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Intracytoplasmic sperm injection (ICSI), developed 30 years ago in our Center, allowed men with low to extremely low sperm quality to become the genetic father of their child. Overall, ICSI is now applied in up to 70% of all ART cycles and has resulted in the birth of millions of children worldwide. ICSI circumvents natural sperm selection mechanisms and concerns related to transgenerational inheritance of male infertility following ICSI could not be fully answered till today, given the young age of the offspring born after ICSI. Since genetic abnormalities account for one third of all cases of male factor infertility, it is essential to identify these genetic abnormalities because of the potential risk of their transmission to the offspring through the use of ART. As ICSI is nowadays performed even in couples with normal semen parameters, the disentanglement of procedure-related and parental background-related factors on the health of the offspring is important. Due to the safety concerns regarding the health of children born after ART, a longitudinal follow-up program was set up in our Center in the 80’s, resulting in data of more than 50 000 pregnancies. To date, more than 25 000 children are conceived after ICSI in our Center. We have not only the worldwide eldest cohort of ICSI offspring but also a uniform cohort of children and young adults conceived by ICSI because of (severe) male factor infertility. We present the results of these prospective studies with focus on the reproductive health in ICSI children between 8 and 22 years and compare these with available data from others. First of all, we will show data on the gonadal function assessed both clinically and by means of biomarkers at pre-pubertal and pubertal ages in ICSI boys. Secondly, we will present findings on the reproductive status of young adult men and women conceived after ICSI between 1992 and 1996: results of hormonal work-up, physical examination and semen parameters for ICSI men and results of hormone levels and antral follicle counts for ICSI women. Finally, we will discuss the findings in these cohorts of children and young adults conceived because of male infertility in view of the available literature regarding reproductive function in offspring conceived following ICSI.

status: published

Study question How are the trophectoderm (TE) key regulators CDX2, TEAD4 and YAP expressed during early human embryogenesis? Summary answer In human embryos, the restriction of TE-specific components to the TE occurs after expansion of the embryo, when the two cell lineages (TE versus inner cells mass (ICM)) are morphologically distinguishable, suggesting a role for other molecules in the initial segregation between the ICM and TE lineages. What is known already In mice, the Hippo Signaling pathway has been described in the first lineage segregation (Nishioka et al. 2009). Differences in cell-cell contacts through differences in cell position lead to activation or inactivation of YAP, which subsequently can activate or inactivate TEAD4 in the nucleus and consequently regulate CDX2 expression in the inner versus outer cells. In the human it is not known how these Hippo signaling pathway components regulate TE specification. Study design, size, duration In this study, human preimplantation embryos wereanalysed for the expression of the transcription factors CDX2 and TEAD4, and the co-activator protein YAP. Both localization within the embryo and the time-specific expression were analysed. Participants/materials, setting, methods The study was approved by the Local and Federal Ethical Committees for research on human embryos. Good quality embryos were obtained at our IVF Laboratory after informed consent of the patients. They were fixed in 4% paraformaldehyde, permeabilized with 0,1% triton and stained for the respective proteins using immunocytochemistry. Main results and the role of chance In cleavage-stage embryos, compaction and early blastocyst stages CDX2 was found weakly cytoplasmic. From the full blastocyst stage onwards, some cells showed nuclear CDX2 expression. In expanded blastocysts CDX2 was found either in the TE nuclei or in the ICM and TE nuclei. In hatching/hatched blastocysts CDX2 was found in the ICM and TE nuclei except for one big hatched blastocyst that started to downregulate CDX2 expression in ICM nuclei. YAP and TEAD4 started to be expressed in the nuclei of some cells of late cleavage-stage and compacted embryos. In expanded, hatching and hatched blastocysts TEAD4 and YAP were expressed in ICM and TE nuclei, except for one big hatched blastocyst in which YAP started to become downregulated in the nuclei of ICM cells. Limitations, reason for caution Due to ethical concerns the amount of material is limited. This work is rather descriptive, but functional studies will be performed in the future. Wider implications of the findings Information about the key players leading to the first lineage segregation in the human embryo and the mechanisms underlying differentiation will contribute to our basic knowledge on human embryogenesis. This fundamental research is unique and crucial to the field of reproductive medicine. Even though a lot is known about this subject in mice, results obtained in the human are different. This study shows that data obtained in mice cannot always be extrapolated to humans. Study funding/competing interest(s) This research is supported by grants from the Scientific Research Foundation- Flanders (FWO-Vlaanderen) and the Research Council (OZR) of the VUB

Background: Hemoglobinopathies are common monogenic diseases forming a major public health problem due to their severity and disabling nature. Genetic identification of the two most frequently observed missense mutations in the beta-globin gene, HbS and HbC, are important epidemiologically and aid in prevention of the sickle cell trait and other serious hemoglobin disorders. Aim: Our increasing patient population of Mediterranean, African and Middle Eastern origin urge for the need of a rapid, inexpensive and high-throughput genetic testing for HbS and HbC variants. Results: Classical high resolution melting analysis with an optimized PCR amplicon of 110 bp allowed a sensitive and reliable identification of the two neighbouring HbS (c.20A>T) and HbC (c.19G>A) mutations. Discriminatory melting profiles were observed for all possible combinations of mutations: HbAA, HbAS, HbAC, HbSS, HbCC and HbSC, and confirmed the results obtained by Hb-chromatography and PCR followed by restriction digestion. Withinthe wild type control population tested, mainly consisting of Belgian and North African individuals, two other aberrant melting patterns were observed. Sequencing of all samples with aberrant melting patterns revealed one polymorphism at position c.9 either in heterozygous or homozygous state. Further testing including samples from over 35 other nationalities did not disclose other melting profiles. Conclusion: This HbS and HbC HRM seems a promising, inexpensive and high-throughput alternative to PCR and restriction digestion analysis, although further validation is needed prior to implementation in a post- and prenatal diagnostic setting.

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No abstract available

Cartilage-hair hypoplasia (CHH) is a rare autosomal recessive disorder, characterised by dwarfism, hypoplastic hair and immunodeficiency. Allogeneic hematopoietic stem cell transplantation (HSCT) has been shown to be an effective treatment for CHH patients. We present a couple, both carriers of different ribonuclease mitochondrial RNA-processing (RMRP) mutations underlying CHH, which relied on In Vitro Fertilisation (IVF) and PGD to select unaffected embryos that were HLA compatible with an affected compound heterozygous sibling. Preclinical workup involved confirmation of the maternal g.126C>T and paternal g.40G>A mutations in genomic DNA of the couple and affected child, together with informativity and segregation testing of RMRP polymorphic markers (STR16GT at 248 kb 5' and STR16AAT at 34 kb 3') and markers in the HLA locus of 4 Mb (MOG3, D6S2700, D6S2670, D6S2443, D6S1560 and D6S1618). (Half)-informative HLA and RMRP markers were selected and combined with the amplicon of the mutations in a single-cell multiplex PCR protocol. The mutations were detected using post-PCR minisequencing reactions. The RMRP markers ensured an accurate diagnosis of the mutations whereas the HLA markers provided indirect preimplantation HLA haplotyping. The couple achieved an ongoing singleton pregnancy in their third IVF/PGD cycle and the PGD results were confirmed in prenatal testing. At birth, hematopoietic stem cells will be collected from the cord blood and used to transplant the affected sibling. This report shows that PGD with HLA matching is an alternative therapeutic option for couples of reproductive age with children affected by CHH requiring a non-urgent HSCT and lacking suitable donors.

