipotent competence prior to any visible morphological differentiation. KRT18 may be used as an early marker of differentiation during human preimplantation development.

Preimplantation genetic diagnosis (PGD) for monogenic diseases has known a considerable evolution since its first application in the early 1990s. Especially the technical aspects of the genetic diagnosis itself, the single-cell genetic analysis, has constantly evolved to reach levels of accuracy and efficiency nearing those of genetic diagnosis on regular DNA samples. In this review, we will focus on the molecular biological techniques that are currently in use in the most advanced centers for PGD for monogenic disorders, including multiplex polymerase chain reaction (PCR) and post-PCR diagnostic methods, whole genome amplification (WGA) and multiple displacement amplification (MDA). As it becomes more and more clear that when it comes to ethically difficult indications, PGD goes further than prenatal diagnosis (PND), we will also briefly discuss ethical issues.

Fragile X syndrome is the most common monogenic cause of mental retardation in boys. It is always characterized clinically by moderate mental retardation and often by a long face with large everted ears and macro-orchidism. The causal mutation is an expansion of a CGG triplet repeat in a 5' exon of the FMR-1 gene in Xq27.3. We report here for the first time a method for preimplantation genetic diagnosis (PGD) for fragile X syndrome based on the amplification of the CGG triplet in the normal allele. Our candidate-patient population, as well as two clinical preimplantation genetic diagnosis (PGD) cycles which led to a pregnancy with an unaffected fetus, are presented in this paper.

STUDY QUESTION What are the aneuploidy rates and incidence of mosaicism in good-quality human preimplantation embryos. SUMMARY ANSWER High-level mosaicism and structural aberrations are not restricted to arrested or poorly developing embryos but are also common in good-quality IVF embryos. WHAT IS KNOWN ALREADY Humans, compared with other mammals, have a poor fertility rate, and even IVF treatments have a relatively low success rate. It is known that human gametes and early preimplantation embryos carry chromosomal abnormalities that are thought to lower their developmental potential. STUDY DESIGN, SIZE AND DURATION The embryos studied came from nine young (age

Objectives Mutations in the APC, NF2 and BRCA1 genes cause adult-onset cancer predisposition syndromes. Prenatal diagnosis (PND) and selective pregnancy termination for adult-onset disorders is emotionally difficult and, in some cases, socially not well accepted. Preimplantation genetic diagnosis (PGD) appears as an attractive alternative to PND, as it ensures the establishment of a pregnancy free of the mutation from the onset, circumventing the potentially difficult decision of termination of pregnancy. Methods Development of single-cell PCRs using Epstein-Barr virus transformed lymphoblasts as single-cell model, followed by clinical application in PGD. Results A total of five duplex-PCRs were developed, three for adenomatous polyposis of the colon (APC), one for neurofibromatosis type 2 (NF2) and one for inherited breast and ovarian cancer caused by BRCA1 mutations. Eleven clinical cycles were performed, resulting in the birth of an unaffected girl. For one of the couples undergoing PGD for NF2, a spontaneous pregnancy ensued after five unsuccessful PGD cycles. The couple underwent chorionic villus sampling (CVS) and the application of the same protocol as used during PGD showed an unaffected fetus. Conclusion In this work, we present the development and clinical application of PGD for three cancer predisposition syndromes. Copyright © 2007 John Wiley & Sons, Ltd.

Human embryonic stem cells (hESC) are considered to be an indefinite source of self-renewing cells that can differentiate into all types of cells of the human body and could be used in regenerative medicine, drug discovery and as a model for studying early developmental biology. hESC carrying disease-causing mutations hold promise as a tool to investigate mechanisms involved in the pathogenesis of the disease. In this report, we describe the behaviour of an expanded CTG repeat in the 3 0 untranslated region of the DMPK gene in VUB03_DM1, a hESC line carrying the myotonic dystrophy type 1 (DM1) mutation compared with the normal CTG repeat in two hESC lines VUB01 and VUB04_CF. Expanded CTG repeats were detected by small amount PCR, small pool PCR and Southern blot analysis in consecutive passages of VUB03_DM1. An important instability of the CTG repeat was detected during prolonged in vitro culture, showing stepwise increases of the repeat number in consecutive passages as well as a higher range of variability. This variability was present in cells of different colonies of the same passage and even within single colonies. The high repeat instability is in contrast to the previously observed stability of the repeat in preimplantation embryos and in fetuses during the first trimester of pregnancy. This in vitro culture of affected hESC represents a valuable model for studying the biology of repeat instability.

X-linked spinal and bulbar muscular atrophy is characterized by adult onset motor neuron disease and results from a defect in the androgen receptor. The disease is caused by a dynamic mutation in the first exon of the androgen receptor gene, involving a CAG trinucleotide repeat. We have developed a single-cell polymerase chain reaction assay for the androgen receptor gene and describe the application of this assay for preimlantation genetic diagnosis (PGD) in a couple at risk, where the female partner is a carrier of 47 repeats. Diagnosis was based on the detection of both normal and expanded alleles. Allele dropout of the expanded allele was observed in only 1 of 25 lymphoblasts of the carrier and of a non-expanded allele in 1 of 20 research blastomeres tested before the actual PGD. One contraction of four repeats was also found in the carrier's lymphoblasts. Neither expansions nor contractions were observed in the blastomeres biopsied from 11 embryos. Two embryos were unaffected, eight were female carriers and one was an affected male embryo. Hum Genet