Durante o cultivo in vitro, as células são submetidas a uma série de estímulos estressores e em geral, a heterogeneidade da resposta a estes estímulos não é levada em consideração. O presente estudo foi baseado em um modelo fatorial (2x2x3) criado para avaliar como embriões com cinética de desenvolvimento distinta respondem à combinação de estresse ambiental e metabólico durante o cultivo in vitro. Para isso, embriões rápidos (4 ou mais células às 22 horas de cultura) e Lentos (2 ou 3 células) foram produzidos in vitro, cultivados em 20% ou 5% O2 e também em suplementações de glicose distintas (0.6, 2 e 5mM), resultando em 12 grupos de estudo. Os embriões em estágio de blastocisto foram avaliados em 95 caracteres, incluindo 82 genes, 7 evidências bioquímicas (consumo de glicose, glutamato e piruvato; produção de lactato e ATP), geração de espécies reativas de oxigênio (EROs), atividade mitocondrial e, finalmente, o conteúdo lipídico. Os dados foram normalizados e reunidos em uma matriz que foi analisada em parcimônia, com 500 repetições e Tree-Bissection Reconection como algoritmo (software TNT) em busca de comportamentos semelhantes entre os grupos. Todos os caracteres foram aditivos e igualmente ponderados. Esta análise resultou em uma única árvore ideal, totalmente resolvida. Os resultados mostram os grupos dispostos em dois arranjos principais de acordo com a tensão de oxigênio, exceto os grupos de embriões lentos cultivados em um ambiente de alta glicose (5mM). Num segundo momento, cada um dos grupos pertencentes aos primeiros arranjos foi novamente analisado. A 5% O2 (mais semelhante ao encontrado no útero), os embriões se agruparam de acordo com a cinética de desenvolvimento, independentemente da concentração de glicose no meio de cultura. Nesta condição, embriões rápidos foram mais capazes de atingir o estágio de blastocisto sem sobrecarregar o Metabolismo. Já a 20% O2, a suplementação de glicose foi mais importante para o agrupamento. Em um ambiente de alta glicose, em especial os embriões lentos, apresentaram metabolismo alterado e bloqueio de desenvolvimento. No entanto, embriões cultivados em baixas concentrações de glicose foram mais capazes de ativar mecanismos adaptativos e superar as injúrias causadas pelo estresse. A plasticidade embrionária é uma característica única e com esse trabalho conseguimos demonstrar como o 13 metabolismo se modula para ajudar os embriões a enfrentar condições estressantes enquanto tentam sobreviver a qualquer custo. / During in vitro culture cells are submitted to many stressful stimuli, however, the heterogeneity of the response to those stimuli is usually not considered. This study was based on a factorial experimental design (2x2x3) created to evaluate how embryos with distinct developmental kinetics respond to the combination of environmental and metabolic stress during in vitro culture. For this purpose, Fast (4 or more cells at 22 hours of culture) and Slow embryos (2-3 cells) were produced in vitro using standard protocols, cultured in 20% or 5% O2 and also in distinct glucose concentrations (0, 2 and 5mM), resulting in 12 groups. Blastocysts were evaluated for 95 characters including 82 genes, 7 biochemical evidences: consumption of glucose, glutamate and pyruvate; production of lactate and ATP; generation of reactive oxygen species (ROS), mitochondrial activity and finally the lipid content. Data were normalized and gathered in a matrix that was analyzed under parsimony, with 500 replicates and Tree-Bissection Reconection as the swapping algorithm (TNT software), searching for similar behaviors. All characters were additive and equally weighted. This analysis resulted in a single optimal tree, fully resolved. Results show the groups arranging in two main clusters according to oxygen tension regardless of developmental kinetics and glucose, except the groups of slow embryos cultured in a high glucose environment (5mM). Each cluster was separately analyzed and at 5% of oxygen (more similar to what is found in the uterus), embryos clustered according to developmental kinetics and independently of glucose concentration in culture media. On that condition, embryos with a faster kinetics were more capable of reaching blastocyst stage without overloading the metabolism. At 20% of oxygen, glucose supplementation seems to be more important for the clustering. In a high glucose environment embryos, in special de slow ones, show altered metabolism and block. However embryos cultured in lower glucose concentrations are more capable of activating adaptive mechanisms and overcome stress injuries. The embryonic plasticity is a unique feature and with this work we were able to demonstrate how metabolism modulates to help the embryos face stressful conditions while trying to survive at any cost.

