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Objectives In a previous mass spectrometry study of our research group, 25 proteins were found to be differentially expressed in cerebrospinal fluid of patients with preeclampsia compared to controls. The objective of the current study was to investigate DNA methylation of the genes encoding for the former mentioned proteins in an independent dataset. Study design In a nested case-control study of the Rotterdam Periconceptional Cohort, placental tissue, umbilical cord white blood cells and human umbilical vein endothelial cells (HUVEC) were obtained of 13 patients with early-onset preeclampsia, 16 patients with late-onset preeclampsia and 83 normotensive controls (27 patients with fetal growth restriction, 20 patients with spontaneous preterm birth and 36 uncomplicated pregnancies). DNA methylation of 783 CpGs in regions of 25 genes was measured. Main outcome measures DNA methylation of selected candidate genes in early- and late-onset preeclampsia compared to fetal growth restriction, spontaneous preterm birth and uncomplicated controls. Results From the 783 CpGs of the 25 selected genes, 15 CpGs were differentially methylated between early-onset preeclampsia and spontaneous preterm birth (3.80 E-5 ≤ p ≤ 0.036). Four CpGs were differentially methylated between early-onset preeclampsia and fetal growth restriction (0.0002 ≤ p ≤ 0.037) and 13 CpGs were differentially methylated between early onset preeclampsia and uncomplicated controls (0.0001 ≤ p ≤ 0.04). Conclusion Differences in DNA methylation were found in placental tissue, umbilical cord white blood cells and HUVEC of patients with early onset preeclampsia compared to (un)complicated controls, but not in patients with late-onset preeclampsia. The genes showing the largest differential methylation encode insulin-like growth factor 2 binding protein and receptor and cadherin 13.

Background.With the introduction of three-dimensional (3D) ultrasound it has become possible to measure volumes. The relative increase in embryonic volume (EV) is much larger than that of the crown-rump length (CRL) over the same time period. We examined whether EV is a better parameter to determine growth restriction in fetuses with structural congenital abnormalities.Study Design, Subjects, and Outcome Measures.CRL and EV were measured using a Virtual Reality (VR) system in prospectively collected 3D ultrasound volumes of 56 fetuses diagnosed with structural congenital abnormalities in the first trimester of pregnancy (gestational age 7+5to 14+5weeks). Measured CRL and EV were converted toz-scores and to percentages of the expected mean using previously published reference curves of euploid fetuses. The one-samplet-test was performed to test significance.Results.The EV was smaller than expected for GA in fetuses with structural congenital abnormalities (−35% p<0.001,z-score −1.44 p<0.001), whereas CRL was not (−6.43% p=0.118,z-score −0.43 p=0.605).Conclusions.CRL is a less reliable parameter to determine growth restriction in fetuses with structural congenital abnormalities as compared with EV. By measuring EV, growth restriction in first-trimester fetuses with structural congenital abnormalities becomes more evident and enables an earlier detection of these cases.

BACKGROUND: Ustekinumab is a fully human anti-p40 monoclonal antibody which neutralizes interleukin (IL)-12 and IL-23, thereby interfering with T-helper (Th)1/Th17 pathways and keratinocyte activation, and is highly effective in the treatment of psoriasis. During ustekinumab treatment, some of our patients noticed reduced koebnerization of noninvolved skin and less new plaque formation. OBJECTIVES: To determine whether ustekinumab improves psoriasis-related gene expression and tape-strip responses in noninvolved skin. METHODS: Before and 4 weeks after ustekinumab treatment, noninvolved skin was tape-stripped. After 5 h, biopsies were taken from untouched and tape-stripped skin. The mRNA expression of psoriasis-related markers such as NGF, GATA3 and IL-22RA1, and several antimicrobial peptides (AMP) was quantified. Leucocyte counts and a broad range of inflammatory serum proteins were analysed to gain insight into the systemic alterations. RESULTS: Four weeks following a single ustekinumab injection, NGF showed a significant decrease, whereas GATA3 and IL-22RA1 expression increased, indicative of reduced responsiveness to epidermal triggering. This was accompanied by an increase of the inflammation-related serum proteins GPNMB, MST1 and TRADD. The baseline and tape-strip-induced mRNA expression of the AMP human beta-defensin-2 (hBD-2), S100A7 and LL-37 remained unaltered. Clinically, after 4 weeks, eight out of 11 patients showed a 50% psoriasis area and severity index (PASI) improvement, which was accompanied by a significant reduction in serum hBD-2 levels. No changes were noted in total leucocytes, C-reactive protein and erythrocyte sedimentation rate. CONCLUSIONS: These findings indicate that ustekinumab reduces psoriasis-related gene expression in noninvolved psoriatic skin, making it more resistant to exogenous triggering, without disturbing its antimicrobial response. In parallel, ustekinumab modulates important circulating inflammation-related proteins.

Objectives To examine whether embryonic volume (EV), as measured using three-dimensional (3D) ultrasound and a virtual reality approach, is a better measure of growth restriction than is crown–rump length (CRL) in aneuploid fetuses. Methods We retrospectively measured CRL and EV in prospectively collected 3D ultrasound volumes of 55 aneuploid fetuses using the Barco I-Space VR system. The gestational age ranged from 11 + 2 to 14 + 4 weeks. We compared our measured data with previously published reference curves for euploid fetuses. Delta-values were calculated by subtracting the expected mean for euploid fetuses of the same gestational age from observed values. The one-sample t-test was used to test the significance of differences observed. Results The CRL measurements of fetuses with trisomy 21 (n = 26), trisomy 13 (n = 5) and monosomy X (n = 5) were comparable with those of euploid fetuses, but in fetuses with trisomy 18 (n = 19) the CRL was 14.5% smaller (P