Mitochondrial DNA (mtDNA) copy number reduction, known as the mitochondrial DNA depletion syndrome (MDS), is a common cause of severe mitochondrial disorders of infancy and early childhood. MDS results from defects in nuclear encoded factors involved in mtDNA maintenance and within the past years mutations in the POLG1, DGUOK, MPV17, PEO1, SUCLG1, TK2, SUCLA2 and TYMP genes have proven to be implicated in the pathogenesis of this disorder. The clinical phenotypes associated with the different gene alterations vary considerately but present either as a hepatocerebral or a myopathic syndrome. We have identified a homozygous p.E85 deletion in exon 3 of the RRM2B gene in a neonate. The patient, born to first-cousin Caucasian parents, presented with lactic acidosis, severe hypotonia, deafness, blindness and hyperammonemia. Muscle biopsy showed RRF, a combined respiratory chain defects and massive subcomplexes of ATP synthase both with traditional spectrophotometry and BN-PAGE. Western blotting using antibodies against selective OXPHOS subunits indicated the preservation of nuclear encoded complex II. She died at 2 months of age. Mutations of the RRM2B gene, encoding the cytosolic R2 subunit of a p53 controlled ribonucleotide reductase (p53R2), have been reported to cause severe depletion of muscle mtDNA by Bourdon et al. 2007. Indeed

Introduction: We investigated the spatiotemporal expression pattern of different DNA methyltransferases (DNMTs): DNMT3L, the de novo DNA methyltransferase regulator, and DNMT1, an enzyme responsible for maintenance of the methylation patterns during DNA replication. There are two forms of DNMT1: DNMT1o, found only in oocytes and cleavage stage preimplantation embryos and the somatic form DNMT1s. DNMT1o lacks the N-terminus of DNMT1s. These proteins are crucial for the establishment and maintenance of epigenetic marks during gametogenesis and embryonic stages. DNMT deficiencies will lead to embryonic developmental defects, cancer and other diseases. Not much is known about the expression and regulation of these proteins in human oocytes and preimplantation embryos and we will compare our results to data obtained in mouse. Material and Methods: Spare human oocytes and preimplantation embryos from infertility treatments at our centre were donated after informed consent of the patients. Oocytes and embryos were stained by immunocytochemistry with antibodies against DNMT3L and two different epitopes of DNMT1 to distinguish both forms. One DNMT1 antibody is specific for the N-terminus, and thus binds only to DNMT1s, the other binds to the C-terminus of both proteins. Immunofluorescence was visualised with a confocal laser scanner for 3D images. Results: DNMT3L was absent in oocytes (n = 13), zygotes (n = 2) and 4-cell stage embryos (n = 9). It was observed in the cytoplasm of blastomeres from the 8-cell stage onwards. In expanding and expanded blastocysts (10/18) DNMT3L was found in both the cytoplasm and nucleus of cells from trophectoderm and inner cell mass. DNMT1o and DNMT1s could not be detected in the majority of human oocytes (n = 37) while a weak DMNT1o expression could be detected in most zygotes (n = 7). DNMT1s was mainly found in the cytoplasm from the 2-cell stage on whereas DNMT1o was constantly present in the nucleus from the 8-cell stage onwards. This pattern seems opposite to mice, where Dnmt1s shows a nuclear localization during all preimplantation embryonic stages and Dnmt1o remains cytoplasmic with only a transient nuclear switch at the 8-cell stage. Conclusion: In human preimplantation embryos there is a gradual increase of DNMT3L in the cytoplasm from the 8-cell stage onwards, which may correspond with the activation of the embryonic genome. After blastocyst expansion DNMT3L appears in the nucleus of the cells where it may assist DNMT3a and DNMT3b in de novo methylation processes. These results are in line with mice where a sharp rise in Dnmt3L was found before the time of implantation, which is the time window for genome-wide de novo methylation processes. In mice, Dnmt3L has been found at early stages of oogenesis. We have not been able to examine this in humans because we do not have access to growing human oocytes yet. The absence of DNMT1o in oocytes of different maturity stages together with its weak expression in zygotes could reflect the superior quality of oocytes suitable for fertilization and bring forward the hypothesis that DNMT1 expression is only associated with maturation competency. Alternatively, maternally stored DNMT1 mRNA transcripts are translated at the zygote stage. The absence of nuclear DNMT1 expression during the first two cleavage divisions may help the passive genome-wide demethylation process that takes place in the early embryo. After the 4-cell stage, the protein was also detected in the nucleus. The differences in spatiotemporal expression patterns between human and mouse for DNMT1 may be due to differences in the demethylation reprogramming process while the similarities found for DNMT3L point to a better homology for the de novo methylation process.

Introduction: According to the literature, the fertilized oocyte is converted into a multicellular embryo by cleavages that occur according to specific planes (Gulyas, 1975). A meridional first cleavage creates two cells with animal and vegetal cytoplasm. During the second cleavage, one cell divides meridionally, producing two identical cells, whereas the other daughter cell divides equatorially, creating one cell with animal cytoplasm and another with vegetal cytoplasm. This way of cleaving results in a tetrahedral arrangement of the four blastomeres (Payne et al., 2004; Edwards et al., 1970). However, also non-tetrahedral shaped 4-cell embryos have been described, assuming two meridionally or equatorially divisions during the second cleavage. Because oocytes might exhibit a polarized ooplasm, the distribution of maternally inherited factors may be different in tetrahedral and non-tetrahedral embryos (Antczak and Van Blerkom, 1997; Edwards and Beard, 1997). This study aimed to examine whether differences in the developmental capacities and pregnancy rates exist between tetrahedral and non-tetrahedral embryos. Material and Methods: The architecture of 1850 4-cell embryos of 540 patients was evaluated. The developmental capacity up to day 5 was studied in 698 4-cell embryos of 148 patients. The embryos were classified as top blastocysts, good blastocysts or bad quality embryos. Pregnancy rate was examined in 113 patients (

Alterations throughout the androgen receptor (AR) gene influence male reproductive development and function and are associated with androgen insensitivity syndrome (AIS), a disease with a prominent genotypic-phenotypic heterogeneity. We determined the AR gene sequence in 21 patients. Ten patients were suspicious of AIS. 4/6 with known 46,XY karyotype had complete AIS (CAIS) and 1/6 had premature amenorrhea. The presence of an Y-chromosome was confirmed in 2 patients by AR-MLPA, whereas karyotypes remained unknown for 2 AIS patients. Three nonsense mutations were discovered at codons 353, 829 and 831 respectively, and a 4bp duplication c.2227_2230dupATGG were found to induce a premature stopcodon at codon 769. Two missense mutations at codon 700 and 860 were detected within the AR ligand binding domain. Suspicion of AR-gene or -exon deletion in 2 patients, was corroborated by AR-MLPA, demonstrating the importance of confirmation methodology. For the remaining 6 patients (few clinical and/or karyotype information was available and) no AR-alterations were found. Carrier status was confirmed in 5 investigated relatives of some of the index cases. Our study has revealed a number of previously undescribed amino acid alterations presumingly causing impairment of the AR protein function. Our findings demonstrate that AR sequencing and MLPA are important tools to support the clinical diagnosis and counseling of AIS. Moreover, the AIS genetic testing may be used for prenatal diagnosis and pre-implantation genetic diagnosis, for which the CAG-repeat in exon 1 can be used as marker.

