Semi-automated morphometric analysis of human embryos can reveal correlations between total embryo volume and clinical pregnancy

what is known already: Morphometric analysis of a large group of embryos has revealed the potential to optimize algorithms for image-analysis systems for the grading of embryos and predicting pregnancy outcomes. study design, size, duration: Oocytes and embryos were obtained from 458 patients who underwent single embryo transfer on Day 3 after IVF/ICSI, between September 2006 and December 2010 at the Leuven University Fertility Center, Belgium. In total, the data set contained 2796 embryos including 458 embryos that were transferred on Day 3. Ongoing pregnancy was defined as the presence of at least one intrauterine gestational sac at 20 weeks. participants/materials, setting, methods: Patients included in this study were younger than 36 years, entering their first (n ¼ 375) or second (n ¼ 83) IVF/ICSI cycle and were only included once. Patients were excluded if the cycle included biopsy for PGD or if donor sperm/donor oocytes were used. Based on the 26 sequential images of the same embryo taken at one time point in different planes, the software calculates the total cytoplasmic volume for each time point, from which any reduction or change in the volume with time can be assessed (which helps interpret the degree of fragmentation) and the size of blastomeres. The diameter of the smallest and largest blastomere and the total volume of each embryo were extracted from the computer-assisted scoring system database and the coefficient of diversity was calculated for Days 1, 2 and 3. A logistic regression analysis was performed to determine the range of embryo volume associated with an increased chance of pregnancy. main results and the role of chance: On Day 3, blastomeres of 8-cell stage embryos were less divergent in size than those of 6-, 7-, 9-cell stage embryos. Although, the coefficients of diversity (ratio of the largest:smallest blastomeres) of implanted embryos tended to be lower than for non-implanted embryos, the difference was only significant for 6-cell stage embryos (P ¼ 0.02). After logistic regression, an association between total embryo volume and pregnancy was observed which had a quadratic nature: both lower and higher volumes were associated with a lower probability of successful pregnancy. A significant association was identified between total embryo volume and pregnancy rate on both Days 2 (P ¼ 0.003) and 3 (P ¼ 0.0003). Diagnostic measures (sensitivity, specificity, positive predictive value, accuracy and c-statistics) of the defined volume range were relatively poor. However, results showed a good negative predictive value [76.86% (95% confidence interval 71.03-82.02) on Day 3]. limitations, reasons for caution: A general disadvantage of studies evaluating the impact of a characteristic on the implantation potential of an embryo is the fact that the best embryo is chosen for transfer. No comparisons can therefore be made with the other embryos. Moreover, the decision process is currently based on a non-automated, standard scoring system, which means that a 'bias' in the selection process is always present. wider implications of the findings: Our results are an important step towards the development of an automated computer-assisted scoring system for the morphological characteristics of human embryos to improve embryo selection for optimizing implantation potential. Total embryo volume appears to be one of the objective characteristics that should be included. study funding/competing interest(s): None. trial registration number: Not applicable

Principles guiding embryo selection following genome-wide haplotyping of preimplantation embryos.