Objectives Virtual reality is a novel method of visualizing ultrasound data with the perception of depth and offers possibilities for measuring non-planar structures. The levator ani hiatus has both convex and concave aspects. The aim of this study was to compare levator ani hiatus volume measurements obtained with conventional three-dimensional (3D) ultrasound and with a virtual reality measurement technique and to establish their reliability and agreement. Methods 100 symptomatic patients visiting a tertiary pelvic floor clinic with a normal intact levator ani muscle diagnosed on translabial ultrasound were selected. Datasets were analyzed using a rendered volume with a slice thickness of 1.5 cm at the level of minimal hiatal dimensions during contraction. The levator area (in cm2) was measured and multiplied by 1.5 to get the levator ani hiatus volume in conventional 3D ultrasound (in cm3). Levator ani hiatus volume measurements were then measured semi-automatically in virtual reality (cm3) using a segmentation algorithm. An intra- and interobserver analysis of reliability and agreement was performed in 20 randomly chosen patients. Results The mean difference between levator ani hiatus volume measurements performed using conventional 3D ultrasound and virtual reality was 0.10 (95% CI, − 0.15 to 0.35) cm3. The intraclass correlation coefficient (ICC) comparing conventional 3D ultrasound with virtual reality measurements was > 0.96. Intra- and interobserver ICCs for conventional 3D ultrasound measurements were > 0.94 and for virtual reality measurements were > 0.97, indicating good reliability for both. Conclusion Levator ani hiatus volume measurements performed using virtual reality were reliable and the results were similar to those obtained with conventional 3D ultrasonography. Copyright © 2012 ISUOG. Published by John Wiley & Sons, Ltd.

Aims Atherosclerotic plaque rupture and subsequent thrombus formation are the major cause of acute cardiovascular events. Local plaque markers may facilitate detection of the vulnerable plaque and help identify the patient at risk for cardiovascular events. Matrix metalloproteinases (MMPs) are prevalent in the arterial wall throughout the arterial system and are associated with local plaque destabilization. We hypothesized that local MMP plaque levels are predictive for atherosclerotic cardiovascular events in other vascular territories. Methods and resultsAtherosclerotic plaques were obtained from 543 patients undergoing carotid endarterectomy (CEA). Plaques were analysed for the presence of macrophages, lipid-core, smooth muscle cells, collagen, calcification, and presence of plaque haemorrhage. MMP-2, MMP-8, and MMP-9 levels were assessed within the plaque. Following CEA, all patients underwent follow-up during 3 years. The primary outcome was defined as the composite of vascular death, non-fatal vascular event, and surgical or percutaneous vascular intervention. In contrast with MMP-2 plaque levels, MMP-8 and MMP-9 levels in the plaque were associated with an unstable carotid plaque composition and clinical presentation at baseline. Increased plaque MMP-8 level (>4.58) was associated with an increased risk for the occurrence of secondary manifestations of atherosclerotic disease during follow-up [hazard ratio 1.76, 95 CI (1.252.48)] (P 0.001), whereas plaque MMP-2 and MMP-9 levels were not predictive for systemic cardiovascular events. ConclusionIn contrast with MMP-2, increased carotid MMP-8 and MMP-9 plaque levels are associated with an unstable plaque phenotype. High collagenase MMP-8 levels in the carotid plaque are associated with the occurrence of systemic cardiovascular outcome during follow-up. © 2011 The Author.

Objectives To evaluate a virtual reality (VR) application for gestational sac fluid volume (GSFV) measurements in first-trimester pregnancies and to study the correlation between different embryonic growth parameters. Methods This was a prospective cohort study analyzing 180 three-dimensional (3D) ultrasound scans of 42 healthy women, performed between 5 + 5 and 12 + 6 weeks' gestational age (GA). The 3D datasets were transferred to the I-Space immersive VR system. The V-Scope application was used to create a ‘hologram’ of the ultrasound image, allowing depth perception and interaction with the rendered objects. Volumes were measured semi-automatically using a segmentation algorithm. In addition to the GSFV, the total gestational sac volume (GSV) and its diameter (GSD) were measured. The GSV was also calculated using the ellipsoid formula. Previously obtained measurements of embryonic volume and crown–rump length (CRL) were included in the study. The outcomes were analyzed using repeated-measures analysis of variance. Results The GSFV was measured in 78 scans, and varied from 434 to 81 491 mm3. A positive correlation between GSFV and GA, CRL and GSD was found. Comparison of the GSD formula constructed in our study in relation to GA with a formula that is commonly used clinically showed an increasing difference with increasing GA either side of 8 + 5 weeks. The GSFV/embryonic volume ratio showed a decrease with GA. The GSV calculated using the ellipsoid formula was on average 19.8% larger compared with the GSV measured in VR. Conclusion New charts for first-trimester GSFV were constructed using VR. These growth charts could be promising tools for studying normal and abnormal embryonic development. Copyright © 2011 ISUOG. Published by John Wiley & Sons, Ltd.

BACKGROUND: The recent introduction of virtual reality (VR) enables us to use all three dimensions in a three-dimensional (3D) image. The aim of this prospective study was to evaluate an innovative VR technique for automated 3D volume measurements of the human embryo and yolk sac in first trimester pregnancies. METHODS: We analysed 180 3D first trimester ultrasound scans of 42 pregnancies. Scans were transferred to an I-Space VR system and visualized as 3D 'holograms' with the V-Scope volume-rendering software. A semi-automatic segmentation algorithm was used to calculate the volumes. The logarithmically transformed outcomes were analysed using repeated measurements ANOVA. Interobserver and intraobserver agreement was established by calculating intraclass correlation coefficients (ICCs). RESULTS: Eighty-eight embryonic volumes (EVs) and 118 yolk sac volumes (YSVs) were selected and measured between 5 +5 and 12 +6 weeks of gestational age (GA). EV ranged from 14 to 29 877 mm 3 and YSV ranged from 33 to 424 mm 3 . ANOVA calculations showed that when the crown-rump length (CRL) doubles, the mean EV increases 6.5-fold and when the GA doubles, the mean EV increases 500-fold (P < 0.001). Furthermore, it was found that a doubling in GA results in a 3.8-fold increase of the YSV and when the CRL doubles, the YSV increases 1.5-fold (P < 0.001). Interobserver and intraobserver agreement were both excellent with ICCs of 0.99. CONCLUSION: We measured the human EV and YSV in early pregnancy using a VR system. This innovative technique allows us to obtain unique information about the size of the embryo using all dimensions, which may be used to differentiate between normal and abnormal human development.

