Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-04-04T13:30:03Z No. of bitstreams: 1 sabinewohlresviana.pdf: 1957296 bytes, checksum: 29394666e22c25c01e78f33c22873af2 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-04-04T15:37:11Z (GMT) No. of bitstreams: 1 sabinewohlresviana.pdf: 1957296 bytes, checksum: 29394666e22c25c01e78f33c22873af2 (MD5) / Made available in DSpace on 2017-04-04T15:37:11Z (GMT). No. of bitstreams: 1 sabinewohlresviana.pdf: 1957296 bytes, checksum: 29394666e22c25c01e78f33c22873af2 (MD5) Previous issue date: 2009-03-09 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Embriões produzidos in vivo e in vitro apresentam diferenças morfológicas e de desenvolvimento as quais podem ser influenciadas pelo de cultivo. Além disso, existem poucos estudos mostrando o efeito do ambiente in vitro sobre os embriões provenientes de diferentes raças bovinas como, por exemplo, Gir (Bos indicus) e Holandesa (Bos taurus). Alguns genes importantes para a formação, sobrevivência e posterior desenvolvimento de embriões bovinos podem ter seu padrão de expressão influenciado por diversos fatores relacionados ao sistema de produção e a raça. O objetivo deste estudo foi avaliar a abundância relativa de transcritos relacionados ao transporte de água e solutos (Aquaporinas), formação da blastocele (Na/KATPases), apoptose (BAX) e resposta a estresse celular (Peroxiredoxina 1) em blastocistos bovinos das raças Gir e Holandesa produzidos in vivo e in vitro para identificar efeitos de sistema de produção e/ou raça. Para cada grupo (Gir-in vivo; Gir-in vitro; Holandês-in vivo; Holandês-in vitro), blastocistos (n=15) divididos em três conjuntos foram utilizados para a extração do RNA, amplificação, e transcrição reversa. Os cDNAs obtidos foram submetidos à PCR em Tempo-Real, utilizando o gene H2a como controle endógeno, e analisados pelo software REST©. Na avaliação de efeito do sistema de produção para a raça Gir, a expressão relativa do gene PRDX1 apresentou-se sub-regulada (p<0,01) assim como nos genes Na/KATPase-β2 e BAX (p<0,05), enquanto os genes AQP3, AQP11 e Na/K-ATPase-α1 não apresentaram diferenças significativas (p>0,05) nas amostras de embriões produzidos in vitro comparados com embriões in vivo. Na raça Holandesa, a expressão relativa do gene BAX apresentou-se sobre-regulada (p<0,01), enquanto o gene Na/K-ATPase-β2 apresentou uma tendência a sub-regulação (p=0,06) e os genes AQP3, AQP11, Na/K-ATPase-α1, PRDX1 não apresentaram diferenças significativas (p>0,05) nas amostras de embriões produzidos in vitro comparados com embriões in vivo. Quando a produção in vivo foi avaliada entre as raças, os embriões da raça Holandesa apresentaram os genes AQP3, Na/K-ATPase-α1, PRDX1, e BAX sub-regulados (p<0,05), enquanto os genes Na/K-ATPase-β2 e AQP11 não demonstraram diferenças significativas na expressão (p>0,05) quando comparados com embriões da raça Gir. Para a produção in vitro, apenas o gene BAX apresentou-se sobre-regulado (p<0,05) nos embriões da raça Holandesa quando comparados com embriões da raça Gir. Com base nestes resultados podese concluir que, para os genes avaliados, existem diferenças na expressão gênica inerentes às raças observadas nos embriões produzidos in vivo e que o sistema de produção in vitro pode influenciar a expressão gênica, atenuando estas variações. A avaliação de outros transcritos é necessária para uma melhor compreensão dos efeitos relacionados aos processos de produção de embriões bovinos em diferentes raças. / Embryos produced in vivo and in vitro show morphological and developmental differences, which can be influenced by culture environment. Nevertheless, there are few studies showing the effect of in vitro environment on embryos from different bovine breeds, such as Gyr (Bos indicus) and Holstein (Bos taurus). Some important genes associated to formation, survival and subsequent development of bovine embryos can be affected by various factors related to the production system and breed. The aim of this study was to evaluate the relative abundance of transcripts associated to water transport (Aquaporins), blastocoel formation (Na/K-ATPases), apoptosis (BAX) and response to cellular stress (Peroxiredoxin 1) in bovine blastocysts from Gyr and Holstein