O momento das primeiras divisões celulares pode predizer o potencial de desenvolvimento de um embrião, incluindo sua capacidade de estabelecer prenhez. Além de diferenças relacionadas ao metabolismo, estresse e sobrevivência, embriões com diferentes velocidades de desenvolvimento apresentam padrões transcricionais distintos, principalmente relacionados ao metabolismo energético e lipídico. Como o padrão transcricional é regulado por fatores epigenéticos este estudo visou caracterizar os mecanismos epigenéticos envolvidos neste fenótipo. Para isto, embriões bovinos foram produzidos in vitro utilizando sêmen sexado (fêmeas) e as 40 horas pós inseminação (hpi) foram classificados como Rápidos (4 ou mais células) ou Lentos (2 ou 3 células), permanecendo em cultivo até o estágio de blastocisto (168 hpi) (Capítulo 1) ou sendo avaliados com 40 hpi, 96 hpi ou 168 hpi (Capítulo 2). No capítulo 1, a análise da metilação global do genoma pelo sistema EmbryoGENE Methylation DNA Array (EDMA) identificou 11.584 regiões diferencialmente metiladas (DMRs) (7.976 regiões hipermetiladas em blastocistos derivados do grupo de clivagem rápida FBL - e 3.608 regiões hipermetiladas em blastocistos derivados de embriões de clivagem lenta - SBL). Os FBL apresentaram mais regiões classificadas como hipermetiladas, distribuídas ao longo do genoma, como nos íntrons, exons, promotores e elementos de repetição, enquanto em SBL, as regiões hipermetiladas estavam mais presentes nas ilhas CpG. As DMRs foram agrupadas em relação aos processos biológicos a que estavam envolvidas, e algumas das vias mais afetadas foram relacionadas à sobrevivência/diferenciação celular e metabolismo energético/lipídico. Os perfis dos transcritos dos genes diferencialmente metilados (DMs) relacionados com estas vias também foram avaliados, e a maior parte revelou mudanças na quantificação relativa. No capítulo 2, foi avaliada ao longo do desenvolvimento a presença de metilações e hidroximetilações do DNA e modificações pós-traducionais de histonas (acetilação e trimetilação da lisina 9 da histona 3 e trimetilação da lisina 27 da histona 3 H3K9ac, H3K9me3 e H3K27me3, respectivamente). A cinética das primeiras clivagens influenciou a maioria das modificações epigenéticas estudadas desde os estágios iniciais até o blastocisto (exceto pela hidroximetilação do DNA e H3K27me3). A análise dos transcritos relacionados a inclusão ou remoção dessas modificações corroborou o padrão de modificações encontrado. É possível concluir que a cinética das primeiras clivagens apresenta relação com modificações epigenéticas nos embriões e que se manterão até o final do desenvolvimento pré-implantacional, resultando em blastocistos de padrão transcricional e metabólico distintos, podendo influenciar na viabilidade embrionária. / The timing of the first cell divisions may predict the developmental potential of an embryo, including its ability to establish pregnancy. Besides differences related to metabolism, stress, and survival, embryos with different speeds of development present distinct patterns of gene expression, mainly related to energy and lipid metabolism. As gene expression is regulated by epigenetic factors, and that includes DNA methylation patterns, in this study we compared the global DNA methylation profile of embryos with different kinetics of development in order to identify general pathways and regions that are most influenced by this phenotype. For this, bovine embryos were produced in vitro using sexed semen (females) and the 40 hours post insemination (hpi) were classified as Fast (4 or more cells) or Slow (2 or 3 cells), remaining in culture until the blastocyst stage (168 hpi) (Chapter 1) or evaluated at 40 hpi, 96 hpi or 168 hpi (Chapter 2). In Chapter 1, the analysis of global DNA methylation by EmbryoGENE DNA Methylation Array (EDMA) identified 11,584 differentially methylated regions (DMRs) (7,976 regions hypermethylated in blastocysts derived from the fast cleavage group -FBL- and 3,608 hypermethylated regions in blastocyst derived from the slow cleavage group -SBL). FBL presented more regions classified as hypermethylated, distributed throughout the genome, as in introns, exons, promoters and repeating elements, whereas in SBL, hypermethylated regions were more present in the CpG islands. DMRs were grouped in relation to the biological processes to which they were involved, and some of the most affected pathways were related to cell survival/differentiation and energy/lipid metabolism. Profiles of transcripts of differentially methylated (DMs) genes related to these pathways were also evaluated, and most presented changes in relative quantification. In Chapter 2, the presence of DNA methylations and hydroxymethylations and post-translational histone modifications (histone 3 lysine 9 acetylation and trimethylation and histone 3 lysine 27 trimethylation - H3K9ac, H3K9me3 and H3K27me3, respectively). The kinetics of the first cleavages influenced most of the epigenetic modifications studied from the initial stages to the blastocyst (except DNA hydroxymethylations and H3K27me3). The analysis of the transcripts related to insertion or removal of these modifications corroborated the pattern of modifications. It is possible to conclude that the kinetics of the first cleavages is related with epigenetic modifications in embryos that will be maintained through the pre-implantation development, resulting in blastocysts with different transcriptional and metabolic patterns, which might influence the embryonic viability.