BACKGROUND This study investigates whether dietary patterns, substantiated by biomarkers, are associated with semen quality. METHODS In 161 men of subfertile couples undergoing in vitro fertilization treatment in a tertiary referral clinic in Rotterdam, the Netherlands, we assessed nutrient intakes and performed principal component factor analysis to identify dietary patterns. Total homocysteine (tHcy), folate, vitamin B12 and B6 were measured in blood and seminal plasma. Semen quality was assessed by sperm volume, concentration, motility, morphology and DNA fragmentation index (DFI). Linear regression models analyzed associations between dietary patterns, biomarkers and sperm parameters, adjusted for age, body mass index (BMI), smoking, vitamins and varicocele. RESULTS The 'Health Conscious' dietary pattern shows high intakes of fruits, vegetables, fish and whole grains. The 'Traditional Dutch' dietary pattern is characterized by high intakes of meat, potatoes and whole grains and low intakes of beverages and sweets. The 'Health Conscious' diet was inversely correlated with tHcy in blood (beta = -0.07, P = 0.02) and seminal plasma (beta = -1.34, P = 0.02) and positively with vitamin B6 in blood (beta = 0.217, P = 0.01). An inverse association was demonstrated between the 'Health Conscious' diet and DFI (beta = -2.81, P = 0.05). The 'Traditional Dutch' diet was positively correlated with red blood cell folate (beta = 0.06, P = 0.04) and sperm concentration (beta = 13.25, P = 0.01). CONCLUSIONS The 'Health Conscious' and 'Traditional Dutch' dietary pattern seem to be associated with semen quality in men of subfertile couples.

Objective To investigate accuracy and reliability of four different ultrasound-related volume-measuring methods. Design Observational study. Setting Both in vitro and in vivo. Population or Sample Ten phantoms for in vitro measurements and 28 pregnancies with gestational ages ranging from 6 to 11 weeks for in vivo measurements were included. Methods Three-dimensional (3D) ultrasound images of phantoms (with known variable contents) and yolk sacs were used to calculate volumes using four different methods: Virtual Organ Computed-Aided AnaLysis (VOCAL), inversion mode, Sono Automatic Volume Calculation (SonoAVC) and V-Scope. V-Scope is a newly developed 3D volume visualisation application using a Barco I-Space virtual reality system. Intra- and interobserver agreement was established by calculating intraclass correlation coefficients (ICC). Main outcome measure Evaluation of accuracy and reliability by comparing the different techniques with true volumes (in vitro) and with each other (in vitro and in vivo). Results In the in vitro study, volume measurements by VOCAL, inversion mode and V-Scope proved to be accurate. SonoAVC measurements resulted in a substantial systematic underestimation. Correlation coefficients of measured versus true volumes were excellent in all four techniques. For all techniques, an intra- and interobserver agreement of at least 0.91 was found. Yolk sac measurements by the different techniques proved to be highly correlated (ICCs > 0.91). Conclusions We demonstrated that VOCAL, inversion mode and V-Scope can all be used to measure volumes of hypoechoic structures. The newly introduced V-Scope application proved to be accurate and reliable.

BACKGROUND: The aim of this study was to demonstrate that Carnegie Stages could be assigned to embryos visualized with a 3D virtual reality system. METHODS: We analysed 48 3D ultrasound scans of 19 IVF/ICSI pregnancies at 7-10 weeks' gestation. These datasets were visualized as 3D 'holograms' in the BARCO I-Space virtual reality system. Embryos were staged according to external morphological features (i.e. mainly limb development). After staging, the crown rump length (CRL) was measured. Stage and CRL were compared with gestational age based on the date of oocyte retrieval and with the classical data on embryology from the Carnegie Collection. RESULTS: Embryonic staging was relatively easy because the I-Space allows depth perception, which helps in the estimation of size and position. The presumed stages corresponded well with the measured CRL. However, in 28 out of 48 cases, the stages seemed to have been reached earlier than previously described for the Carnegie Collection. CONCLUSIONS: The I-Space, tentatively named Virtual Embryoscopy, is a promising non-invasive tool for early pregnancy evaluation. Combining embryonic growth with embryonic development opens a new area to study the relationship between embryonic growth, development and morphology, as well as second and third trimester pregnancy complications.

Objective The utility of a virtual reality system was examined in the visualization of three-dimensional (3D) ultrasound images of fetal ambiguous genitalia. Methods In 2005, fetal ambiguous genitalia were diagnosed in four patients referred to our department for prenatal ultrasound assessment. The patients were examined by two-dimensional (2D) and 3D ultrasound and, subsequently, the volumes obtained on 3D ultrasound were visualized in the BARCO I-Space virtual reality system. This system projects stereoscopic images on three walls and the floor of a small 'room', allowing several viewers to see a 3D 'hologram' of the data being visualized. The results of 2D and 3D ultrasound examination and the virtual reality images of the I-Space were compared with diagnoses made postpartum. Results In all cases, prenatal diagnosis was unclear based on 2D ultrasound alone. Surface rendering of 3D data provided an impression of ambiguity, but diagnosis based on these data proved incorrect at birth in three cases. Conclusions based on the evaluation of 3D volumes in virtual reality best fitted the postpartum diagnosis in all cases. Conclusions This study suggests that by evaluation of the genitals in the I-Space, a better impression of genital ambiguity can be established. Binocular depth perception appeared particularly useful in distinguishing either a micropenis or enlarged clitoris from labia minora, since it helps in the estimation of size and position. Therefore, we see potential for the application of virtual reality not only for the evaluation of fetal ambiguous genitalia, but in all those cases where depth perception would improve the visualization of anatomical structures. Copyright (C) 2008 ISUOG. Published by John Wiley & Sons, Ltd.

Objective To establish The reliability of three-dimensional (3D) ultrasound measurements in early pregnancy using a virtual reality system (the Barco I-Space). Methods The study included 28 pregnancies with gestational ages ranging from 6 to 14 (median., 10) weeks. 3D volumes were acquired and offline measurements were made, where possible, of the yolk sac diameter, crown-rump length, biparietal diameter, head circumference and abdominal circumference, using specialized 3D imaging software (4DView). The datasets were then transferred to the Barco I-Space, a virtual reality system that allows the observer to perceive depth and interact with volume-rendered (ultrasound) data. The 3D rendered volumes were measured using a virtual pointer, controlled by a wireless joystick. For intraobserver variability, 3D and virtual reality volumes were measured twice by one operator. For interobserver variability, another operator performed the same measurements once. All measurements were repeated three times and their mean values were used for comparisons. Results All intraclass correlation coefficients (ICCs) comparing 4D View measurements with I-Space measurements were > 0.97. Intra- and interobserver ICCs for the 4D View measurements were > 0.96 and for the I-Space ones were > 0.98, representing good agreement. Conclusions The application of virtual reality is a novel method of visualizing 3D ultrasound data and perception of the depth in the I-Space offers possibilities for measuring non-planar structures. We have demonstrated that earl), pregnancy measurements in the I-Space are reliable. New areas of embryonic and fetal biometry can now be explored using ibis technique, which we tentatively name virtual embryoscopy'. Copyright (C) 2008 ISUOG. Published by John Wiley, & Sons, Ltd.