Mitochondrial disorders of oxidative phosphorylation (OXPHOS disorders) affect ~1/5000 individuals in the general population and present with a surprisingly wide range of multisystemic and neuromuscular phenotypes. The m.14487T>C mutation is a known pathogenic mtDNA mutation resulting in an amino acid substitution (p.M63V) in NADH dehydrogenase 6 (ND6), a complex I subunit of the mitochondrial respiratory chain, and has thus far been found in isolated cases with infantile Leigh syndrome and progressive dystonia. We report here adult and late-onset phenotypes as it was seen in a 5-generation Belgian family with 12 affected family members. Clinical and mutation load data were available for 9 family members, while biochemical analysis of the respiratory chain was performed in 3 muscle biopsies. Heteroplasmic m.14487T>C levels (36-52 % in leukocytes, 97-99 % in muscle) were found in patients with progressive myoclonic epilepsy (PME) and dystonia or progressive hypokinetic-rigid syndrome. Patients with infantile LS were homoplasmic (99-100 % in leukocytes, 100 % in muscle). We found lower mutation loads (between 8 and 35 % in blood) in adult patients with clinical features including migraine with aura, Leber Hereditary Optic Neuropathy (LHON), sensorineural hearing loss and Diabetes Mellitus type 2. Despite homoplasmic mutation loads, complex I catalytic activity was only moderately decreased in muscle tissue. Conclusions: The m.14487T>C mutation resulted in a broad spectrum of phenotypes in our family. This is the first report of PME as an important neurological manifestation of an isolated mitochondrial complex I defect.

Introduction: Incontinentia pigmenti (IP) is a rare X-linked dominant genodermatosis that is lethal in males. Females can survive because of X-inactivation mosaicism. The most frequent mutation is a deletion of exons 4-10 of the gene, NEMO that causes IP. Materials and Methods: Molecular analysis was performed in 99 female patients referred for NEMO gene analysis. Results: The patients were first studied for the presence or absence of the exon 4-10 deletion by PCR: 46 patients carried this deletion. Upon request of the referring physician, 10 of the 53 patients without a deletion were further sequenced, and in 7 a mutation (six resulting in nonsense codons and one in a missense codon) was found. From 30 patients with a proven mutation DNA samples from the mothers were available for testing and in 7 of them a mutation was found (7/30 or 23%). In the end, no mutation was found in 46/99 patients. Conclusion: Our results are in agreement with other studies that show that the exon 4-10 NEMO deletion is a major mutation causing IP, and that many mutations occur de novo. However, the deletion frequency in our patients is lower than has been described in literature (46% versus 70-90%). This can probably be attributed to the fact that we performed deletion screening for each request for IP mutation analysis without any selection. Clinical data are being collected to verify this hypothesis.

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Preimplantation genetic diagnosis (PGD) is the genetic analysis of a single (or two) blastomere(s) from an in vitro fertilized embryo and is offered to couples to avoid the transmission of heritable genetic disorders to their potential offspring. Fluorescence in situ hybridization (FISH) is the current standard technique to detect chromosomal imbalances, however it requires a long, labor-intensive and indication-specific preparation. Moreover, the number of fluorescent labels is limited and couples carrying complex chromosomal aberrations cannot be helped. Array-based PGD can overcome all these shortcomings and will increase the accuracy. As proof-of-principle, we applied the genome-wide 1Mb resolution BAC array in the PGD-clinic for a couple that suffered multiple miscarriages and could not be helped using the current FISH technique since one of the partners carried a complex chromosomal aberration including a reciprocal translocation between chromosomes 6 and 14 and an insertion between chromosomes 2 and 3. In concurrence with the ethical committee, we decided to check only the copy number status of the chromosomes involved in the translocation and insertion. Consequently, whole chromosome or segmental imbalances of other autosomes did not affect the transfer policy. The chromosomal imbalances detected following array screening were confirmed on the remaining blastomeres using locus-specific FISH. Two array-based PGD cycles with single embryo transfer were performed, which resulted in a clinical pregnancy. The embryo that give rise to the pregnancy was normal diploid (or balanced) for both the translocation and the insertion region, but carried however a trisomy 8 and nullisomy 9 in one blastomere, while the second blastomere was completely normal diploid. Besides the successful implementation of single cell arrays into the fertility clinic, we thus proved that at least some mosaic embryos, which are currently all discarded, can result in a clinical pregnancy. Finally, the application of single cell arrays on human cleavage stage IVF embryos revealed new insights in the segregation patterns during early embryogenesis. Whereas mosaic FISH results would have been classified as potential FISH errors, whole-chromosome array analysis shows that despite the occurrence of meiotic aberrations, mosaic results can be explained by additional mitotic non-disjunctions. In conclusions, FISH can be replaced by genome-wide single cell array analysis in PGD for the detection of chromosomal imbalances as small as 10 Mb.

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Introduction: It is still unclear if the in-vitro procedure and more specifically the ICSI procedure, in humans can permanently and detrimentally affect metabolic function in the offspring. Some surveys point to an increased risk for impaired glucose tolerance and increased adiposity during childhood. To investigate whether ICSI conception leads to an altered body fat accumulation and distribution in the offspring during adolescence, skinfold measurements were compared between children born after ICSI and after spontaneous conception (SC). Patients and Methods: Preliminary body composition data of 118 ICSI (50% boys) and 162 SC (47 % boys) children were analysed at the age of 14 years. ICSI teenagers, all singletons and born after 32 weeks of gestation, were part of a previously published ICSI cohort followed since birth at the Medical Genetics Department of UZ Brussel, Belgium. Spontaneously conceived peers were recruited from schools in the surroundings. Skinfold thickness was measured by a Harpenden calliper at the biceps and triceps site (as markers for peripheral adiposity) and at the subscapular and suprailiac site (as markers for central adiposity). BMI, the sum of skinfolds and the sum-derived estimate of body fat (expressed as percentage) were calculated as an index for total adiposity. The subscapular/triceps skinfold ratio was measured as an index of body fat distribution. Results: Mean age of the ICSI teens was 14.2 ± 0.5 years and 14.3 ± 0.4 years of the SC teens (p = 0.005). Pubertal development, assessed by Tanner staging, was also different between the ICSI and SC population (p = 0.009). As expected, boys had a significant lower mean sum of central (20.1 ± 10.4 mm), peripheral (19.5 ± 10.0 mm) and total (39.6 ± 19.4 mm) skinfolds and percentage body fat (19.9 ± 5.2 %) compared to girls (26.9 ± 10.0 mm; 26.7 ± 8.7; 53.7 ± 18.0 mm; 28.3 ± 3.7 %) in both study groups (p 0.05). Controlling for child characteristics at the moment of examination (age, gender, height and pubertal stage) did not change the results. Unadjusted mean sum of peripheral skinfolds did not differ between ICSI (24.3 ± 10.2 mm) and SC children (22.5 ± 9.9 mm) (p > 0.05). However, a significant difference in mean sum of peripheral skinfolds ( + 2.3 mm in the ICSI group) was found between ICSI and SC children after correction for the auxological confounding variables (p = 0.047). The effect size of 2.3 mm difference in peripheral skinfolds between the ICSI and the SC group is in accordance with a previous report on body composition in IVF children (Ceelen et al., 2007). Conclusion: Preliminary results of skinfold measurements indicate that ICSI conceived teenagers are more prone to peripheral adiposity, but not to central and total adiposity compared to spontaneously conceived peers. However, before we can draw firm conclusions on the role of the in vitro procedure on body composition and long term health outcome, more subjects available in this worldwide eldest cohort of ICSI offspring have to be examined.

