64 research outputs found

    Human Blastocyst\u27s Zona Pellucida Segmentation via Boosting Ensemble of Complementary Learning

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    Characteristics of Zona Pellucida (ZP), particularly its thickness, is a key indicator of human blastocyst (day-5embryo) quality. Therefore, ZP segmentation is an important step towards achieving automatic embryo qualityassessment. In this paper, a novel approach based on boosting ensemble of hybrid complementary learning isproposed to segment Zona Pellucida in human blastocyst images. First, an inner-ZP localization method isproposed to separate the ZP from the heavily textured area inside a blastocyst. Then, a deep Hierarchical NeuralNetwork (HiNN) is proposed to segment ZP area. The hierarchical nature of the proposed network enableslearning features with respect to their spatial location in the embryo. Finally, a Self-supervised Image-SpecificRefinement (SISR) strategy is proposed as a complementary step to boost the performance. The proposed systemis a hybrid approach in the sense that the HiNN learns the intra-correlation among images, while the SISR takesinto account the inter-correlation within the query image. Experimental results confirm that the proposed method is capable of identifying ZP area with average Precision, Recall, Accuracy and Jaccard Index of 85.2%, 92.0%, 95.6% and 78.1%, respectively. The proposed HiNN system outperforms state of the art by 4.9% in Precision, 11.2% in Recall, 3.6% in Accuracy and 10.7% in Jaccard Index

    Aneuploidy in Health and Disease

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    Aneuploidy means any karyotype that is not euploid, anything that stands outside the norm. Two particular characteristics make the research of aneuploidy challenging. First, it is often hard to distinguish what is a cause and what is a consequence. Secondly, aneuploidy is often associated with a persistent defect in maintenance of genome stability. Thus, working with aneuploid, unstable cells means analyzing an ever changing creature and capturing the features that persist. In the book Aneuploidy in Health and Disease we summarize the recent advances in understanding the causes and consequences of aneuploidy and its link to human pathologies

    Effects of ovarian stimulation on oocyte development and embryo quality

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    Ovarian stimulation plays a pivotal role in assisted reproductive therapies, to increase the number of embryos available for treatment; however, there is no clear consensus from meta-analyses in the literature which, if any, of the preparations in use are superior in terms of clinical outcomes. The aim of this thesis was to examine the effect of common human gonadotrophin preparations with different half lives and LH activity (hMG, rFSH and Pergoveris) on embryo quality and resulting offspring, compared to non- stimulated negative controls and positive PMSG treated controls, using the mouse model. The studies in this thesis indicated that an LH ceiling threshold is evident during folliculogenesis, where the use of long acting LH preparations resulted in higher numbers of fragmented oocytes, absent of cumulus cells (P<0.001), reduced expression of the pro and anti-angiogenic factors, MYHII and PEDF in cumulus cells (P<0.05), increased embryonic developmental arrest (P<0.001) and perturbed IGF2 (P<0.05) and VEGFA gene expression in resulting blastocysts (P<0.01), compared to negative controls. Use of preparations containing LH bioactivity resulted in offspring with altered total body weight trajectories and internal organ weight abnormalities (P<0.05), which were, in some instances, compounded by in vitro culture. In addition, we elucidated a relationship between FSH half life differences between urinary and recombinant preparations and embryo quality. The urinary human gonadotrophin preparation, hMG, could yield developmentally competent embryos at lower concentrations, than the recombinant Pergoveris treatment. In addition to FSH, these preparations contain LH and both low doses of preparations composed of short half life rFSH and rLH and high doses of preparations containing long acting LH bioactivity, resulted in the highest rates of developmental arrest. These groups were observed to have complete absence of H19 expression. The results of this thesis provide clear evidence that ovarian stimulation does negatively impact the embryo and subsequent offspring and provides support for an LH ceiling threshold, above which detrimental effects occur, both on in vitro embryo development and in vivo foetal development, which later effects postnatal growth