In human-assisted reproduction, embryos are cultured in an environment designed to mimic the natural environment of embryo development. To maintain the optimal culture environment, the culture media is covered with oil. Unfortunately, several studies have shown that mineral oils used in embryo cultivation contains toxic substances that have negative effects on embryo development. Before these culture oils are used in production they are washed and filtered. This procedure reduces the concentration of toxic substances to an approved level.The aim of this study was to compare three different paraffin oils effect on embryo development. The study includes two oils from Nidacon (Oil A, Oil B) and one from Vitrolife (Ovoil). To compare the effect on embryo development, time points for important embryonic development stages was noted: first division after thawing, morula, early blastocyst, blastocyst, expanded blastocyst and hatching blastocyst. In addition, blastocyst classification was done on day 5 and 6 according to Gardner and Schoolcraft´s blastocyst classification system.A total of 47 human day-2 cryopreserved embryos were divided in three groups and cultured for 4 days in EmbryoSlides overlaid with Oil A, Oil B or Ovoil respectively. The results showed no significant difference in effect on embryo development regarding morphokinetics and blastocyst classification between the three examined paraffin oils. In conclusion, the results indicated the same quality and toxicity level between the three examined paraffin oils.

Durant la division cellulaire, la ségrégation des chromosomes et le partage du cytoplasme sont essentiels pour maintenir l'intégrité génomique. Cependant, les erreurs de ségrégation sont fréquentes chez l'embryon préimplantatoire de mammifère et entraînent un gain ou une perte de chromosomes, appelé aneuploïdie. L'aneuploïdie est préjudiciable au développement et est la principale cause de pertes de grossesse. La mitose est coordonnée par cycle cellulaire, notamment la Cycline-B. Comprendre comment la destruction de la Cycline-B contrôle la sortie de la mitose des embryons pourrait expliquer pourquoi l'aneuploïdie est courante en clinique de fertilité. Nous avons étudié la destruction de la Cycline-B en fonction du stade de développement et de l'aneuploïdie. La littérature suggère que l’aneuploïdie perturbe le cycle cellulaire conduisant les cliniques de fertilité à utiliser la durée du cycle cellulaire et la morphologie (morphocinétique) pour prédire la santé de l'embryon. Cependant, la prédiction de la ploïdie par morphocinétique reste à démontrer. Notre objectif était de savoir comment l'aneuploïdie affecte le cycle cellulaire et le développement de l'embryon. Après une micro-injection de CyclineB1:GFP (Cycline-B) et H2B:RFP (chromosomes), les embryons de souris furent imagés par microscopie confocale. Des cellules aneuploïdes furent générées chimiquement pour évaluer leurs morphocinétiques. Curieusement, l'apparition de la Cycline-B après nuclear envelope breakdown a été devancée avec la progression du développement indépendamment de la taille des cellules. De plus, les erreurs de ségrégation ont peu impacté le développement et la destruction de la Cycline-B. Nous concluons que la morphocinétique est un outil prédictif peu fiable pour identifier les embryons aneuploïdes. / During cell division, it is essential that chromosome segregation during mitosis, and the partitioning of the cytoplasm at cytokinesis occur in successive timing to maintain genomic integrity. However, segregation errors are frequently observed in the early mammalian embryo, causing daughter cells to inherit whole chromosome gains and losses, termed aneuploidy. Aneuploidy is detrimental to development, being the leading cause of pregnancy loss and developmental disorders. The timing of mitosis is coordinated by the cell cycle component, Cyclin B. Understanding how Cyclin B destruction temporally controls mitotic exit in embryos could help elucidate why aneuploidy is common in IVF clinics. We investigate how Cyclin B destruction changes in different developmental stages and the presence of aneuploidy. Literature suggests