In severe forms of Diamond-Blackfan anemia, preimplantation genetic diagnosis (PGD) of histocompatibility leukocyte antigen-compatible embryos for enabling the next sibling in the family to be a stem-cell transplantation donor constitutes the sole lasting cure capable of terminating the enduring need for iterative transfusions. We report here an open collaboration between two renowned institutions to provide a family desiring this treatment even though they resided where the preimplantation genetic diagnosis procedure is banned.

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Mitochondrial DNA (mtDNA) copy number reduction, known as the mitochondrial DNA depletion syndrome (MDS), is a common cause of severe mitochondrial disorders of infancy and early childhood. MDS results from defects in nuclear encoded factors involved in mtDNA maintenance and within the past years mutations in the POLG1, DGUOK, MPV17, PEO1, SUCLG1, TK2, SUCLA2 and TYMP genes have proven to be implicated in the pathogenesis of this disorder. The clinical phenotypes associated with the different gene alterations vary considerately but present either as a hepatocerebral or a myopathic syndrome. We have identified a homozygous p.E85 deletion in exon 3 of the RRM2B gene in a neonate. The patient, born to first-cousin Caucasian parents, presented with lactic acidosis, severe hypotonia, deafness, blindness and hyperammonemia. Muscle biopsy showed RRF, a combined respiratory chain defects and massive subcomplexes of ATP synthase both with traditional spectrophotometry and BN-PAGE. Western blotting using antibodies against selective OXPHOS subunits indicated the preservation of nuclear encoded complex II. She died at 2 months of age. Mutations of the RRM2B gene, encoding the cytosolic R2 subunit of a p53 controlled ribonucleotide reductase (p53R2), have been reported to cause severe depletion of muscle mtDNA by Bourdon et al. 2007. Indeed

Introduction: The zygote is the ultimate totipotent cell. Totipotency is lost during preimplantation development, however it is unknown when and in which cells. According to the inside-outside hypothesis of Tarkowski et al. (1967), the outer cells of a morula become trophectoderm (TE) cells, whereas the inner cells become inner cell mass (ICM) cells. The first morphologically visible sign of differentiation becomes apparent at the fifth day of preimplantation development during blastulation when the two lineages can be distinguished. Blastomeres of 2-cell, 4-cell and 8-cell stage human embryos are thought but not proven to be totipotent. In order to allow the embryo to overcome perturbations in its organization such as cell loss due to fragmentation, cryodamage and biopsy for preimplantation genetic diagnosis or disturbance of the cleavage planes, the blastomeres should be flexible and capable to develop into ICM and TE. This has been called regulative development. The developmental decision to become ICM or TE is crucial. We aimed (1) to investigate regulative development and (2) to determine when the decision to develop into ICM or TE becomes irreversible. Material and Methods: Embryos were created in vitro with the approval of the Belgian Federal Committee on medical and scientific research on embryos in vitro and the Local Ethical Committee using gametes from consenting donors. In addition, embryos which were diagnosed to carry a genetic disease were used after informed consent. Only top quality embryos were used. Embryos were biopsied in Ca2 + /Mg2 + free medium using laser biopsy. Blastomeres were aspirated using a pipette with an inner diameter of 60-80 µm and put into an empty zona pellucida (ZP). Reconstituted embryos were cultured individually in sequential medium in microdroplets under oil up to day 6 of preimplantation development. They were analysed by immunocytochemistry and confocal microscopy for the expression of NANOG (ICM) and HLA-G (TE) (data not discussed). Results: In order to investigate regulative development in vitro, we disturbed the organization of the embryo by changing the position of the blastomeres at the 4-, 8- and compacting/compacted stage. Seven tetrahedron 4-cell stage embryos (day 2), eight 8-cell stage embryos (day 3) and eight compacting/compacted embryos (14-28 cells on day 4) were disaggregated and the blastomeres were randomly transferred into an empty ZP. Six, eight and seven reconstituted embryos respectively developed into good quality blastocysts with TE and ICM. In order to determine at which stage the decision to become ICM or TE is irreversibly taken, we investigated the potency of the outer cells to develop into blastocysts with TE and ICM. Seven compacting/compacted embryos were manipulated by removing a limited number of outer cells (5-7) in order not to confuse with inner cells and these cells were transferred into an empty ZP. Six out of these ZPs with outer cells only developed into blastocysts with TE but none of them appearedto have an ICM. Six out of the seven original embryos (few remaining outer + all inner cells) developed into good quality blastocysts. In order to exclude the possibility that 5-7 outer cells is not enough mass for the development of an inner cell population within the embryo, outer cells of two distinct embryos were transferred into an empty ZP (12-14 cells). Four embryos reconstituted with outer cells only developed into good quality blastocyst with TE and ICM, as well as the eight original embryos. Conclusions: Interference with the localisation at the 4-cell, 8-cell and compacting/compacted stage does not interfere with blastocyst formation, indicating that human preimplantation development is highly regulative. The decision by the outer cells to become TE is not irreversibly taken at the compacting/compaction stage of human preimplantation development.

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Objectives Our primary aim was to gain more insight into maturation arrest of spermatogenesis (MA), and its relationship with mutations in genes essential for meiosis. Yet, we also investigated the possibility that mutations in human meiosis genes are causing a milder phenotype than predicted from mouse studies. We speculated that in such cases meiosis could potentially be completed with the production of mature germ cells but with abnormal chromosomal constitution. After fertilization, miscarriages might be a consequence. Methods Patients with MA (n=40) or couples with recurrent miscarriages (n=23) were analyzed for the presence of mutations in three genes (SYCP3, MSH4 and DNMT3L) by PCR and sequencing. Results Among patients with MA, five changes were observed that also predict an alteration at the amino acid level. However, since all changes were also present in men with normozoospermia in equal frequencies, these changes are likely single nucleotide polymorphisms. Among the46 patients with recurrent miscarriages, two additional changes were detected that predict an alteration at the amino acid level. One change was also detected in controls. However, the second heterozygous change detected in a conserved functional domain of the SYCP3 gene (p.I183T ) was absent in >200 controls. Conclusions Despite our small patient group, these preliminary results confirm our prediction that abnormalities in meiosis genes might be involved in the formation of gametes with abnormal chromosomal constitution. Yet, further research is necessary to determine the impact and frequency of such changes before implementing mutation screening in genetic counseling.

