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

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

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

Mechanisms and effects of cell density of embryos in culture in in vitro fertilization processes in embryonary development

Thesis to obtain the Master Degree in Biomedical EngineeringBackground and Objectives: The objective of achieving higher pregnancy rates in the area of human reproduction has promoted the search for new insights that could lead to a better embryonic development. The culture of embryos in groups aims to promote embryotrophic interaction, mediated by the secretion of paracrine and autocrine factors that induce mutual embryonic development, being crucial to evaluate the presence of factors that lead this evolution to fully understand the process. The present study aimed to explore what crucial knowledge could be obtained for the medically assisted reproduction area through the use of mid-infrared spectroscopy in the analysis of culture media spent by murine embryos and to evaluate retrospectively ovodonation cycle data for evaluation of the benefit of group human embryo culture (CG) versus individual culture (IC). In this way, it would be possible to take a position regarding the theory of the benefit of group culture and to understand whether the FTIR spectroscopy tool would be useful in evaluating the spectral profile of media spent by embryos. Methods: This thesis is divided in three distinct methodological chapters: I) the analysis and comparison of the FTIR spectral profile of different commercial human embryo culture media. In order to increase the accuracy of this tool, pre-processing methods were carried out as atmospheric and baseline correction, normalizations and derivatives that minimize distortions and resolve overlapping bands. II) the analysis of the spectral profile of spent culture media of grouped murine embryos: the molecular profile of 47 culture media spent by murine embryos up to the state of two cells was studied, with a density variation between 3 to 114 embryos per drop were evaluated before and after fertilization. III) the retrospective analysis to assessthe impact of group culture on human embryonic development rates using data from Ginemed’s IVF unit: An analysis was performed on each ovodonation cycle to compare the results of individually culture embryos and those culture in groups. Results: In chapter I), it was observed through the pattern recognition method of Principal Component Analysis (PCA) and the pre-processing methods applied that FTIR spectroscopy is sensitive enough to recognize different molecular profiles between culture media, highlighting differences at aminoacids (aa) and lipids molecules. In chapter II), The analysis of culture media spent of murine embryos showed different spectral profiles between media analyzed individually, without being able to establish any link between variables (species, number of embryos, pre/post fertilization). In chapter III), the retrospective comparison of culture of human embryos in individual and groups, revealed similar developmental rates to the blastocyst stage between both strategies; i.e. it was not detected a positive effect of group culture. Conclusions: The FTIR spectroscopy enables to acquire the molecular profile of human embryo culture media. Despite this, it was not possible to acquire by PCA of spectral data of spent media from the murine embryos patterns associated to species, number of embryos, etc. In the human embryo population and conditions evaluated it was not possible to observe advantages of culture the embryos in group in relation to culture embryos individually. It is proposed as future work, to evaluate other spectral processing techniques and increase the dimension of the human embryos evaluated.Enquadramento e Objetivos: O objetivo de atingir melhores taxas de gravidez na área da reprodução humana tem promovido a procura de novas e melhoradas técnicas. A cultura de embriões em grupo visa promover a interação embriotrófica e avaliar a importância da secreção de fatores parácrinos e autócrinos que induzam um desenvolvimento embrionário mútuo mais promissor, sendo para isso crucial avaliar a presença de fatores que impulsionem este desenvolvimento. O presente trabalho teve como objetivo explorar que conhecimentos cruciais poderiam ser obtidos para a área de reprodução medicamente assistida através do uso da espectroscopia do infravermelho médio na análise de meios de cultura gastos por embriões de murinos e avaliar retrospectivamente dados de ciclos de ovodoação para avaliação do benefício da cultura de embriões humanos em grupo (CG) versus cultura individual (CI). Desta forma, seria possível assumir uma posição relativamente à teoria do benefício da cultura em grupo e perceber se a ferramenta FTIR seria útil na avaliação do perfil espectral dos meios gastos por embriões. Métodos: A presente dissertação foi estruturada fundamentalmente em três capítulos metodológicos distintos: I) a análise e comparação do perfil FTIR espectral de diferentes meios de cultura de embriões humanos comerciais. Com o intuito de assegurar a precisão desta ferramenta, foram aplicados métodos de pré-processamento como correções atmosféricas e de linha de base, normalizações e derivativas que minimizam distorções e resolvem bandas sobrepostas. II) a análise do perfil espectral de meio de cultura gasto por embriões de murinos agrupados: o perfil molecular de 47 meios de cultura gastos por embriões murinos até ao estado de duas células foi avaliado, com uma variação de densidade entre 3 a 114 embriões por gota, antes e após fertilização. III) a análise retrospetiva com o intuito de avaliar o impacto da cultura em grupo nas taxas de desenvolvimento embrionário, através de dados obtidos na clínica Ginemed Lisboa: realizou-se uma análise a cada ciclo de ovodoação para comparar os resultados obtidos na cultura individual e em grupo de embriões humanos. Resultados: No capítulo I) observou-se através do método padrão de reconhecimento – Análise de Componentes Principais – e com a aplicação dos métodos de pré-processamento que a ferramenta FTIR revelou sensibilidade suficiente para reconhecer que as amostras obtidas de um dos meios de cultura analisados apresentam um perfil molecular que se diferencia dos restantes, destacando diferenças em aminoácidos e moléculas lipídicas. No capítulo II, a análise do meio de cultura gasto por embriões murinos apresentou perfis espectrais diferentes entre meios de cultura analisados mas sem qualquer ligação entre grupos que permitisse estabelecer uma distinção entre as variáveis (espécie, número de embriões por gota, estado pré-fertilização vs pós-fertilização). No capítulo III) a comparação retrospetiva da cultura de embriões individual e em grupos revelou taxas de desenvolvimento embrionário semelhantes em ambas as estratégias aplicadas; o efeito benéfico da cultura em grupo não foi detetado nesta população específica através do protocolo utilizado. Conclusões: A ferramenta FTIR demonstrou a sua eficiência na avaliação do perfil molecular de meios de cultura de embriões humanos. Ainda assim, com recurso a esta técnica não foi possível adquirir através da análise de PCA, dados espectrais dos meios gastos pelos embriões de murinos com padrões associados a espécies, densidade embrionária ou outra variável. Na população de embriões humanos e condições avaliadas não foi possível observar quaisquer vantagens na cultura de embriões em grupo relativamente à cultura de embriões individual. Propõe-se como futuro projeto a avaliação de outras técnicas de pré-processamento e o aumento da dimensão da população avaliada.info:eu-repo/semantics/publishedVersio