    Effects of ovarian stimulation on oocyte development and embryo quality

    Get PDF
    Ovarian stimulation plays a pivotal role in assisted reproductive therapies, to increase the number of embryos available for treatment; however, there is no clear consensus from meta-analyses in the literature which, if any, of the preparations in use are superior in terms of clinical outcomes. The aim of this thesis was to examine the effect of common human gonadotrophin preparations with different half lives and LH activity (hMG, rFSH and Pergoveris) on embryo quality and resulting offspring, compared to non- stimulated negative controls and positive PMSG treated controls, using the mouse model. The studies in this thesis indicated that an LH ceiling threshold is evident during folliculogenesis, where the use of long acting LH preparations resulted in higher numbers of fragmented oocytes, absent of cumulus cells (P<0.001), reduced expression of the pro and anti-angiogenic factors, MYHII and PEDF in cumulus cells (P<0.05), increased embryonic developmental arrest (P<0.001) and perturbed IGF2 (P<0.05) and VEGFA gene expression in resulting blastocysts (P<0.01), compared to negative controls. Use of preparations containing LH bioactivity resulted in offspring with altered total body weight trajectories and internal organ weight abnormalities (P<0.05), which were, in some instances, compounded by in vitro culture. In addition, we elucidated a relationship between FSH half life differences between urinary and recombinant preparations and embryo quality. The urinary human gonadotrophin preparation, hMG, could yield developmentally competent embryos at lower concentrations, than the recombinant Pergoveris treatment. In addition to FSH, these preparations contain LH and both low doses of preparations composed of short half life rFSH and rLH and high doses of preparations containing long acting LH bioactivity, resulted in the highest rates of developmental arrest. These groups were observed to have complete absence of H19 expression. The results of this thesis provide clear evidence that ovarian stimulation does negatively impact the embryo and subsequent offspring and provides support for an LH ceiling threshold, above which detrimental effects occur, both on in vitro embryo development and in vivo foetal development, which later effects postnatal growth

    Derivation of human embryonic stem cells to study early development and genetic disease

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    Stem cells are unique cells that have both the capacity for self-renewal and, depending on their origin, the ability to form at least one, and sometimes many, specialised cell types of all three embryonic germ lineages - germ cells (endoderm, mesoderm and ectoderm), extra-embryonic tissue and trophoblast. Since the derivation of the fi�rst human embryonic stem cell (hESC) line in 1998, there has been substantial interest in the potential of these cells both for regenerative medicine and cell therapy, and as disease models for monogenic disorders. Aside from the need to improve derivation efficiency and further the understanding of the basic biology of these cells, the ability to work with hESC opens up three broad research areas. The �first is the development of clinical grade culture systems with the aim of producing cell lines suitable for subsequent manipulation for therapy. The second is the opportunity to use these cells as a tool to study the earliest determinative events in mammalian development, such as the origins of patterning in the mammalian embryo. The third is the use of hESCs carrying clinically relevant genetic mutations as models for disease research and therapeutic target identifi�cation. The development of several methods of embryo manipulation tailored to the morphology of the blastocyst is described here, which resulted in the derivation of seven lines from four di�fferent procedures and provided the tools for subsequent research. Acknowledging that each laboratory in isolation is unlikely to derive sufficient lines to draw signifi�cant conclusions regarding manipulation methodology and culture parameters, an international collaboration was initiated with the aim of standardising the reporting of derivation and thus obtaining the maximum information from the generation of each new hESC line. To address the need for the development of clinical grade culture systems, alternative feeder cells were assessed for their suitability in hESC culture and derivation. Modi�fied human foreskin fi�broblasts and human amniotic epithelial cells (hAECs) were investigated, as both cell types can be fully qualifi�ed and validated. Whilst both were able to support the culture of existing lines, only the hAECs showed promise in supporting derivation. In addition, analysis of in-house and commercially available media showed that neither were physiologically optimal for the growth of inner cell mass (ICM) cells or putative hESC, as metabolite concentrations were in excess and subsequent catabolite levels exceeded known toxic levels. The timing and mechanisms establishing patterning and future polarity in the mammalian embryo remains a subject of intense debate. Here, the potential of single blastomeres to generate hESC was used as an assessment of pluripotency. The determination of the most appropriate day for attempting derivation was performed by assessing blastomere development and pluripotent marker expression, and the predicted success of derivation was considered in the light of division patterns. Putative stem-like cells were visible in several cultures. Furthermore, isolated blastomeres from two-, four-, and eight-cell embryos were analysed for the quantitative expression of multiple target genes known to be associated with lineage formation and the stem cell state. Analysis suggested that broad changes in gene expression were occurring with development stage. However, no consistent grouping structure for cells within embryos was observed, and no convincing pattern was seen when considering the individual embryo variance scores. Several approaches are discussed to diff�erentiate between the biological and methodological variability in this experimental design. The suitability of hESC as models for genetic disease was studied following the derivation of two lines carrying Huntington disease (HD). Subsequent di�fferentiation using a stromal co-culture neural induction protocol resulted in the establishment of a stable, highly proliferative cell population which was simple to culture and bank. The cells were of an astroglial phenotype, and therefore highly suited for subsequent studies regarding HD pathophysiology, as glial cells are severely aff�ected in HD. During diff�erentiation the CAG repeat size increased from 46 to 70, showing the salient feature of somatic instability of the huntingtin gene. Therefore this cell population provides a valuable tool in the study of disease pathogenesis and transmission