Introduction: Oocyte donation (OD) has become well established. As with any other reproductive techniques, assessment of possible associated obstetric and perinatal risk remains paramount. To evaluate the pure obstetrical and perinatal impact of conceiving through OD we compared an OD-group with a matched control group of autologeous oocytes (AO). Secondly, we evaluate differences in obstetrical and perinatal outcome between OD-pregnancies for which an appropriate AO-match was available, versus OD-pregnancies at an advanced maternal age and therefore without a match. Materials and Methods: All singleton (n = 210) and twin (n = 87) pregnancies beyond 20 weeks of gestation conceived after OD at our centre between 1999 and 2008 were considered for this study. Controls were selected from the patient population undergoing in-vitro fertilization with AO. As for the OD-pregnancies, all controls were conceived with the intra cytoplasmic sperm injection (ICSI) technique. A match for maternal age ( ± 12 months), parity, plurality (singleton or twin) and gender of the children could be found for 205 OD-pregnancies resulting in 262 live births. Obstetrical outcome data from these 205 OD-pregnancies and their matched control pregnancies were analysed by multivariate analysis. Matched groups were compared using paired t tests for continuous variables and McNemar test for categorical variables. We performed conditional logistic regression analyses, further adjusting for paternal age, age of the oocyte donor and for the number of embryos transferred on the obstetric and perinatal outcomes. Obstetrical and perinatal outcome of OD-pregnancies without available match were analysed using Student t tests for continuous variables and Fisher's exact test for categorical variables. We also conducted linear and binary logistic regression analyses adjusting for baseline characteristics of interest. Results: OD is associated with a increased risk of first trimester bleeding (OR:1.493 CI:1.036-2.15) and pregnancy induced hypertension (PIH) (OR:1.502 CI: 1.024-2.204) as seen by conditional logistic regression of the matched groups after further adjusting for paternal age, age of the donor and number of transferred embryos. No differences in gestational age, mean birth weight and length, head circumference or Apgar scores were observed between the two matched groups. Logistic regression of obstetrical outcome parameters in all OD-pregnancies (n = 297) found a significant relation between preterm labour, the incidence of caesarean section and the presence of a twin pregnancy. Matching could not be performed for 152 live births resulting from 92 singleton pregnancies and 30 twin OD-pregnancies. These oocyte recipients without an available AO-match in our ICSI-population were significantly (p 1500g) birth weight babies between the older and younger OD-population in singletons and twin pregnancies. Conclusion: This is the first large follow-up study that demonstrates that OD is associated with an increased risk for first trimester bleeding and PIH independent of the recipients' parity, plurality or age of the recipient, donor or partner. However, no difference in perinatal outcome is observed after OD. A shorter gestational age and a lower birth weight and length were observed in OD-recipients of an advanced maternal age as compared to OD-recipients at an age were ICSI-pregnancies with AO available.

Mitochondrial disorders of the oxidative phosphorylation (OXPHOS) system affect ~1/5000 individuals in the general population and present with a surprisingly wide range of multisystemic and neuromuscular phenotypes. The m.14487T>C mutation is a known pathogenic mtDNA mutation resulting in an amino acid substitution (p.M63V) in NADH dehydrogenase 6 (MT ND6), a complex I subunit of the mitochondrial respiratory chain. Thus far it has been found in isolated cases with infantile Leigh syndrome and progressive dystonia. We report here adult and late-onset phenotypes as it was seen in a 5-generation Belgian family with 12 affected family members. Clinical and mutation load data were available for 9 family members, while biochemical analysis of the respiratory chain was performed in 3 muscle biopsies. Heteroplasmic m.14487T>C levels (36-52 % in leukocytes, 97-99 % in muscle) were found in patients with progressive myoclonic epilepsy (PME) and dystonia or progressive hypokinetic-rigid syndrome. Patients with infantile LS were homoplasmic (99-100 % in leukocytes, 100 % in muscle). We found lower mutation loads (8-35 % in blood) in adult patients with clinical features including migraine with aura, Leber Hereditary Optic Neuropathy (LHON), sensorineural hearing loss and Diabetes Mellitus type 2. Despite homoplasmic mutation loads, complex I catalytic activity was only moderately decreased in muscle tissue of these patients. Conclusions: The m.14487T>C mutation resulted in a broad spectrum of phenotypes in our family. This is the first report of PME as an important neurological manifestation of an isolated mitochondrial complex I defect.

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Introduction: Accumulating evidence suggests that the preimplantation environment has an influence on postnatal development and cardiometabolic health later in life. To evaluate if the in-vitro procedure in humans has longterm consequences on the cardiovascular functioning, we compared blood pressure at the age of 8 and 10 years in children born after intra cytoplasmic sperm injection (ICSI) with children born after spontaneous conception (SC). Material & Methods: Longitudinal questionnaire data and parameters collected at physical examination including longitudinal blood pressure measurements of 8-year-old ICSI children were compared with results of peers born after SC. At the age of 10 years, 108 of the intial recruited150 ICSI children were re-examined and 93 out of the 147 SC children. All children were singletons born after 32 weeks gestation. Blood pressure measurements were taken using a portable manual sphygmometer, at the same time in the protocol i.e. after the child had been seated for a least five minutes. The measurements were performed at the non-dominant arm in the sitting position after the child had been seated for a least five minutes by a qualified investigator. Results: Blood pressure measurements were available at 8 years in 100 ICSI and 90 SC children and at 10 years in 104 ICSI and 92 SC children. Birth weight, birth weight SDS, birth length and gestational age did not differ between the ICSI population and the SC group. Anthropometric findings such as height and weight in ICSI and SC children were comparable, at the age of 8 as well at the age of 10 years. Systolic and diastolic blood pressure were higher in ICSI children than in SC children at the age of 8 years (98 mmHg versus 94 mmHg; P , 0.001 and 59 mmHg versus 55 mmHg; P ¼ 0.001 respectively). The difference remained after correcting for birth characteristics (weight, gestational age), maternal factors (age, nulliparity, level of education) and current physical characteristics (age, BMI, pubertal score). Ten-year old ICSI children had a comparable systolic but a lower diastolic blood pressure compared to their spontaneous conceived peers (99 mmHg versus 99 mmHg; P ¼ 0.5 and 65 mmHg versus 68 mmHg; p ¼ 0.002 respectively), although the effect of lower diastolic blood pressure in 10-year-old children attenuated after adjusting for confounders. Conclusion: ICSI conception is associated with a 5 mmHg increase in blood pressure at 8 years, but this finding could not be confirmed at age 10. Blood pressure levels in childhood are known to be predictive of those in later life. Our findings warrant long-term follow-up of ICSI conceived individuals to assess possible effects of periconception events on cardiovascular health in later life. Currently, ICSI children are examined at the age of 14-15 years.