STUDY QUESTION How to select and prioritize embryos during PGD following genome-wide haplotyping? SUMMARY ANSWER In addition to genetic disease-specific information, the embryo selected for transfer is based on ranking criteria including the existence of mitotic and/or meiotic aneuploidies, but not carriership of mutations causing recessive disorders. WHAT IS KNOWN ALREADY Embryo selection for monogenic diseases has been mainly performed using targeted disease-specific assays. Recently, these targeted approaches are being complemented by generic genome-wide genetic analysis methods such as karyomapping or haplarithmisis, which are based on genomic haplotype reconstruction of cell(s) biopsied from embryos. This provides not only information about the inheritance of Mendelian disease alleles but also about numerical and structural chromosome anomalies and haplotypes genome-wide. Reflections on how to use this information in the diagnostic laboratory are lacking. STUDY DESIGN, SIZE, DURATION We present the results of the first 101 PGD cycles (373 embryos) using haplarithmisis, performed in the Centre for Human Genetics, UZ Leuven. The questions raised were addressed by a multidisciplinary team of clinical geneticist, fertility specialists and ethicists. PARTICIPANTS/MATERIALS, SETTING, METHODS Sixty-three couples enrolled in the genome-wide haplotyping-based PGD program. Families presented with either inherited genetic variants causing known disorders and/or chromosomal rearrangements that could lead to unbalanced translocations in the offspring. MAIN RESULTS AND THE ROLE OF CHANCE Embryos were selected based on the absence or presence of the disease allele, a trisomy or other chromosomal abnormality leading to known developmental disorders. In addition, morphologically normal Day 5 embryos were prioritized for transfer based on the presence of other chromosomal imbalances and/or carrier information. LIMITATIONS, REASONS FOR CAUTION Some of the choices made and principles put forward are specific for cleavage-stage-based genetic testing. The proposed guidelines are subject to continuous update based on the accumulating knowledge from the implementation of genome-wide methods for PGD in many different centers world-wide as well as the results of ongoing scientific research. WIDER IMPLICATIONS OF THE FINDINGS Our embryo selection principles have a profound impact on the organization of PGD operations and on the information that is transferred among the genetic unit, the fertility clinic and the patients. These principles are also important for the organization of pre- and post-counseling and influence the interpretation and reporting of preimplantation genotyping results. As novel genome-wide approaches for embryo selection are revolutionizing the field of reproductive genetics, national and international discussions to set general guidelines are warranted. STUDY FUNDING/COMPETING INTEREST(S) The European Union's Research and Innovation funding programs FP7-PEOPLE-2012-IAPP SARM: 324509 and Horizon 2020 WIDENLIFE: 692065 to J.R.V., T.V., E.D. and M.Z.E. J.R.V., T.V. and M.Z.E. have patents ZL910050-PCT/EP2011/060211-WO/2011/157846 (‘Methods for haplotyping single cells’) with royalties paid and ZL913096-PCT/EP2014/068315-WO/2015/028576 (‘Haplotyping and copy-number typing using polymorphic variant allelic frequencies’) with royalties paid, licensed to Cartagenia (Agilent technologies). J.R.V. also has a patent ZL91 2076-PCT/EP20 one 3/070858 (‘High throughout genotyping by sequencing’) with royalties paid

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

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

Single-cell chromosomal imbalances detection by array CGH

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

Prevalence and association of asthma and allergic sensitization with dietary factors in schoolchildren: data from the french six cities study

International audienceBackground: The prevalence of asthma and allergy has recently risen among children. This increase in prevalence might be related to various factors, particularly diet. The aim of this study is to assess the prevalence and association of asthma and allergic sensitization with dietary factors in the French Six Cities Study. Methods: Cross-sectional studies were performed among 7432 schoolchildren aged 9-11 years in Bordeaux, Clermont-Ferrand, Creteil, Marseille, Reims, and Strasbourg. Parental questionnaires, based on the International Study on Asthma and Allergies in Childhood (ISAAC), were used to collect information on allergic diseases and potential exposure factors including a food frequency questionnaire to evaluate dietary habits. Skin prick testing to common allergens for allergic sensitization and bronchial hyper-responsiveness (BHR) testing to exercise were performed. Confounders control was performed with multiple logistic regressions. Results: Asthma symptoms, asthma and allergic sensitization were more prevalent in boys than in girls and were more prevalent in the South than in the North of France. After adjustment for confounders, fruit juice intake was associated with a low prevalence of lifetime asthma (ORa [95 % CI]; 0.73 [0.56-0.97]), butter intake was positively associated with atopic wheeze (1.48 [1.07-2.05]) and having lunch at the canteen 1-2 times/week compared to never or occasionally was associated with a lower prevalence of past year wheeze (0.71 [0.52-0.96]), lifetime asthma (0.76 [0.60-0.96]) and allergic sensitization (0.80 [0.67-0.95]). Meat intake was inversely related to past year wheeze among atopic children (0.68 [0.50-0.98]) while fast food consumption and butter intake were associated with an increase prevalence of asthma (2.39 [1.47-3.93] and 1.51 [1.17-2.00] respectively). Fish intake was associated with a lower prevalence of asthma among non-atopic children (0.61 [0.43-0.87]. None of the dietary factors was associated with BHR. Conclusions: Diet is associated with wheeze, asthma and allergic sensitization but not with BHR in children. These results provide further evidence that adherence to a healthy diet including fruits, meat and fish seems to have a protective effect on asthma and allergy in childhood. However, prospective and experimental studies are needed to provide causal evidence concerning the effect of diet on asthma and atopy