    Technological forecasting and regulatory assessment: an application to assisted reproductive technologies

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    Tésis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias Económicas y Empresariales, Departamento de Estructura Económica y Economía del Desarrollo, Fecha de lectura: 08-11-2019The aim of this thesis is to assess the trajectories of artificial reproductive technology (ART) and its pace of diffusion by identifying the factors that influence this process. This general objective can be split into the following specific objectives. First, to conduct a technology forecasting exercise in order to better understand the potential developments in IVF, PGD, and genetic engineering. Second, to identify the factors affecting regulation and priority setting regarding ART and to review the responses to technological and market developments in the field, through a regulatory assessment and a comparative analysis between Israel and Spain. Third, to assess the regulatory trends that may (or not) lead to the “geneticization” of reproduction (i.e. the shift of reproduction into the lab due to the ability to select or design genetic traits of embryos). These three sub-objectives define the structure of this thesis around three chapters, which have been drafted as independent papers to be published in scientific journal

    MicroRNAs in the bovine ovary and placentas derived from in vivo, in vitro and nuclear transfer pregnancies

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    MicroRNAs are the major class of gene regulating molecules playing pivotal roles at post-transcriptional level. Identification and expression profiling are the initial steps to understand their regulation of biological processes. Despite increasing efforts in miRNAs characterization in different species, little is known in the bovine reproductive tissues especially in ovary and placenta. Two subsequent studies were carried out to the expression of miRNAs in bovine ovary and Day-50 placenta derived from different sources of pregnancy. The first study aims to identify and characterize miRNAs in bovine ovary through cloning, expression analysis and target prediction. The constructed miRNA library revealed cloning of 50 known and 24 novel miRNAs. Among these, 38 were new miRNAs which were derived from 43 distinct loci with characteristic secondary structure. Most of the miRNAs were cloned multiple times and thereby reflecting their expression level and potential role in the ovary. Analysis of identified miRNAs in different intra-ovarian structures and other tissues reveals their stage and tissue specific expression patterns. Furthermore, in silico target prediction and Gene Ontology analysis of the targets genes identified several biological processes and pathways underlying the ovarian function. Results of this study suggest the presence of miRNAs in the bovine ovary; thereby elucidate their potential role in regulating diverse mechanisms underlying the ovarian functionality. The second study aimed to elucidate the difference in expression profile of miRNAs in the placenta at day 50 derived from Somatic cell nuclear transfer (SCNT), in vitro production (IVP) and artificial insemination (AI) pregnancies by quantifying 377 miRNAs. The study reveals a massive deregulation of miRNAs which were poorly reprogrammed and affected as large chromosomal cluster as well as miRNA families in the NT and IVP placenta compared to that of AI. Furthermore, cell specific localization miRNAs in the expanded blastocysts and expression profiling in different developmental stages of embryos and placenta identified that the major deregulation of miRNAs arises at day 50 of NT and IVP pregnancies. This deregulation were found to be less dependent on global DNA methylation, rather aberrant miRNA processing molecules were evidenced. Among them, observed down regulation of AGO2 could be a reason for global down regulation of miRNAs in the NT or IVP placenta. Identified deregulation of miRNAs might associate to the abnormal placentogenesis in NT or IVP pregnancies, which are the results of aberrant genetic and epigenetic modification. Result of this study will help to move one step closer towards improving the efficiency of nuclear transfer pregnancy. Altogether, the present study has discovered miRNAs in the bovine ovary and elucidated the pattern of expression of miRNAs along with their regulatory mechanism in the placenta derived from pregnancies of various origins.Untersuchung von MicroRNAs in bovinen Ovarien und Plazenten aus geklonten, in vivo und in vitro erzeugten Trächtigkeiten MicroRNAs gehören zur großen Klasse der genregulierenden Moleküle und spielen eine bedeutende Rolle auf der posttranskriptionellen Ebene. Die Identifikation und die Erstellung von Expressionsprofilen sind die ersten Schritte für ein besseres Verständnis der miRNA und ihrer Beteiligung an der Regulierung von biologischen Prozessen. Trotz der zunehmenden Charakterisierung der miRNA in verschiedenen Spezies, sind kaum Untersuchungen in bovinen Ovarien und Plazenten bekannt. In zwei Studien wurde die miRNA Expression in bovinen