breed produced in vivo and in vitro to identify breed and/or production system effects. For each group (Gyr-in vivo; Gyr-in vitro; Holstein-in vivo; Holstein-in vitro), blastocysts (n=15) distributed in 3 pools were used for RNA extraction, followed by RNA amplification and reverse transcription. The cDNAs obtained were submitted to Real-Time PCR, using the H2a gene as endogenous control, and analyzed with REST software©. For the production system in the Gyr embryos, the relative expression of PRDX1 gene was down-regulated (p<0.01) as well as Na/K-ATPase-β2 and BAX genes (p<0.05), and the AQP3, AQP11 and Na/K-ATPase-α1 genes were not significantly different (p>0.05) when in vitro embryos were compared with in vivo produced ones. In Holstein embryos, BAX was up-regulated in in vitro embryos (p<0.01), while AQP3, AQP11, Na/K-ATPaseα1, Na/K-ATPase-β2 and PRDX1 genes were not significantly different (p>0.05) from in vivo produced embryos. When the breed effect was evaluated, Holstein in vivo produced blastocysts presented AQP3, Na/K-ATPase-α1, PRDX1, and BAX genes down-regulated (p<0.05) while Na/K-ATPase-β2 and AQP11 genes were not significantly different (p>0.05) from Gyr embryos. For the in vitro produced embryos, only Bax was up-regulated (p<0.05) in Holstein embryos compared with Gyr embryos. Based on those results, for the evaluated genes, it is possible to conclude that are inherent breed differences in gene expression observed in in vivo produced embryos and that the in vitro production system influences in the relative expression in both breeds, attenuating the inherent breed differences. Other genes should be evaluated for a better understanding of these breeds differences.

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.

The preimplantation stages of mammalian development are dedicated to the differentiation of two extraembryonic epithelia, the trophectoderm (TE) and the primitive endoderm (PrE), and their segregation from the pluripotent embryonic lineage, the epiblast. The TE and PrE are responsible for implantation into the uterus and for producing the tissues that will support and pattern the epiblast as it develops into the foetus. PrE and epiblast are formed in a two step process that involves random cell fate specification, mediated by fibroblast growth factor (FGF) signalling, and cell sorting through several mechanisms. In the present work I have addressed aspects of both steps of this process. Chimaera assays showed that epiblast precursors transplanted onto a recipient embryo rarely differentiate into PrE, while PrE precursors are able to switch their identity and become epiblast. Transient stimulation or inhibition of the FGF4-ERK pathway in the chimaeras can modify the behaviour of these cells and restore the plasticity of epiblast precursors. This work shows that epiblast precursors are refractory to differentiation signals, thus ensuring the preservation of the embryonic lineage. I have also found that atypical Protein Kinase C (aPKC) is a marker of PrE cells and that pharmacological inhibition of aPKC impairs the segregation of PrE and epiblast precursors. Furthermore, it affects the survival of PrE cells and can alter the subcellular localisation of the PrE transcription factor GATA4. These data indicate aPKC plays a central role for the sorting of the PrE and epiblast populations and links cell position within the embryo to PrE maturation and survival. Lastly, I have found that aPKC can directly phosphorylate GATA4 in vitro. Knockdown of GATA4 affects cell position within the embryo, whereas aPKC knockdown reduces the number of GATA4-positive cells. These results indicate GATA4 plays an important role in cell sorting during preimplantation development and suggest phosphorylation by aPKC could determine its presence in the nuclei of PrE cells. My work, in the light of the current knowledge, supports a model where the earliest cell fate decisions during mammalian development depend on cellular interactions and not on inherited cell fate determinants. This robust mode of development underlies the plasticity of the preimplantation embryo and ensures the formation of the first mammalian cell lineages, critical for any further progression in mammalian development.