Melanoma is the most lethal form of skin cancer and successful treatment of metastatic melanoma remains challenging. BRAF/MEK inhibitors only show a temporary benefit due to rapid occurrence of resistance, whereas immunotherapy is mainly effective in selected subsets of patients. Thus, there is a need to identify new targets to improve treatment of metastatic melanoma. To this extent, we searched for markers that are elevated in melanoma and are under regulation of potentially druggable enzymes. Here, we show that the pro-proliferative transcription factor FOXM1 is elevated and activated in malignant melanoma. FOXM1 activity correlated with expression of the enzyme Pin1, which we found to be indicative of a poor prognosis. In functional experiments, Pin1 proved to be a main regulator of FOXM1 activity through MEK-dependent physical regulation during the cell cycle. The Pin1-FOXM1 interaction was enhanced by BRAFV600E, the driver oncogene in the majority of melanomas, and in extrapolation of the correlation data, interference with\ Pin1 in BRAFV600E-driven metastatic melanoma cells impaired both FOXM1 activity and cell survival. Importantly, cell-permeable Pin1-FOXM1-blocking peptides repressed the proliferation of melanoma cells in freshly isolated human metastatic melanoma ex vivo and in three-dimensional-cultured patient-derived melanoids. When combined with the BRAFV600E-inhibitor PLX4032 a robust repression in melanoid viability was obtained, establishing preclinical value of patient-derived melanoids for prognostic use of drug sensitivity and further underscoring the beneficial effect of Pin1-FOXM1 inhibitory peptides as anti-melanoma drugs. These proof-of-concept results provide a starting point for development of therapeutic Pin1-FOXM1 inhibitors to target metastatic melanoma.Oncogene advance online publication, 17 August 2015; doi:10.1038/onc.2015.282.

Testicular germ cell tumours (GCTs) of adolescents and adults can be subdivided into seminomas (referred to as dysgerminomas of the ovary) and non-seminomas, all referred to as type II GCTs. They originate from carcinoma in situ (CIS), being the malignant counterparts of primordial germ cells (PGCs)/gonocytes. The invasive components mimic embryogenesis, including the stem cell component embryonal carcinoma (EC), the somatic lineage teratoma (TE), and the extra-embryonic tissues yolk sac tumour (YST) and choriocarcinoma (CH). The other type is the so-called spermatocytic seminomas (SS, type III GCT), composed of neoplastic primary spermatocytes. We reported previously that the miRNAs hsa-miR 371-373 cluster is involved in overruling cellular senescence induced by oncogenic stress, allowing cells to become malignant. Here we report the first high-throughput screen of 156 microRNAs in a series of type II and III GCTs (n = 69, in duplicate) using a quantitative PCR-based approach. After normalization to allow inter-sample analysis, the technical replicates clustered together, and the previous hsa-miRNA 371-373 cluster finding was confirmed. Unsupervised cluster analysis demonstrated that the cell lines are different from the in vivo samples. The in vivo samples, both normal and malignant, clustered predominantly based on their maturation status. This parallels normal embryogenesis, rather than chromosomal anomalies in the tumours. miRNAs within a single cluster showed a similar expression pattern, implying common regulatory mechanisms. Normal testicular tissue expressed most discriminating miRNAs at a higher level than SE and SS. Moreover, differentiated non-seminomas showed overexpression of discriminating miRNAs. These results support the model that miRNAs are involved in regulating differentiation of stem cells, retained in GCTs.

Megalencephaly (MEG), or enlargement of the brain, can either represent a familial variant with normal cerebral structure, or a rare brain malformation associated with developmental delay and neurological problems. MEG has been split into two subtypes: anatomical and metabolic. The latter features a build-up inside the cells owing to metabolic causes. Anatomical MEG has been detected in many different conditions, including many overgrowth syndromes. In 2004 Mirzaa et al. reported five non-consanguineous patients with a new MCA/MR syndrome characterized by severe congenital MEG with polymicrogyria (PMG), postaxial polydactyly (POLY) and hydrocephalus (HYD). The authors argued that these findings identified a new and distinct malformation syndrome, which they named MPPH. We report on a new case of MPPH, the first to be described after the original series (Mirzaa et al., 2004).

International audience; DNA repair-deficient trichothiodystrophy (TTD) results from mutations in the XPD and XPB subunits of the DNA repair and transcription factor TFIIH. In a third form of DNA repair-deficient TTD, called group A, none of the nine subunits encoding TFIIH carried mutations; instead, the steady-state level of the entire complex was severely reduced. A new, tenth TFIIH subunit (TFB5) was recently identified in yeast. Here, we describe the identification of the human TFB5 ortholog and its association with human TFIIH. Microinjection of cDNA encoding TFB5 (GTF2H5, also called TTDA) corrected the DNA-repair defect of TTD-A cells, and we identified three functional inactivating mutations in this gene in three unrelated families with TTD-A. The GTF2H5 gene product has a role in regulating the level of TFIIH. The identification of a new evolutionarily conserved subunit of TFIIH implicated in TTD-A provides insight into TFIIH function in transcription, DNA repair and human disease.