aneuploidy disrupts the cell cycle, leading IVF clinics to use cell cycle timings and morphology (morphokinetics) to predict embryo health. However, whether morphokinetics predicts embryo ploidy is uncertain. We seek to investigate how aneuploidy affects the cell cycle and embryo development. We used live-cell confocal imaging and microinjection of CyclinB1:GFP and H2B:RFP mRNA to visualise Cyclin B and chromosomes during mitosis in the 2-, 4- and 8-cell stage mouse embryo. Secondly, we pharmacologically-induced aneuploidy to assess aneuploid morphokinetics. Interestingly, we observe a developmental trend, independent of cell size, where Cyclin B onset begins progressively sooner after NEBD at the 2-, 4- and 8-cell stage. Additionally, chromosome segregation errors had little impact on Cyclin B destruction and development. Finally, we find morphokinetics to be a poor predictive tool in identifying aneuploid embryos.

Philosophiae Doctor - PhD / The aim of this thesis is first, to evaluate various culture conditions to improve embryo development, and secondly, to analyze spent culture media for any biomarkers that may be predictive of embryo health. Single-step and sequential culture media were compared in both Planer and EmbryoScope™ incubators. Single-step media resulted in better blastocyst development compared to sequential media and the EmbryoScope™ incubation system showed slight improvements in embryo development than the Planer system. The benefits of supplementing the culture medium with either insulin or insulin-like growth factor 1 (IGF-1) or culturing in a 2% O2 environment, using two different strains of mice (hybrid and C57), as well as the suitability of these strains for quality control were compared. In insulin, hybrid embryos were slower to blastulate and had a lower blastocyst rate, whereas C57 embryos were slower to the morula and faster to blastocyst stages, and lower blastocyst rate than the controls. IGF-1 showed no difference in time-lapse morphokinetics (TLM) or blastocyst rates compared to controls in both hybrid and C57 embryos. Under 2% O2, hybrid embryos showed no significant difference in TLM up to the 8-cell stage, but slowed down afterwards, resulting in blastocysts with significantly lower cell counts than the 6% O2 group. The C57 embryos were slower to reach morula and expanded blastocyst, and had lower blastocyst rates in 2%O2 vs 6%O2. The C57 strain had significant slower overall embryo development for all time points than hybrid embryos in insulin, IGF-1 and ultra-low O2, as well as lower blastocyst rates. Measurement of growth differentiation factor 9 (GDF-9) and oxidation-reduction potential (ORP) in spent media as markers for embryo health were evaluated. Day 5 human blastocysts yielded higher pregnancy rates and GDF-9 levels in spent media compared to Day 6 blastocysts, but TLM parameters showed no impact on pregnancy outcome. In Day 6 blastocysts, the non-pregnant group showed significantly faster embryo development compared to the clinically pregnant group up to the 8-cell stage and start of blastulation. GDF-9 did not show any significant differences between non-pregnant and pregnant groups of Day 5 or Day 6 embryo transfers. ORP in spent media from good quality Day 3 embryos that developed into blastocysts were significantly higher than from those that did not, with no difference in control medium ORP. Spent media from arrested embryos showed lower ORP than their corresponding controls. Arrested embryos had slower development at syngamy, morula, blastulation and blastocyst stages. The single step medium in the EmbryoScope™ is the preferred choice for embryo culture. Insulin or IGF-1 media supplementation or 2% O2 culture did not provide any benefit to embryo development. The C57 mouse strain is more sensitive and may be better to detect changes in culture conditions, and therefore better model for quality control assays. GDF-9 values decrease from Day 5 to Day 6 which gives new insight to understanding the role of GDF-9 during embryogenesis. ORP in spent media indicate that embryos that developed into blastocysts did not contribute to ROS, but maintained ORP balance.