Neste estudo foi analisada a expressão gênica e protéica, uterina e embrionária da quimiocina Ccl25 e de seu receptor Ccr9 nas fases iniciais da implantação embrionária (dias 3,5, 4,5, 5,5 e 7,5 de gestação). Por meio de reações imunohistoquímicas e de citometria de fluxo, foram identificadas as populações celulares envolvidas nesta expressão. Também, foram realizados ensaios de quimiotaxia com o silenciamento da expressão de Ccl25 (ODNs-Antisense) nas células trofoblásticas, para a avaliação das atividades desta quimocina. Nossos resultados sugerem o estabelecimento de uma comunicação embrião (células trofoblásticas) - endométrio (células do sistema immunológico), via Ccl25/Ccr9. Esses achados são relevantes para a compreensão das interações blastocisto/sistema immunológico materno no estabelecimento dos mecanismos imunoreguladores durante a implantação embrionária. / In this study, we analyzed the gene and protein, uterine and embryonic expression of Ccl25 chemokine and its receptor Ccr9 in early stages of embryo implantation (days 3.5, 4.5, 5.5 and 7.5 of gestation). By using immunohistochemistry and flow cytometric assays, cell populations involved in this expression were identified. In addition, chemotaxis assays were also performed after silencing of Ccl25 (ODNs-antisense) in trophoblast cells. . Our results suggest the establishment of an endometrium (immune cells) - embryo (trophoblast cells) dialogue via Ccl25/CCR9. These findings are relevant for understanding the interactions between blastocyst and maternal immune system in the establishment of immunoregulatory mechanisms during implantation.

Infertility is a widespread problem around the world, although the available treatment options constantly improve through research and methodological development. A common treatment option mainly for biologically caused infertility is in-vitro fertilization, where in a menstrual cycle multiple oocytes are stimulated to mature and are then fertilized in a laboratory. This often results in multiple good quality embryos which can be cryopreserved through vitrification and used in a later cycle to increase the success chance of the treatment. The purpose of this pilot study was to evaluate vitrification and warming kits from Kitazato and Irvine Scientific regarding results and procedures. Human cleavage stage embryos (n=76) were thawed and cultured to the blastocyst stage. The blastocysts were scored according to Gardner and cryopreserved with vitrification and warming kits from either Kitazato (n=20) or Irvine Scientific (n=20). The warmed blastocysts were controlled after 2 and 4 hours for re-expansion and freeze injuries. The data was analysed with Fisher’s exact test and considered statistically significant if two-tailed p-value &lt;0.05. The results showed no significant difference between the kits after 2 respectively 4 hours regarding re-expansion (p=0.432; p=0.492) or freeze injury (p=1.000; p=0.476). A significant difference was observed between group AB (with higher Gardner-score) and group C (with lower Gardner-score) in the degree of freeze injury (p=0.048; p=0.034), regardless of vitrification kit used. The details of the procedures differed somewhat between the kits, both having pros and cons, although overall procedures were equivalent. Further evaluation is needed before a change in method can be conducted.

abstract: Prior to the first successful allogeneic organ transplantation in 1954, virtually every attempt at transplanting organs in humans had resulted in death, and understanding the role of the immune mechanisms that induced graft rejection served as one of the biggest obstacles impeding its success. While the eventual achievement of organ transplantation is touted as one of the most important success stories in modern medicine, there still remains a physiological need for immunosuppression in order to make organ transplantation work. One such solution in the field of experimental regenerative medicine is interspecies blastocyst complementation, a means of growing patient-specific human organs within animals. To address the progression of immune-related constraints on organ transplantation, the first part of this thesis contains a historical analysis tracing early transplant motivations and the events that led to the discoveries broadly related to tolerance, rejection, and compatibility. Despite the advancement of those concepts over time, this early history shows that immunosuppression was one of the earliest limiting barriers to successful organ transplantation, and remains one of the most significant technical challenges. Then, the second part of this thesis determines the extent at which interspecies blastocyst complementation could satisfy modern technical limitations of organ transplantation. Demonstrated in 2010, this process involves using human progenitor cells derived from induced pluripotent stem cells (iPSCs) to manipulate an animal blastocyst genetically modified to lack one or more functional genes responsible for the development of the intended organ. Instead of directly modulating the immune response, the use of iPSCs with interspecies blastocyst complementation could theoretically eliminate the need for immunosuppression entirely based on the establishment of tolerance and elimination of rejection, while also satisfying the logistical demands imposed by the national organ shortage. Although the technology will require some further refinement, it remains a promising solution to eliminate the requirement of immunosuppression after an organ transplant. / Dissertation/Thesis / Masters Thesis Biology 2020

A live birth from a somatic cell nuclear transfer (SCNT) embryo represents a small percentage of donor cells that survived the reprogramming gauntlet. The inability to reprogram histone modifications in the donor cell line could add to the reprogramming deficiencies associated with SCNT. The effects of two histone modifications associated with transcriptional activation (H3K4m3 and H4K16ac) and two histone modifications associated with repressing transcription (H3K9m2 and H3K27me3) were evaluated in the context of their association to three genes known to contribute to maintaining totipotency: Nanog, Oct4, and Sox2. A µChIP assay was utilized using antibodies specific for each histone modification followed by real time PCR (qPCR) analysis to quantify the percentage of each gene associated with each particular histone modification. Gene expression analysis was followed by immunofluorescence and protein analysis. Results of these analyses suggest that gene association to certain histone modifications did not accurately predict gene expression in bovine blastocyst embryos. Of the three genes studied, only Oct4 expression differed significantly between in vitro fertilized (IVF; control) and SCNT blastocysts. Protein levels detected through immunofluorescence correlated directly with the gene expression analysis. Nanog and Sox2 expression profiles of IVF and SCNT bovine blastocysts are similar, yet the histone modification profiles associated with all three genes differ significantly. Altered expression levels in developmentally important genes will likely result in abnormal activity of the associated cellular pathway. Aberrant histone modifications, along with abnormal Oct4 expression, may contribute to the low percentage of SCNT embryos that result in live offspring.