Study question Are first trimester trophoblast volume (TV) and placental bed vascular volume (PBVV) different in IVF or IVF/ICSI pregnancies in comparison with spontaneously conceived pregnancies? Summary answer Any possible abnormal placentation in IVF or IVF/ICSI pregnancies in comparison with spontaneously conceived pregnancies is not detected by a difference in PBVV or TV at an early gestational age (GA). What is known already Assisted reproductive technology pregnancies have been associated with an increased risk of placenta-related adverse pregnancy outcomes. It is unclear whether these effects originate from infertility or from the technique itself. Study design, size, duration We performed a retrospective cohort study in which 154 pregnant patients qualified for participation. Participants/materials, setting, methods Out of 154 pregnant patients, 84 conceived spontaneously and 70 conceived after IVF or IVF/ICSI. We determined the TV at 10 weeks GA by Virtual Organ Computer-aided AnaLysis measuring application and the PBVV at 12 weeks GA by the virtual reality operating system of BARCO I-Space in both subgroups. The investigators were blinded to the mode of conception during the measurements. Analysis was limited to singleton pregnancies with only one sac ever detectable. Main results and the role of chance There were no differences in TV (mean 42.7, SD 15.9 versus mean 41.2, SD 13.9, P = 0.70) and PBVV (mean 27.6, SD 16.9 versus mean 24.8, SD 19.9, P = 0.20) between IVF or IVF/ICSI pregnancies and spontaneously conceived pregnancies. There was a significant correlation between TV and PBVV (rs = 0.283, P = 0.004). Limitations, reasons for caution The limitations of the present study concern the small size of the study groups. Wider implications of the findings IVF or IVF/ICSI does not seem to be associated with abnormal placentation. Study funding/competing interests This study was financially supported by the Erasmus Trustfonds, the Meindert de Hoop foundation and the Fonds NutsOhra. No competing interests are declared.

Introduction Multiple Myeloma (MM) is a heterogeneous disease with diverse gene expression patterns (GEP) across patients. This has led to the development of various signatures allowing virtual karyotyping, defining different clusters of patients, and prognostication by high risk signatures (e.g. EMC92/SKY92). Several GEP datasets exist, but may have scaling/offset differences (batch effects) in the data, e.g. due to differences in reagents used, location, etc. Batch wise normalization approaches can reduce batch effects, and have allowed successful validation of those signatures across independent datasets. Batch wise normalization requires groups of patients that have a similar distribution of clinical characteristics, and hence cannot be applied on single patients. Here we demonstrate the validity of applying GEP algorithms on single patients using the MMprofiler, enabling the application of GEP in a routine clinical setting Materials and Methods The MMprofiler GEP assay is a standardized assay from bone marrow to data analysis and result reporting. It was used for 77 MM patients that were enrolled in the HOVON87/NMSG18 trial (73 patients) or HOVON95/EMN02 trial (4 patients). A representative reference set of 30 HOVON patients was selected from which normalization parameters were derived, to be used for normalization of a single sample against this HOVON reference dataset. The remaining 47 samples served as an independent set of samples. In addition, we have also used the publicly available GEP data from 247 patients (MRC-IX trial) as independent samples. This MRC-IX dataset has been produced using different reagents and sample work-up procedures. Therefore, it is likely that a batch effect will exist relative to the HOVON reference dataset, which may influence correctness of single sample analyses The GEP data from the 47 and 247 independent samples were normalized using two approaches. Firstly, by batch wise mean variance normalization (i.e. across the 47 and 247 patient batches separately). And secondly, by single sample normalization using the normalization parameters from the initial 30 HOVON samples. Subsequently, several classifiers (EMC92/SKY92 etc.) were applied to the data, and their results were compared between the two normalization approaches Results Figure 1 shows the EMC92/SKY92 scores that were obtained after batch normalization (x-axis) and single sample normalization (y-axis). For the 47 HOVON samples there is a high degree of concordance with data points close to the identity line (y=x). Only 2 out of the 47 samples would switch assignment, which is not unexpected since those 2 samples are really close to the threshold (e.g. might also switch due to technical variation). For the MRC-IX dataset, based on single sample normalization more patients would be predicted as high risk (87 (35.2%) instead of 52 (21.0%), see Figure 1), which is caused by a positive offset (i.e. intersect with the y-axis) due to the batch effect For the Virtual t(4;14) classifier, both datasets have a very high concordance with 0 out 47 HOVON samples, and 5 out of 247 MRC-IX samples (but really close to the threshold) switching assignment (see Figure 1). Hence, even in the presence of a potential batch effect in the MRC-IX dataset, the single sample predictions are accurate. These data suggest that single sample normalization of microarray GEP is possible but requires the strict standardization of the MMprofiler assay and algorithms Conclusions Scores for the EMC92/SKY92 signature were nearly equivalent when derived from the data following single sample normalization and batch normalization in the Skyline generated data. In the external dataset, a much higher discrepancy was found, highlighting the need to use highly standardized methods to generate Affymetrix GeneChip results. Further validation of this method is planned, and will include replicate runs systematically controlled for various conditions.

XPC-hHR23B protein complex is specifically involved in nucleotide excision repair (NER) of DNA lesions on transcriptionally inactive sequences as well as the nontranscribed strand of active genes. Here we demonstrate that not only highly purified recombinant hHR23B (rhHR23B) but also a second human homolog of the Saccharomyces cerevisiae Rad23 repair protein, hHR23A, stimulates the in vitro repair activity of recombinant human XPC (rhXPC), revealing functional redundancy between these human Rad23 homologs. Coprecipitation experiments with His-tagged rhHR23 as well as sedimentation velocity analysis showed that both rhHR23 proteins in vitro reconstitute a physical complex with rhXPC. Both complexes were more active than free rhXPC, indicating that complex assembly is required for the stimulation. rhHR23B was shown to stimulate an early stage of NER at or prior to incision. Furthermore, both rhHR23 proteins function in a defined NER system reconstituted with purified proteins, indicating direct involvement of hHR23 proteins in the DNA repair reaction via interaction with XPC.

Enzymatic photoreactivation is a DNA repair mechanism that removes UV-induced pyrimidine dimer lesions by action of a single enzyme, photolyase, and visible light. Its presence has been demonstrated in a wide variety of organisms, ranging from simple prokaryotes to higher eukaryotes. We have isolated a human gene encoding a 66-kDa protein that shows clear overall homology to known bacterial photolyase genes. The human gene product is more similar to plant blue-light receptors within class I photolyases than to higher eukaryote class II photolyases. Northern blot analysis showed two transcripts with constitutive expression in all tissues examined and an elevated expression in testis. In situ hybridization with a cDNA-derived probe localized this human gene to chromosome 12q23-q24.1. Southern analysis of the cloned human gene suggests a wide distribution of the gene family in various species.