Ovarien und in Plazenten am Tag 50 der Trächtigkeit, in unterschiedlich erzeugten Schwangerschaften untersucht. Das Ziel der ersten Studie war die Identifizierung und Charakterisierung von miRNAs in klonierten bovinen Ovarien, Expressionsanalysen sowie Target-Vorhersagen. Die konstruierte miRNA-Bibliothek ergab 50 bekannte und 24 neue miRNAs. Unter diesen waren 38 neue bovine miRNAs die von 43 eindeutigen Loci abgeleitet werden konnten, die eine charakteristische sekundäre Struktur zeigten. Durch die mehrfache Klonierung der meisten miRNAs, konnte ihr Expressionsniveau in den Ovarien erfasst werden. Analysen von miRNAs in unterschiedlichen intra-ovariellen Strukturen sowie anderen Geweben zeigten ihr phasen- und gewebsspezifisches Expressionsmuster. Des Weiteren konnte durch bioinformatische Auswertungen und Gen Ontology Analysen der Target Gene verschiedene biologische Prozesse und Pathways der Ovarienfunktion identifiziert werden. Die Ergebnisse dieser Studie deuten auf das Vorhandensein von miRNAs in bovinen Ovarien hin und verdeutlichen die potenzielle Bedeutung in der Regulierung diverser Mechanismen der Ovarienfunktion. Die zweite Studie sollte die Unterschiede von Expressionsprofilen von miRNAs in Plazenten am Tag 50 der Trächtigkeit von SCNT, IVP und AI Schwangerschaften durch Quantifizierung von 377 miRNAs aufklären. Die Studie zeigte eine starke reduzierte Expression und eine geringe Reprogrammierung der miRNAs in NT und IVP im Vergleich zu AI. Diese miRNAs gehören vermutlich zu einer miRNA Familie in einem chromosomalen Cluster. Des Weiteren zeigten zellspezifische Lokalisationen von miRNAs in der expandierenden Blastozyste und Expressionsprofile von unterschiedlichen Entwicklungsstadien im Embryo und in der Plazenta, dass die wichtigsten Deregulierungen von miRNAs am Tag 50 der Trächtigkeit in NT und IVP entstehen. Diese Deregulierung erwies sich als weniger abhängig von einer globalen DNA-Methylierung, vielmehr wurde eine Abweichung in den miRNA-Prozessgenen belegt. Von diesen könnte die reduzierte Expression von AGO2 die Ursache für die globale Deregulierung der miRNAs in NT oder IVP Plazenten sein. Die identifizierten Deregulationen der miRNAs könnten im Zusammenhang mit abnormaler Plazentogenese in NT oder IVP Trächtigkeiten stehen. Die Ursachen für Abnormalitäten in der Plazentogenese liegen in genetischen und epigenetischen Modifikationen. Zusammenfassend konnte durch diese Studien miRNAs in bovinen Ovarien identifiziert werden und Expressionsmustern der miRNAs sowie ihre regulierenden Mechanismen in der Plazenta von unterschiedlichen Trächtigkeiten beschrieben werden

    Mechanical Manipulation and Characterization of Biological Cells

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    Mechanical manipulation and characterization of an individual biological cell is currently one of the most exciting research areas in the field of medical robotics. Single cell manipulation is an important process in intracytoplasmic sperm injection (ICSI), pro-nuclei DNA injection, gene therapy, and other biomedical areas. However, conventional cell manipulation requires long training and the success rate depends on the experience of the operator. The goal of this research is to address the drawbacks of conventional cell manipulation by using force and vision feedback for cell manipulation tasks. We hypothesize that force feedback plays an important role in cell manipulation and possibly helps in cell characterization. This dissertation will summarize our research on: 1) the development of force and vision feedback interface for cell manipulation, 2) human subject studies to evaluate the addition of force feedback for cell injection tasks, 3) the development of haptics-enabled atomic force microscope system for cell indentation tasks, 4) appropriate analytical model for characterizing the mechanical property of mouse embryonic stem cells (mESC) and 5) several indentation studies on mESC to determine the mechanical property of undifferentiated and early differentiating (6 days under differentiation conditions) mESC. Our experimental results on zebrafish egg cells show that a system with force feedback capability when combined with vision feedback can lead to potentially higher success rates in cell injection tasks. Using this information, we performed experiments on mESC using the AFM to understand their characteristics in the undifferentiated pluripotent state as well as early differentiating state. These experiments were done on both live as well as fixed cells to understand the correlation between the two during cell indentation studies. Our results show that the mechanical property of undifferentiated mESC differs from early differentiating (6th day) mESC in both live and fixed cells. Thus, we hypothesize that mechanical characterization studies will potentially pave the way for developing a high throughput system with force feedback capability, to understand and predict the differentiation path a particular pluripotent cell will follow. This finding could also be used to develop improved methods of targeted cellular differentiation of stem cells for therapeutic and regenerative medicine