The development of oocytes of differing quality retrieved using transvaginal follicular aspiration (TVFA) and following DNA injection was examined. Eight cows were subjected to twice weekly TVF A for 16 wk. Oocytes retrieved were graded and placed in an in-vitro maturation, fertilization and co-culture (IVMIIVFIIVC) program. Two thirds of oocytes were injected with DNA. Good quality oocytes from slaughtered cows (SH) were obtained once monthly and processed the same way. Good quality TVF A oocytes had a higher mean development score than poor quality oocytes, but not different from that of good quality SH oocytes. Good quality TVF A oocytes produced more viable embryos (31.7% blastocysts) than poor quality oocytes or SH oocytes (12.8% and 20.4% blastocysts, respectively). Embryo development following injection of DNA was the same for oocytes for each source-quality group (TVF A-good, 8.4; TVF A-poor, 5.5; SH-good, 6.3 % blastocysts). Development of good quality TVFA oocytes increased during the last 9 wk of the 16 wk collection period. Poor oocyte development increased slightly to 9 wk and then decreased. Development of TVF A oocytes injected with DNA did not vary during the experiment. However, development of controls increased from a mean score of2.50 at wk 1 to 4.17 at wk 16. Oocytes from TVFA produced more PCR positive blastocysts (95.0%) than SH oocytes (61.5%). More calves were born from the transfer of embryos injected with DNA from TVF A oocytes (3/5) than from SH oocytes (116), although not statistically significant. One calf was PCR positive in bone-marrow, but was negative in other tissues. The use of oocytes obtained by TVF A may improve the efficiency of producing transgenic cattle. / Master of Science

In vitro systems like those used for in vitro embryo production are invaluable for our understanding of embryogenesis and the processes that regulate it. However, extensive research has also highlighted that in vitro produced embryos negatively differ from their in vivo counterparts in various ways. Not surprisingly, there is ~20% decrease in pregnancy success from pregnancies established using in vitro produced embryos. Therefore, much research has relied on attempting to produce a better in vitro embryo that more closely resembles their in vivo counterparts. Our laboratory has investigated this by supplementing a cytokine, interleukin-6 (IL6), during in vitro embryo culture. My dissertation work expands upon those initial efforts by answering more detailed questions related to the biological role of IL6 during cattle embryogenesis. In the work presented herein, IL6 supplementation during in vitro culture was able to transform the transcriptome of resulting conceptuses post embryo transfer. The transcriptome of these conceptuses included an abundance of genes associated with survival. Indeed, we witnessed IL6-treated conceptuses resulted in a 20% increased survival rate and were longer than their non-treated counterparts. In the second research project, we employed CRISPR-Cas9 genome editing technology to understand the embryo phenotype after part of the IL6 receptor responsible for signal transduction, interleukin-6 signal transducer (IL6ST), is disrupted. We discovered that IL6ST is required for development before the blastocyst stage. In addition, IL6ST disrupted blastocysts, presumed to contain wildtype, presented with severe, abnormal morphology. Not only did this group of embryos have decreased ICM and TE cell numbers, but they also had an increased occurrence of cells within the TE region that were negative for its traditional marker, CDX2. This suggests IL6ST is likely involved in a pathway responsible for determining cell fate identity at the blastocyst stage. Collectively, IL6 in cooperation with IL6ST, is a key controller of embryogenesis in cattle. / Doctor of Philosophy / There are major events that an embryo must successfully advance from to continue development to form into an organism capable of survival after birth. Over 30% of pregnancies in cattle and humans will fail within the first 30 days of gestation. This time period coincides with several key developmental events that ultimately modify the morphology of the growing embryo. Our laboratory primarily focuses on embryo development around the blastocyst stage. If an embryo advances to this stage, it has a greater likelihood of maintaining viability. Therefore, my dissertation research has focused on early embryonic development from the time of first cleavage (~day 2 of gestation) through embryo elongation (~day 15 of gestation), which encompasses the blastocyst stage. Within this time frame, I have been investigating embryonic effects after supplementation of a protein, interleukin-6 (IL6). Previously, our laboratory has identified IL6 to cause favorable impacts on the developing embryo, but its mode of action was unknown. Therefore, my dissertation research has investigated the mechanistic actions of IL6, and its beta receptor subunit, interleukin-6 signal transducer (IL6ST). In my first research project, we discovered that supplementing IL6 during in vitro embryo culture resulted in increased embryo elongation and survival. In my second research project, we found IL6ST is an absolute requirement for embryo survival to the blastocyst stage. Together, these results indicate IL6 is a very important protein needed for sustained pregnancy viability.