Study Questio: NIs it possible to evaluate first trimester brain ventricle development in human pregnancies using an innovative virtual reality (VR) application and to analyze the relation of the embryonic volume (EV) and brain ventricle fluid volume (BVFV) with gestational age (GA), crown-rump length (CRL) and the Carnegie stage? Summary Answer: Volumetry and staging of the human embryo using a VR application make it possible to obtain unique information about in-vivo embryonic normal and abnormal development and about the sizes of the ventricles and body. What Is Known Already: Human brain development is complex and has a rapidly changing anatomy during the first trimester of pregnancy. New insights will enable early detection of cerebral pathology.STUDY Design: , SIZE, DURATIONIn a prospective cohort study, we weekly performed three-dimensional (3D) ultrasound examinations in 112 uncomplicated pregnancies between 6 + 0 and 12 + 6 weeks GA.MATERIALS, SETTING, METHODSThe examinations resulted in 696 3D ultrasound scans that were transferred to the I-Space VR system and analyzed using V-Scope volume rendering software. V-Scope is used to create a 'hologram' of the ultrasound image and allows depth perception and interaction with the rendered objects. The CRL measurements were performed with a tracing tool, and the volume measurements were automatically performed with a segmentation algorithm. The embryos were staged according to the internal and external characteristics of the Carnegie staging system. All longitudinal outcomes were analyzed using repeated measures ANOVA. Main Results and the Role of Chance: CRL could be measured in 91% of the datasets and ranged from 2.5 to 79.0 mm. EV could be measured in 66% of the datasets and ranged from 2.4 to 23 812.0 mm, whereas the BVFV could be measured in 38% of the datasets and ranged from 10.4 to 226.3 mm. Finally, in 74% of the datasets, the embryos were staged according to the Carnegie criteria, starting as early as stage 12. Reference charts of volumes versus GA, CRL and stage were constructed. There was no significant relationship between the CRL or EV and the birthweight.LIMITATIONS, REASONS FOR CAUTIONSThe low success rate is a limitation of this study that can be explained mainly by non-targeted scanning of the embryonic head. Wider Implications of the Findings: The I-Space VR system and the V-Scope software enable automatic EV and BVFV measurements and 3D observations of embryonic development in the first trimester. This allows in-vivo staging of human embryos based on both internal and external morphological characteristics. Study Funding, Competing Interests None. © 2013 The Author.

The aim aim of the study was to investigate whether first trimester chorionic villous vascularisation is different in women who smoked cigarettes before and during pregnancy in comparison with women who did not smoke. Placentas of smoking (>10 cigarettes/day, n = 13) and non-smoking women (n = 13), scheduled for a legal termination of a viable first trimester pregnancy for social indications, were retrieved. Placental tissues of 3-5 mm(3) were whole mount CD31 immunofluorescence stained. Images of the CD31 immunofluorescence and contour of the villi were captured using an Optical Projection Tomography scanner. An immersive BARCO virtual reality system was used to create an enlarged interactive 3-dimensional hologram of the reconstructed images. Automatic volume measurements were performed using a flexible and robust segmentation algorithm that is based on a region-growing approach in combination with a neighbourhood variation threshold. The villous volume, vascular volume and vascular density were measured for the total chorionic villous tree as well as for its central and peripheral parts. No differences in maternal age and gestational age were found between non-smoking and smoking women. No differences were found in the total, central and peripheral villous tree volume and vascular volume. The central (13.4% vs. 9.5%, p=0.03) and peripheral (8.4% vs. 6.4%, p=0.02) villous tree vascular densities were increased in the smoking women as compared with the non-smoking women. In conclusion, chorionic villous vascularisation is already altered in first trimester of pregnancy in women who smoked cigarettes before and during pregnancy. (C) 2012 Elsevier Ltd. All rights reserved

The nucleotide excision repair (NER) disorder xeroderma pigmentosum (XP) is characterized by sun (UV) sensitivity, predisposition to skin cancer, and extensive genetic heterogeneity. Recently, we reported the cloning and analysis of three human NER genes, XPC, HHR23A, and HHR23B. The previously cloned XPC gene is involved in the common XP complementation group C, which is defective in excision repair of non-transcribed sequences in the genome. The XPC protein was found to be complexed with the product of HHR23B, one of the two human homologs of the Saccharomyces cerevisiae NER gene RAD23. Here we present the chromosomal localization by in situ hybridization using haptenized probes of all three genes. The HHR23A gene was assigned to chromosome 19p13.2. Interestingly, the HHR23B and XPC genes, the product of which forms a tight complex, were found to colocalize on band 3p25.1. Pulsed-field gel electrophoresis revealed that the HHR23B and XPC genes possibly share a MluI restriction fragment of about 625 kb. Potential involvement of the HHR23 genes in human genetic disorders is discussed.

Partial monosomy 21 has been reported, but the phenotypes described are variable with location and size of the deletion. We present 2 patients with a partially overlapping microdeletion of 21q22 and a striking phenotypic resemblance. They both presented with severe psychomotor delay, behavioral problems, no speech, microcephaly, feeding problems with frequent regurgitation, idiopathic thrombocytopenia, obesity, deep set eyes, down turned corners of the mouth, dysplastic ears, and small chin. Brain MRI showed cerebral atrophy mostly evident in frontal and temporal lobes, widened ventricles and thin corpus callosum in both cases, and in one patient evidence of a migration disorder. The first patient also presented with epilepsy and a ventricular septum defect. The second patient had a unilateral Peters anomaly. Microarray analysis showed a partially overlapping microdeletion spanning about 2.5 Mb in the 21q22.1–q22.2 region including the DYRK1A gene and excluding RUNX1. These patients present with a recognizable phenotype specific for this 21q22.1–q22.2 locus. We searched the literature for patients with overlapping deletions including the DYRK1A gene, in order to define other genes responsible for this presentation.