Systems Biology: A Therapeutic Target for Tumor Therapy

Tumor-related activities that seem to be operationally induced by the division of function, such as inflammation, neoangiogenesis, Warburg effect, immune response, extracellular matrix remodeling, cell proliferation rate, apoptosis, coagulation effects, present itself from a systems perspective as an enhancement of complexity. We hypothesized, that tumor systems-directed therapies might have the capability to use aggregated action effects, as adjustable sizes to therapeutically modulate the tumor systems’ stability, homeostasis, and robustness. We performed a retrospective analysis of recently published data on 224 patients with advanced and heavily pre-treated (10% to 63%) vascular sarcoma, melanoma, renal clear cell, cholangiocellular, carcinoma, hormone-refractory prostate cancer, and multivisceral Langerhans’ cell histiocytosis enrolled in nine multi-center phase II trials (11 centers). Each patient received a multi-targeted systems-directed therapy that consisted of metronomic low-dose chemotherapy, a COX-2 inhibitor, combined with one or two transcription modulators, pioglitazone +/− dexamethasone or IFN-alpha. These treatment schedules may attenuate the metastatic potential, tumor-associated inflammation, may exert site-specific activities, and induce long-term disease stabilization followed by prolonged objective response (3% to 48%) despite poor monoactivity of the respective drugs. Progression-free survival data are comparable with those of reductionist-designed standard first-line therapies. The differential response patterns indicate the therapies’ systems biological activity. Understanding systems biology as adjustable size may break through the barrier of complex tumor-stroma-interactions in a therapeutically relevant way: Comparatively high efficacy at moderate toxicity. Structured systems-directed therapies in metastatic cancer may get a source for detecting the topology of tumor-associated complex aggregated action effects as adjustable sizes available for targeted biomodulatory therapies

Antineoplastic effects of rosiglitazone and PPARγ transactivation in neuroblastoma cells

Neuroblastoma (NB) is the most common extracranial solid tumour in infants. Unfortunately, most children present with advanced disease and have a poor prognosis. In the present study, we evaluated the role of the peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone (RGZ) in two NB cell lines (SK-N-AS and SH-SY5Y), which express PPARγ. Rosiglitazone decreased cell proliferation and viability to a greater extent in SK-N-AS than in SH-SY5Y. Furthermore, 20 μM RGZ significantly inhibited cell adhesion, invasiveness and apoptosis in SK-N-AS, but not in SH-SY5Y. Because of the different response of SK-N-AS and SH-SY5Y cells to RGZ, the function of PPARγ as a transcriptional activator was assessed. Noticeably, transient transcription experiments with a PPARγ responsive element showed that RGZ induced a three-fold increase of the reporter activity in SK-N-AS, whereas no effect was observed in SH-SY5Y. The different PPARγ activity may be likely due to the markedly lower amount of phopshorylated (i.e. inactive) protein observed in SK-N-AS. To our knowledge, this is the first demonstration that the differential response of NB cells to RGZ may be related to differences in PPARγ transactivation. This finding indicates that PPARγ activity may be useful to select those patients, for whom PPARγ agonists may have a beneficial therapeutic effect