Um Projeto Temático de Pesquisa foi desenvolvido no Laboratório de Poluição Ambiental do Departamento de Patologia da Faculdade de Medicina da Universidade de São Paulo com o objetivo de avaliar os efeitos da exposição aguda/crônica ao ar ambiente de um grande centro urbano sobre a saúde. Dentro deste projeto, uma linha de pesquisa foi dedicada ao estudo dos efeitos dessa exposição sobre a saúde reprodutiva feminina. Evidências de estudos epidemiológicos e experimentais implicam os fatores ambientais na infertilidade humana e resultado obstétrico adverso. Contudo, poucos estudos foram conduzidos até o presente para avaliar um possível efeito da exposição à poluição ambiental particulada sobre a saúde reprodutiva feminina. Portanto, o objetivo dos projetos da minha linha de pesquisa é fornecer dados que possam demonstrar os possíveis efeitos da exposição crônica à poluição ambiental particulada sobre a função ovariana e o desenvolvimento embrionário inicial. O objetivo do primeiro projeto desta tese foi avaliar diferentes metodologias utilizadas para a coloração diferencial das linhagens celulares do blastocisto, um método mais adequado para a avaliação de sua qualidade e normalidade. As células de blastocistos intactos de camundongo obtidos através de fertilização in vitro (FIV) foram permeabilizadas e coradas utilizando-se diferentes concentrações de um detergente (TX-100; 0,5% ou 1%) e de iodeto de propídeo (IP; 50 g/mL ou 100 g/mL) e depois disso, incubadas durante a noite em uma solução contendo diferentes fixadores (etanol, metanol, paraformaldeído PFA1% ou 4%) e bisbenzimida. Para a avaliação da qualidade de coloração e contagem diferencial dos núcleos, os blastocistos foram montados em lâminas de vidro e analisados em um microscópio de epifluorescência. O escore de qualidade de coloração foi significativamente diferente (p<0,05) entre todas as soluções fixadoras, sendo maior para o etanol, seguido pelo metanol, PFA1% e PFA4%. Mudanças da concentração do IP e o uso de diferentes soluções de fixação revelaram efeitos significativos na contagem de células da massa celular interna (MCI) e na razão MCI/trofectoderma (TE). Concentrações diferentes do detergente utilizado para a permeabilização celular apresentaram efeitos significativos sobre a contagem de células TE e razão MCI/TE. Concluímos que o protocolo que utiliza TX-100 1% para a permeabilização celular, 50 g/mL de IP para coloração das células TE e etanol como solução de fixação representa o método mais eficiente para a coloração diferencial e contagem das células das linhagens celulares do embrião no estágio de blastocisto. No segundo projeto que compõe esta, o objetivo foi avaliar os efeitos da exposição pré e/ou pós-natal ao ar ambiente sobre a fertilização, desenvolvimento embrionário e segregação das linhagens celulares em blastocistos pré-implantacionais, utilizando o modelo de FIV de camundongo. Fêmeas de camundongo com idade de seis semanas tiveram a ovulação estimulada e foram expostas no período pré- e/ou pós-natal ao ar filtrado (AF-AF), ar filtrado ar ambiente (AF-AA) ou ar ambiente ar ambiente (AA-AA) em câmaras de exposição 24 horas por dia, sete dias na semana, durante nove semanas. Os pontos de avaliação reprodutivos analisados incluíram a duração da gestação, tamanho e peso da prole, índice de nascidos vivos, razão sexual, resposta ovariana à estimulação, taxa de fertilização, desenvolvimento embrionário, taxas de formação e de eclosão dos blastocistos, contagem celular total e proporção da alocação celular à MCI e TE. A duração da gestação, tamanho e peso da prole, índice de nascidos vivos e razão sexual foram similares nos diferentes grupos de exposição. A resposta ovariana não foi afetada pelo protocolo de exposição. Um efeito multivariável para a exposição pré e/ou pós-natal ao material particulado fino ambiente sobre a FIV, o desenvolvimento embrionário e a