Background: Innovative imaging techniques, using up-to-date ultrasonic equipment, necessitate specific biometry. The aim of our study was to test the possibility of detailed human embryonic biometry using a virtual reality (VR) technique. Methods: In a longitudinal study, three-dimensional (3D) measurements were performed from 6 to 14 weeks gestational age in 32 pregnancies (n = 16 spontaneous conception, n = 16 IVF/ICSI). A total of 125 3D volumes were analysed in the I-Space VR system, which allows binocular depth perception, providing a realistic 3D illusion. Crown-rump length (CRL), biparietal diameter (BPD), occipito-frontal diameter (OFD), head circumference (HC) and abdominal circumference (AC) were measured as well as arm length, shoulder width, elbow width, hip width and knee width. Results: CRL, BPD, OFD and HC could be measured in more than 96% of patients, and AC in 78%. Shoulder width, elbow width, hip width and knee width could be measured in more than 95% of cases, and arm length in 82% of cases. Growth curves were constructed for all variables. Ear and foot measurements were only possible beyond 9 weeks gestation. Conclusions: This study provides a detailed, longitudinal description of normal human embryonic growth, facilitated by a VR system. Growth curves were created for embryonic biometry of the CRL, BPD, HC and AC early in pregnancy and also of several 'new' biometric measurements. Applying virtual embryoscopy will enable us to diagnose growth and/or developmental delay earlier and more accurately. This is especially important for pregnancies at risk of severe complications, such as recurrent late miscarriage and early growth restriction.

Objective The objective of this study was to test the hypothesis whether a maternal dietary pattern is associated with the risk of spina bifida (SB) in the offspring. Design Case–control study. Setting Eight clinic sites in the Netherlands, 1999–2001. Sample A total of 50 mothers of children with SB and 81 control mothers. Methods Maternal food intakes were obtained by food frequency questionnaires at the standardised study moment of 14 months after the birth of the index child. Principal component factor analysis (PCA) and reduced rank regression (RRR) were used to identify dietary patterns. Main outcome measures Maternal biomarkers were used as response measures in the RRR analysis and composed of serum and red blood cell (RBC) folate, serum vitamin B12 and total plasma homocysteine. The strength of the use of the dietary pattern in association with SB risk was estimated by odds ratios and 95% CI with the highest quartiles of the dietary pattern as reference. Results A predominantly Mediterranean dietary pattern was identified by both PCA and RRR. Those dietary patterns were highly correlated (r = 0.51, P < 0.001) and characterised by joint intakes of fruit, vegetables, vegetable oil, alcohol, fish, legumes and cereals and low intakes of potatoes and sweets. We observed a significantly increased risk of SB offspring in mothers with a weak use of the Mediterranean dietary pattern, OR 2.7 (95% CI 1.2–6.1) and OR 3.5 (95% CI 1.5–7.9). The Mediterranean dietary pattern was correlated with higher levels of serum and RBC folate, serum vitamin B12 and lower plasma homocysteine. Conclusion The Mediterranean dietary pattern seems to be associated with reduction in the risk of offspring being affected by SB.

First trimester human villous vascularization is mainly Studied by conventional two-dimensional (213) microscopy. With this (2D) technique it is not possible to observe the spatial arrangement of the haemangioblastic cords and vessels, transition of cords into vessels and the transition of vasculogenesis to angiogenesis. The Confocal Laser Scanning Microscopy (CLSM) allows for a three-dimensional (31)) reconstruction of images of early pregnancy villous vascularization. These 3D reconstructions, however, are normally analyzed on a 2D medium, lacking depth perception. We performed a descriptive morphologic study, using an immersive Virtual Reality system to utilize the full third dimension completely. This innovative 3D technique visualizes 3D datasets as enlarged 3D holograms and provided detailed insight in the spatial arrangement of first trimester villous vascularization, the beginning of lumen formation within various junctions of haemangioblastic cords between 5 and 7 weeks gestational age and in the gradual transition of vasculogenesis to angiogenesis. This innovative immersive Virtual Reality system enables new perspectives for vascular research and will be implemented for future investigation. (C) 2009 Elsevier Ltd. All rights reserved

With the flood of data across all aspects of the pharmaceutical industry, information visualization is emerging as a critical component of discovery, development, and business. But it's a new class of visualizations that is having the greatest impact. Higher-level summaries that can provide a framework for understanding the immense volumes of data and that reveal unexpected relationships have come to the forefront. And, the ability to use these visualizations to cross-domains and data types provides the ability to integrate analyses and support fast, effective decisions.

hHR23B was originally isolated as a component of a protein complex that specifically complements nucleotide excision repair (NER) defects of xeroderma pigmentosum group C cell extracts in vitro and was identified as one of two human homologs of the Saccharomyces cerevisiae NER gene product Rad23. Recombinant hHR23B has previously been shown to significantly stimulate the NER activity of recombinant human XPC protein (rhXPC). In this study we identify and functionally characterize the XPC-binding domain of hHR23B protein. We prepared various internal as well as terminal deletion products of hHR23B protein in a His-tagged form and examined their binding with rhXPC by using nickel-chelating Sepharose. We demonstrate that a domain covering 56 amino acids of hHR23B is required for binding to rhXPC as well as for stimulation of in vitro NER reactions. Interestingly, a small polypeptide corresponding to the XPC-binding domain is sufficient to exert stimulation of XPC NER activity. Comparison with known crystal structures and analysis with secondary structure programs provided strong indications that the binding domain has a predominantly amphipathic alpha-helical character, consistent with evidence that the affinity with XPC is based on hydrophobic interactions. Our work shows that binding to XPC alone is required and sufficient for the role of hHR23B in in vitro NER but does not rule out the possibility that the protein has additional functions in vivo.

A protein complex which specifically complements defects of XP-C cell extracts in vitro was previously purified to near homogeneity from HeLa cells. The complex consists of two tightly associated proteins: the XPC gene product and HHR23B, one of two human homologs of the Saccharomyces cerevisiae repair gene product Rad23 (Masutani et al., EMBO J. 13:1831-1843, 1994). To elucidate the roles of these proteins in "genome-overall" repair, we expressed the XPC protein in a baculovirus system and purified it to near homogeneity. The recombinant human XPC (rhXPC) protein exhibited a high level of affinity for single-stranded DNA and corrected the repair defect in XP-C whole-cell extracts without extra addition of recombinant HHR23B (rHHR23B) protein. However, Western blot (immunoblot) experiments revealed that XP-C cell extracts contained excess endogenous HHR23B protein, which might be able to form a complex upon addition of the rhXPC protein. To investigate the role of HHR23B, we fractionated the XP-C cell extracts and constructed a reconstituted system in which neither endogenous XPC nor HHR23B proteins were present. In this assay system, rhXPC alone weakly corrected the repair defect, while significant enhancement of the correcting activity was observed upon coaddition of rHHR23B protein. Stimulation of XPC by HHR23B was found with simian virus 40 minichromosomes as well as with naked plasmid DNA and with UV- as well as N-acetoxy-2- acetylfluorene-induced DNA lesions, indicating a general role of HHR23B in XPC functioning in the genome-overall nucleotide excision repair subpathway.