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Antithrombin (AT) is the major inhibitor of thrombin and other serine proteases involved in coagulation. Hereditary AT deficiency is the most severe form of thrombophilia with first thrombotic events often presenting at young age. Thrombotic risk varies with the type of deficiency. Types and subtypes are defined by plasmatic assays in combination with molecular analysis of the AT gene. In this report, we describe two cases in which sequencing analysis could not identify the causative mutation. Genetic testing included complete sequencing of all exons and intron/exon junctions of the AT gene and multiplex ligation-dependent probe amplification (MLPA), a technique which allows exon quantification. The first patient was a 44-year old woman with a strong personal and familial history of thrombosis. Sequencing revealed a type II Reactive Site mutation at nucleotide position g.13268G>T (p.Ala384Ser), while plasma levels of AT antigen and activity rather suggested type I deficiency. Our second patient was a 13-year old girl with a first event of deep vein thrombosis and positive familial history. Sequencing could not identify any mutation. MLPA analysis revealed in our first patient a large deletion comprising exons 3A and 3B and in the second patient a deletion of exon 5. Both deletions were confirmed by long range PCR. Large deletions/insertions form only a small proportion of all mutations causing hereditary AT deficiency. Southern blot for the detection of large deletions is very time consuming. MLPA, on the other hand, is a much faster and easier method. We suggest performing MLPA whenever inherited AT deficiency is suspected but sequencing fails to identify the molecular defect.

Introduction: Fluorescent in situ hybridization (FISH) is currently the main method used for aneuploidy preimplantation genetic screening (PGS). The main limitation of FISH analysis is that it usually includes only 5-10 chromosome- specific probes, compared to the 23 chromosome pairs of the human cell. This fact along with the high incidence of mosaicism in human embryos could be the explanation for the low success rates of PGS. The development of microarray technology combined with whole genome amplification (WGA) has recently emerged as a promising solution for comprehensive aneuploidy screening of the whole genome of a single cell, since array-CGH allows for the analysis of the full chromosome content and the diagnosis of small genomic imbalances. This study aims to evaluate the use of single-cell array-CGH for the molecular karyotyping of pre-implantation embryos in a pre-clinical setting, to investigate if by analyzing one blastomere we can identify a karyotypically fully normal embryo and to determine the levels of mosaicism in human embryos. Materials and Methods: The study design is blinded, including two operators. Nine good quality supernumerary IVF/ICSI frozen embryos donated for research were thawed at day 3 of embryo development, were disaggregated and all the blastomeres (n ¼ 42) numbered randomly by the first operator. The blastomeres were then subjected to multiple displacement amplification (MDA) to amplify the whole genome of each single cell. The second operator carried out the array-CGH using BAC/PAC arrays of 1 MB resolution (VIB, Leuven). As a reference, genomic DNA was used that was extracted from peripheral blood of male and female donors. Both test and reference DNA samples were labeled by random primer labeling using Cy5- and Cy3-labeled dCTPs. The labeled DNAs were co-precipitated and the slides hybridized in a humid chamber at 378C for 24 to 72 hours. The slides were then washed and scanned and the log2 of the Cy5 to Cy3 fluorescent intensity ratios were calculated and plotted. Results: Array CGH was carried out for the 35 blastomeres that were successfully amplified by MDA and array data were analysed. Before decoding, an attempt was made to assign the single blastomeres to the embryo they were assumed to come from. After decoding of the samples, the following results were obtained: all the blastomeres of two embryos were of normal karyotype. One embryo of which five blastomeres were analysed showed four cells with a normal karyotype 46,XX and one blastomere with 47,XX,þ13. The remaining 24 blastomeres came from six embryos and they were totally chaotic, since that all blastomeres in each embryo showed an abnormal and different karyotype precluding classification per originating embryo prior to decoding. Conclusions: These preliminary results show that array-CGH can be reliably used at the single cell level and diagnose whole chromosome aneuploidies, and moreover identifies chromosomal aberrations such as partial duplication or deletion of chromosome arms. Our results confirm the highprevalence of mosaicism in well developing human embryos and that a variety of different abnormalities can co-exist in an embryo. Most importantly, we could blindly identify the ten normal blastomeres from three embryos. If these nine embryos had been analyzed for PGS, we would have correctly identified the two fully normal embryos, but not the mosaic 46,XX/47,XX,þ13. We are currently analyzing a larger cohort of embryos and we will confirm the diagnosis by multiplex PCR on all the trisomic chromosomes.

Introduction: PGD (Preimplantation Genetic Diagnosis) combined with HLA (Human Leukocyte Antigen) matching is one of the recent applications in reproductive medicine which is used to select HLA compatible embryos in order to cure a severely affected sibling with a transplantation using haematopoietic stem cells from the newborn umbilical cord blood. PGD-HLA emerges numerous ethical and psychological questions. Materials and methods: The number of children born after PGD-HLA is very scarce. In order to evaluate the psychological well-being of the child conceived after PGD-HLA, we visited 3 families spread over Europe and interviewed them about the initial motives to opt for this treatment, how the pregnancy/ treatment was experienced, the openness about this project towards family, friends and the PGD-HLA conceived child and how the PGD-HLA conceived child is experienced by the parents and sibling(s). The degree of contentment about the guidance of the fertility centre before, during and after the treatment was also addressed. Results: All three children were too young (between 18-24 months) to participate in the interview. The parents informed the broader social circle about their project, the sibling(s) were also very aware of the fact that the youngest sibling is a compatible donor. In 2/3 of the families the PGD-HLA children will be informed about the nature of their conception once they start asking questions or can understand. One of the PGD-HLA children was born two weeks after his ill sibling deceased. In this family the PGD-HLA child was the only living child in the family. Because of this reason and because of the different socio-cultural background (Muslims) of this family they did not see the need to inform the child about the motives for his conception. In the two other cases additional bone marrow transplantations were necessary to cure the affected sibling. All parents stated they had had the desire to have another child and the PGD-HLA child was not only conceived to be the donor for his brother or sister. Moreover, all three children seemed very well integrated in their families and were raised and loved in the same way as their sibling(s) according to the parents. The mothers, told that any problems or inconveniences during the treatment/pregnancy faded as compared to what their ill child had to suffer and in one case the ill child was worried about the well-being of the PGD-HLA conceived sibling because additional transplants were necessary an it did not want her PGD-HLA sibling to suffer for her. All three families were content with the guidance/treatment from the hospital. Even the couple that had lost the child before the PGD-HLA conceived child was born was satisfied about the services of the centre since they were pregnant after the first attempt. Conclusion: It is impossible to make solid conclusions on the basis of only 3 follow-up interviews. In the future, assessments of these children and their families on the basis of a multi-method approach (interviews, questionnaires, formal childassessment) are recommended. All 3 families had their own particular story introducing many psychological issues that can only be addressed in time. It is clear that with the birth of a PGD-HLA conceived child the medical issues and the coexisting psychological issues are not solved and a long journey with an insecure outcome seems to be ahead. In general, the PGD-HLA treatment did not seemed to adversely affect the psychological well-being of these 3 children so conceived.