    Regulation of cell fate choice in the mouse blastocyst stage embryo

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    Geeniekspressiooni andmete integreerimine teiste ‘oomika’ andmetega kirjeldamaks endomeetriumi retseptiivsuse bioloogilisi mehhanisme

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    Väitekirja elektrooniline versioon ei sisalda publikatsiooneMaailma Terviseorganisatsiooni statistika väidab, et umbes 10% püsisuhetes olevatest naistest on ühel või teisel põhjusel viljatud. Naise viljakust mõjutab välja palju erinevaid faktoreid ning setõttu on viljatuse põhjuste leidmine tihti väga keeruline. Viljatust põhjustavateks faktoriteks võivad olla üldine terviseseisund, erinevad haigused, geneetiline taust, väliskeskkonna ja eluviisiga seotud tegurid. Ühe näitena võib tuua embrüo pesastumist (implantatsioon) emaka limaskesta (endomeetriumi), mis võib toimuda ainult kindla lühikese perioodi vältel (implantatsiooni aken), kui endomeetrium on embrüo suhtes kõige vastuvõtlikum. Implantatsiooni akna periood on aga iga naise jaoks erinev, ning on määratud erinevate bioloogiliste protsesside poolt. Kunstliku viljastamise (IVF) läbiviimise jaoks on kriitiline teada täpset implantatsiooni akna aega, sellega seotud mehhanisme ja nende vastastikust mõju. Selleks, et uurida mehhanismide omavahelisi seoseid, panime paariviisiliselt kokku erinevaid geneetilise regulatsiooni andmekihte, milleks olid RNA, mikroRNA ja DNA metülatsiooni admed, ja mida koos nimetatakse ‘oomika’ andmekihtideks. Kokkuvõtvalt näitavad antud töö tulemused, et, võrreldes ühe ‘oomika’ andmekihi uurimisega, ‘oomika’ andmekihtide kombineerimine aitab paremini mõista endomeetriumi retseptiivsusega seotud bioloogilisi protsesse ning vältida valepositiivseid tulemusi. Antud tööga me rõhutame süsteemibioloogia ning paljude andmekihtide samaaegse kasutamise olulisust naise reproduktiivsuse bioloogiliste mehhanismide uurimisel.According to the World Health Organization, over 10% of females in a stable relationship are suffering from involuntary infertility/subfertility worldwide. Untangling the reasons for this is difficult because female reproduction is a sophisticated matter and can be affected by many factors such as health, accompanying diseases, genetic background, environment, and lifestyle. As a specific example, embryo implantation – its attachment to the uterine lining (endometrium) – occurs only during a relatively short period of time, called the window of implantation (WOI), when the endometrium is most receptive to an embryo. This is critical for a commonly used fertility treatment of in vitro fertilizaton (IVF) – and to make matters more complex, the WOI is not the same for everyone, but adjusted by an interlocking system of biological regulation mechanisms. Thus, to provide successful IVF, it is important to know these exact regulation mechanisms – and, since they interact with one another, to understand how they work together, not just individually. We used pairwise integration of data from different layers of genetic regulation, such as RNA, microRNA, and DNA methylation, called together the ‘omics’ layers, and showed the advantage of the data integration approach over the usage of just a single ‘omics’ layer. As a result, we obtained the lists of novel potential biomarkers that could regulate WOI, validated some previously known receptivity biomarkers, and showed that integration of different ‘omics’ layers helps to avoid false-positive results. With our work, we encourage other researchers in the female reproduction field to integrate several data layers for further studieshttps://www.ester.ee/record=b535138
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