Background Craniosynostosis, the premature fusion of the cranial sutures, is the second most common craniofacial malformation. A genetic aetiology can be identified in about 21% of cases, including mutations of TWIST1 that cause Saethre-Chotzen syndrome and are associated with coronal synostosis. By contrast, the cause of non-syndromic craniosynostosis is largely unknown. Methods We undertook exome sequencing of seven individuals with bilateral coronal synostosis, identifying mutations of TCF12 in three samples. We sequenced TCF12 in a further 347 patients with craniosynostosis. We performed mutation testing in extended families and determined the effects of mutations on mRNA expression. We examined the genetic interaction between loss-of-function mutations of Tcf12 and Twist1 in mice. Findings Heterozygous TCF12 mutations were present in 38 of the 347 unrelated patients with craniosynostosis. These included 22 (32%) of 69 with isolated bilateral coronal synostosis, and 14 (10%) of 141 with unilateral coronal synostosis, but none with premature fusion of only the metopic, sagittal, or lambdoid sutures (p −6 ). An additional two patients had either bilateral or right coronal synostosis in addition to sagittal synostosis. 14 cases arose de novo, but vertical transmission was demonstrated in 23 families. 16 mutation-positive individuals (47%) had craniosynostosis or suspicious clinical features, but 19 (53%) of 35 mutation-positive relatives were non-penetrant for craniosynostosis. TCF12 mutations were associated with diminished mRNA expression, indicating haploinsufficiency. With appropriate surgical correction the clinical outcome was usually good; six of 66 individuals had learning disability. Mice doubly heterozygous for Twist1 and Tcf12 mutations had severe bilateral coronal synostosis. Interpretation Mutations of TCF12 are a frequent and specific cause of coronal craniosynostosis; mutation testing is indicated in the assessment of these patients. Genetic interaction of Tcf12 and Twist1 is crucial for coronal suture development. Funding National Institute for Health Research Biomedical Research Centre, Oxford (BRC)/Oxford University Clinical Academic Graduate School (OUCAGS), and Oxfordshire Health Services Research Committee (OHSRC), Oxford Craniofacial Unit Charitable Fund; Thames Valley Comprehensive Local Research Network; The Dutch Center for Translational Molecular Medicine; Carolien Bijl Foundation; US National Institutes for Health; Wellcome Trust.

Complementation group C of xeroderma pigmentosum (XP) represents one of the most common forms of this cancer-prone DNA repair syndrome. The primary defect is located in the subpathway of the nucleotide excision repair system, dealing with the removal of lesions from the non-transcribing sequences ('genome-overall' repair). Here we report the purification to homogeneity and subsequent cDNA cloning of a repair complex by in vitro complementation of the XP-C defect in a cell-free repair system containing UV-damaged SV40 minichromosomes. The complex has a high affinity for ssDNA and consists of two tightly associated proteins of 125 and 58 kDa. The 125 kDa subunit is an N-terminally extended version of previously reported XPCC gene product which is thought to represent the human homologue of the Saccharomyces cerevisiae repair gene RAD4. The 58 kDa species turned out to be a human homologue of yeast RAD23. Unexpectedly, a second human counterpart of RAD23 was identified. All RAD23 derivatives share a ubiquitin-like N-terminus. The nature of the XP-C defect implies that the complex exerts a unique function in the genome-overall repair pathway which is important for prevention of skin cancer.

karyotype and feto-pathological examination reports were also collected. Results: Eighteen patients were followed in our center for limb defects discovered during first trimester ultrasound. The mean age at diagnosis was of 12 GW+3 days. −6 (33%) presented isolated limb abnormality, and had a normal karyotype (100%). Only one fetus had an incomplete prenatal diagnosis with an oligodactyly of the left foot discovered on pathological examination after termination of the pregnancy. −12 fetuses (67%), had limb defects which were a part of polymalformative syndrome. 7 (58%) had a normal karyotype while 4 (33%) had an aneuploidy (in 2 of 4 of this group: nuchal translucency was thin) and in 1 karyotype was not determined. Post-natal examination was obtained in 12 of 18 fetuses: all the prenatally described abnormalities were confirmed. However in 4 cases (33,3%) pathological examination revealed small undiagnosed finger and toes defects as syndactylies. Conclusions: Limbs defects can be visualized in first trimester ultrasound: in our study, the specificity of first trimester detection of fetal limbs is high. They are frequently associated with other morphological abnormalities (67%) or aneuploidy (22%). Their prognosis is severe: 72% of the cases were terminated (2IUFD and 11 TOP), even when isolated (50% TOP). Also the morphological examination of the fetus is an important time of the scan in the first trimester in addition to measurement of the nuchal translucency.

Objective: The aim of this study was to evaluate real-time three dimensional (3D) determined volumes of the yolk sac and fetal body, including the limbs, in first trimester pregnancies using a virtual reality (VR) application, and to analyse relations with gestational age (GA) and crown-rump length (CRL). Methods: We analyzed 180 3D first trimester ultrasound scans of 42 spontaneous and IVF/ICSI pregnancies. Only scans with high quality images were included and transferred to a Barco ISpace VR system and visualized as 3D ‘holograms’ with V-Scope volume rendering software. An implemented segmentation algorithm calculated the yolk sac volume (YSV) and fetal volume (FV) semiautomatically (A). The logarithmically transformed outcomes were analyzed using repeated measurements ANOVA. Interobserver agreement was established by calculating intraclass correlation coefficients (ICC). Results: In total 118 YSV and 88 FV were measured with GA between 5 + 5 and 12 + 6 weeks. YSV ranged from 33 mm3 to 424 mm3 and FV ranged from 14 mm3 to 29877 mm3. Reference charts were constructed according to GA and CRL (B). ANOVA calculations showed that when the CRL doubles, the mean FV increases 6.5 fold and when the GA doubles the mean FV increases approximately 500 fold (p