Antithrombin (AT) is a major physiological inhibitor of haemostasis. Patients with inherited AT deficiency are at significantly increased risk for venous thromboembolism. The AT gene SERPINC1 is located on chromosome 1q23-25 and a large number of different mutations underlying AT deficiency have been reported. The single nucleotide polymorphism (SNP) rs2227589 in the SERPINC1 gene has recently been found in strong association with venous thrombosis in a well-characterised setting of case-control studies of first deep vein thrombosis. We have analysed DNA samples from 83 index patients and 43 related family members with inherited AT deficiency. We could identify the causal mutation for AT deficiency in all families by sequencing all seven exons and intronic junction regions and by multiplex ligation-dependent probe amplification. Genotyping for the intronic SNP rs2227589 in the SERPINC1 gene was performed by digestion with BsaHI of a PCR fragment containing the SNP. The risk allele (T) for the SNP was absentin 59 of the 83 index patients (71%), heterozygously present in 23 (28%) and homozygously in one (1%). Nearly all patients had a history of venous thromboembolism. Analyses in the related AT deficient family members and in normal controls are ongoing. In the mentioned study on gene variants associated with DVT, the risk allele (T) of the SNP had a minor allele frequency of about 10% in controls and 12% in cases. We found a clearly higher frequency of the risk allele in our AT deficient patient group. We are currently collecting detailed information on clinical presentation and risk factors of the thrombotic episodes in our patient group in order to study interactions with clinical variables.

Aims : Mitochondrial ATP synthase or complex V couples the proton gradient, generated by the respiratory chain, to ATP synthesis. Assembly of this enzyme requires the activities of several accessory proteins. Here, we report on the functional effect of a p.W94R mutation, present in the ATPAF2 gene of an infant with a complex V decreased activity. The ATPAF2 gene is the human (hu) orthologue of the yeast (y) ATP12 gene. Methods : Multi and single copy plasmid constructs containing the wild type ATPAF2 or ATP12 gene, as well as the human mutant p.W94R or the yeast mutant counterpart p.W103R were generated. These constructs were introduced in an ATP12 respiratory deficient yeast strain. ATP synthase activity of individual yeast transformants was assayed by growth studies on a non-fermentable carbon source and by Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) stained for ATP synthase catalytic activity. WT plasmid constructs rescue the respiratory defect of a y delatp12 mutant strain. Results : Growth on a non-fermentable carbon source was strongly impaired for the human W94R mutant, while the growth of the yeast W103R mutant strain was normal. These results were confirmed by BN-PAGE studies and measurements of the complex V activity of the plasmid constructs in yeast cells. Conclusions : Our yeast complementation studies showed clearly that the p.W94R mutant of the huATPAF2p does not confer respiratory competence to an delatp12 yeast strain and is the cause of the complex V dysfunction and early death of our 14 months-old patient. Additional studies with delatp12delfmc1 double mutants, and at 37°C, the altered yeast protein was no longer able to rescue the respiratory deficient phenotype of the S.cerevisiae host strain.

Introduction: Recently we have shown that blastomeres of a 4-cell stage human embryo were able to develop into blastocysts with an inner cell mass (ICM) and trophectoderm (TE). We therefore hypothesized that blastomeres are potentially totipotent and may not yet be allocated to either ICM or TE at this early stage of preimplantation development. To provide further evidence that blastomeres are pluripotent at this stage, we derived human embryonic stem cells (hESC) from single blastomeres of 4-cell stage embryos. Material & methods: Mature oocytes were donated for research by couples treated at our IVF centre who had given their written informed consent. Embryos were obtained after ICSI using spermatozoa of a consenting donor. Three embryos showing less than 10% fragmentation and regular sized blastomeres underwent zona hatching using laser pulses and the blastomeres were removed by aspiration using a pipette with an inner diameter of 50 _m. All blastomeres were individually cultured in sequential medium asused in our IVF programme. On day 4, the blastomere-derived morulas were plated on inactivated mouse embryonic fibroblasts (MEFs) and further cultured in the same serum-free medium as for the hESC derivation and culture. Results: Eleven out of twelve morulas were plated and eight attached to the MEFs. Eventually, one blastomere-derived morula without microscopically visible signs of cavitation produced an outgrowth. When the outgrowth showed a compact colony-like structure with cells of stem-cell-like morphology (day 22 post splitting), it was mechanically passaged onto fresh MEFs. Routine passaging was performed as described for blastocyst-derived hESC. The putative hESC line was shown to express the typical markers of stemness by immunocytochemistry and/or RT-PCR, i.e. POU5F1, NANOG, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, DNMT3B, GDF3, LIN 28, NPM 1, REX-1, SOX-2 and Alkaline Phosphatase. The morphology and growth rate of the colonies was similar to blastocyst-derived hESC. At passage 10, cells were harvested for molecular karyotyping. We observed a 46,XX karyotype, however, with a deletion of chromosome 18q23 and a duplication of chromosome 7q33. In order to demonstrate the pluripotency of this putative hESC line, cells were injected into NOD-SCID mice. After 9 weeks, a teratoma was formed containing cells from the three germ layers including squamous epithelium (ectoderm), cartilage (mesoderm) and gastro-intestinal epithelium (endoderm). The hESC line is currently at passage 45 and was successfully thawed and passaged after cryopreservation. Conclusions: ESC have already been derived from single blastomeres of both mouse and human embryos at the 8-cell stage, but with a low success rate (4% and 2% respectively), indicating that some of these blastomeres are pluripotent at this stage. However, this derivation required the use of a co-culture system involving aggregation or merging microdrops with established ESC lines respectively. Here we describe hESC derivation starting from blastomere-derived morulas that simply attached to MEFs. One out of twelve single blastomeres developed into a hESC line (8% derivation rate). This does not prove that the four cells are pluripotent. Knowing the low derivation rate in general, it will be difficult/impossible to derive hESC from four single blastomeres. Interestingly, the formation of ICM cells has not been essential for the derivation of this particular hESC line. Further research is required to determine whether this blastomere-derived hESC line is functionally different from blastocyst-derived hESC lines.