A longitudinal study of the experiences and psychological well-being of Indian surrogates

Study question: What is the psychological well-being of Indian surrogates during and after the surrogacy pregnancy? Summary answer: Surrogates were similar to a matched group of expectant mothers on anxiety and stress. However, they scored higher on depression during and after pregnancy. What is known already: The recent ban on trans-national commercial surrogacy in India has led to urgent policy discussions regarding surrogacy. Whilst previous studies have reported the motivations and experiences of Indian surrogates no studies have systematically examined the psychological well-being of Indian surrogates, especially from a longitudinal perspective. Previous research has shown that Indian surrogates are motivated by financial payment and may face criticism from their family and community due to negative social stigma attached to surrogacy. Indian surrogates often recruited by agencies and mainly live together in a “surrogacy house.” Study design, size, duration: A longitudinal study was conducted comparing surrogates to a matched group of expectant mothers over two time points: (a) during pregnancy (Phase1: 50 surrogates, 70 expectant mothers) and (b) 4–6 months after delivery (Phase 2: 45 surrogates, 49 expectant mothers). The Surrogates were recruited from a fertility clinic in Mumbai and the matched comparison group was recruited from four public hospitals in Mumbai and Delhi. Data collection was completed over 2 years. Participants/materials, setting, methods: Surrogates and expectant mothers were aged between 23 and 36 years. All participants were from a low socio-economic background and had left school before 12–13 years of age. In-depth faceto-face semi-structured interviews and a psychological questionnaire assessing anxiety, stress and depression were administered in Hindi to both groups. Interviews took place in a private setting. Audio recordings of surrogate interviews were later translated and transcribed into English. Main results and the role of chance: Stress and anxiety levels did not significantly differ between the two groups for both phases of the study. For depression, surrogates were found to be significantly more depressed than expectant mothers at phase 1 (p = 0.012) and phase 2 (p = 0.017). Within the surrogacy group, stress and depression did not change during and after pregnancy. However, a non-significant trend was found showing that anxiety decreased after delivery (p = 0.086). No participants reported being coerced into surrogacy, however nearly all kept it a secret from their wider family and community and hence did not face criticism. Surrogates lived at the surrogate house for different durations. During pregnancy, 66% (N = 33/50) reported their experiences of the surrogate house as positive, 24% (N = 12/50) as negative and 10% (N = 5/50) as neutral. After delivery, most surrogates (66%, N = 30/45) reported their experiences of surrogacy to be positive, with the remainder viewing it as neutral (28%) or negative (4%). In addition, most (66%, N = 30/45) reported that they had felt “socially supported and loved” during the surrogacy arrangement by friends in the surrogate hostel, clinic staff or family. Most surrogates did not meet the intending parents (49%, N = 22/45) or the resultant child (75%, N = 34/45). Limitations, reasons for caution: Since the surrogates were recruited from only one clinic, the findings may not be representative of all Indian surrogates. Some were lost to follow-up which may have produced sampling bias. Wider implications of the findings: This is the first study to examine the psychological well-being of surrogates in India. This research is of relevance to current policy discussions in India regarding legislation on surrogacy. Moreover, the findings are of relevance to clinicians, counselors and other professionals involved in surrogacy. Trial registration number: N/A

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

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

Effects of ovarian stimulation on oocyte development and embryo quality

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