Introduction Trinucleotide repeat (TNR) instability is the mechanism responsible for numerous inherited human disorders, including Huntington's disease (HD) and myotonic dystrophy (DM1). The TNRs have different patterns of instability according to the disorder as well as the cell type investigated. It is known that the mismatch repair (MMR) machinery is involved in TNR instability, and particularly MSH2 and MSH3 seem to be required for the intergenerational and somatic CTG and CAG expansions. Most of these results have been obtained from studies on transgenic mice, due to the lack of suitable human models. The aim of this work is to study the behaviour of the TNRs causing HD and DM1 in human embryonic stem cells (hESCs), both in the undifferentiated and differentiated state, and its relationship to the MMR machinery, in order to evaluate the potential of these cells as a model in the human. Materials and methods HESC lines were established in our laboratory using spare IVF/PGD embryos. VUB03_DM1 carries a CTG expansion in the DMPK1 gene and VUB05_HD carries a CAG expansion in the huntingtin gene. VUB02 was used as a control line. The lines were kept in standard culture conditions, on mouse embryonic fibroblasts, and samples were taken at different passages. A well-established protocol was used to differentiate hESCs into osteoprogenitor-like cells (OPL). Whenever possible, samples for RNA and DNA extraction were taken after each passage. TNR stability was studied by PCR followed by southern blotting and/or analysis on a DNA sequencer. The MMR machinery was studied by quantitative real-time PCR (qRT-PCR) of the MSH2, MSH3, MLH3 and PMS2 genes. Results While the HD repeat showed a stable pattern in undifferentiated hESCs over more than 80 passages, the DM1 repeat was unstable from the first passages onwards, the TNR mostly increasing in size, leading to a mosaic culture. In the OPLs, the HD TNR remained stable, whereas the DM1 repeat showed a tendency towards a clonal growth (only a few alleles remained detectable), and a stabilization in the behaviour of the repeat. The results of the qRT-PCR showed a downregulation of all the studied genes in the OPLs, in comparison to the undifferentiated hESCs. Conclusions The data on VUB05_HD show that the behaviour of this TNR in hESC follows the same pattern as its somatic behaviour in vivo, in which only striatal neurons show TNR instability. Further experiments on in-vitro differentiated neuronal tissue from hESC are planned. In the case of DM1, the results suggest a correlation between the downregulation of the MMR machinnery and the stabilization of the TNR, which will be further studied by siRNA experiments.

Introduction Human embryonic stem cells (hESCs) are a renewable source of different cell types that could be used for research and cell therapy, and are therefore often kept in culture for long periods of time. It is important to guarantee the general stability of these cells, to ensure the reproducibility and reliability of the results obtained by using them. Recently, several research groups have reported the appearance of culture-induced genetic abnormalities, such as aberrant karyotypes. Although performing a karyotype is part of the routine tests carried out on a putative hESC line during its initial characterization, these findings indicate that the genetic constitution of hESC lines must be regularly followed up. G-banding is the standard method for karyotyping the hESCs, but this was proven difficult and inefficient. We therefore decided to evaluate the use of array-based comparative genomic hybridization to regularly karyotype our hESC lines. Materials and methods Thirteen hESC lines were used in this study, all of them derived in our laboratory using human preimplantation embryos donated for research. Genomic DNA was phenol/chloroform extracted from the cell cultures and used to perform CGH on 1 MB BAC/PAC arrays. Results The following results were obtained (p stands for passage number, b for number of passages after a freeze-thaw procedure). VUB01 carried a monosomy 18 (45, XY, -18) at p55b14 and was 46, XY, +20q11.21 at p135 and p276. This region was amplified as shown by FISH, and contains the DNMT3B gene. VUB02 had a normal karyotype, 46, XY, at p21b15, p109b77 and p219b187. VUB04 was 46, XX, +5q14.2-ter, -18q21.2-ter at p117. A G-banding showed a derivative chromosome 18. VUB05_HD had a normal 46, XY karyotype at p51b21, p58b28, p65b35 and p75b43. VUB07 was 46, XX, +20q11.21 at p77b7, and 46, XX, +3q26.33-q27.2, +20q11.21 at p122b52. These regions contain the genes DNMT3B and SOX2. VUB08_MFS carried a trisomy 22 at p49b11 (47, XX, +22). VUB11_FXS had a normal female karyotype at p45. VUB13_FXS had a normal female karyotype at p28 and p30, but mutated to 46, XX, +5q21.3-ter, -18q12.1-ter at p43, and finally to 45, X, +5q21.3-ter, -18q12.1-ter at p45. G-banding showed a derivative chromosome 18. VUB15, VUB17 and VUB19_DM1 showed normal karyotypes, once 46, XY, twice 46, XX, and at p18, p7 and p5, respectively. VUB20_CMT1A showed a duplication in 5q13.2 at p7 and p18 (46, XX, +5q13.2), that proved to be a polymorphism inherited from the female donor. VUB26 showed a derivative chromosome 18 at p10 (46, XX, +7q33-ter, -18q23-ter), which was visualized by G-banding. Conclusions Molecular karyotyping has proven cost-effective and very accurate. Not only were aneuploidies detected, but small aberrations were also detected, such as the amplifications of the stemness genes DNMT3B and SOX2, which would have passed undetected by G-banding. These amplifications are likely to provide the cells with a better adaptation to the in vitro culture system, and have already proven to influence the results of differentiation experiments in our laboratory. The recurrent appearance of the derivative chromosome 18 is striking, and further experiments are planned to study its biological significance.

Aims : Mitochondrial ATP synthase or complex V couples the proton gradient, generated by the respiratory chain, to ATP synthesis. Assembly of this enzyme requires the activities of several accessory proteins. Here, we report on the functional effect of a W94R mutation, present in the ATPAF2 gene of an infant with a complex V decreased activity. The ATPAF2 gene is the human (h) orthologue of the yeast (y) ATP12 gene. Methods : Multi and single copy plasmid constructs containing the wild type ATPAF2 or ATP12 gene, as well as the human mutant W94R or the yeast mutant counterpart W103R were generated. These constructs were introduced in an ATP12 respiratory deficient yeast strain. ATP synthase activity of individual yeast transformants was assayed by growth studies on a non-fermentable carbon source and by Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) stained for ATP synthase catalytic activity. WT plasmid constructs rescue the respiratory defect of a y _atp12 mutant strain. Results : Growth on anon-fermentable carbon source was strongly impaired for the human W94R mutant, while the growth of the yeast W103R mutant strain was normal. These results were confirmed by BN-PAGE studies and measurements of the complex V activity of the plasmid constructs in yeast cells. Conclusions : Our yeast complementation studies showed clearly that the W94R mutant does not confer respiratory competence to an _atp12 yeast strain and is the cause of the complex V dysfunction in our patient.

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BACKGROUND: Sexual reproduction provides the means for preserving genetic identity and in turn, genetic variability may affect the ability to reproduce. This review aims to summarize current research on genetic diagnosis and genetic causes of reproductive disorders. METHODS: Searches were done by subject in Medline and other databases, and each subject summary was presented to the Workshop Group and omissions or disagreements were resolved by discussion. RESULTS: Single-gene defects are most likely to be found among patients with hypogonadotropic hypogonadism, which may be due to defects in the KAL genes or the gonadotrophin-releasing hormone receptor genes. With premature ovarian failure there is an increased risk of having a premutation of the Fragile X syndrome gene. Complex genetic inheritance may explain the variable familial links in polycystic ovary syndrome and endometriosis, but no definitive genetic pathways are as yet known. With recurrent miscarriage, genetic defects causing thrombophilias are 2-fold more likely. Chromosome abnormalities account for approximately 60% of all spontaneous abortions, and the most common type, trisomy, is closely associated with advanced maternal age. Three percent of couples have a balanced chromosome abnormality, but live birth rates are better with natural conception than with preimplantation genetic diagnosis. CONCLUSIONS: Understanding of the methods used for genetic diagnosis and research is becoming a standard requirement for the clinical practice of reproductive medicine