coloração diferencial dos blastocistos foi observado. A contagem celular na MCI e a razão MCI/TE dos blastocistos produzidos no protocolo AF-AF foram significativamente maiores do que aquelas em blastocistos produzidos nos protocolos FA-AA e AA-AA. Nenhuma diferença na contagem celular total foi observada. Baseando-se nessas observações, nosso estudo sugere que a exposição ao material particulado fino presente no ar ambiente de um grande centro urbano pode afetar negativamente a saúde reprodutiva feminina através da alteração da especificação das linhagens celulares do embrião no estágio de blastocisto. Finalmente, o propósito do terceiro projeto que compõe esta tese foi de avaliar os efeitos da exposição pré e/ou pósnatal ao ar ambiente no final da vida reprodutiva sobre a fertilização, desenvolvimento embrionário e segregação das linhagens celulares em blastocistos pré-implantacionais, utilizando o modelo de FIV de camundongo. Fêmeas de camundongo com idade de cinco meses tiveram a ovulação estimulada e foram expostas no período pré e/ou pós-natal ao ar filtrado (AF-AF), ar filtrado ar ambiente (AF-AA) ou ar ambiente ar ambiente (AA-AA) em câmaras de exposição 24 horas por dia, sete dias na semana, durante seis meses. Os pontos de avaliação reprodutivos foram os mesmos que aqueles utilizados no segundo projeto. A duração da gestação, tamanho e peso da prole, índice de nascidos vivos e razão sexual foram similares nos diferentes grupos de exposição. A resposta ovariana não foi afetada pelo protocolo de exposição. Um efeito multivariável para a exposição pré e/ou pós-natal ao material particulado fino ambiente sobre coloração diferencial dos blastocistos, mas não sobre a FIV e o desenvolvimento embrionário, foi observado. A contagem celular na MCI e a razão MCI/TE dos blastocistos produzidos no protocolo AF-AF foram significativamente maiores do que aquelas em blastocistos produzidos nos protocolos FA-AA e AA-AA. A contagem celular do TE dos blastocistos produzidos no protocolo FA-FA foi significativamente menor do que aquela em blastocistos produzidos nos protocolos FA-AA e AA-AA. A contagem celular total foi similar entre os grupos. Nosso estudo sugere que a exposição à poluição ambiental particulada de um grande centro urbano não altera a função ovariana, mas pode afetar negativamente a saúde reprodutiva feminina no período final do menacme, através da alteração da especificação das linhagens celulares do embrião no estágio de blastocisto / A thematic research project to evaluate the health effects of acute/chronic exposure to ambient air in a large urban center was developed at the Air Pollution Laboratory in the Department of Pathology at the University of São Paulo School of Medicine. Within this project a specific research line was committed to the study of the effects of this exposure on female reproductive health. Evidence from epidemiological and experimental studies implied environmental factors as possible contributors to human infertility and poor obstetric outcome. However, very few studies evaluating a possible effect of exposure to particulate air pollution on female reproductive health have been conducted so far. Thus, the aim of the projects in my research line was to provide data that could show the possible effects of chronic exposure to particulate air pollution on ovarian function and early embryo development. The objective of the first project was to assess different methodologies used in cell lineage differential staining of the blastocyst, a method for more accurate evaluation of its quality and normality. Cells of zona-intact mouse blastocysts obtained from in vitro fertilization (IVF) were permeabilized and stained using different concentrations of a detergent (TX-100; 0.5% or 1%) and propidium iodide (PI; 50 g/mL or 100 g/mL) followed by overnight incubation in a solution containing different fixatives (ethanol, methanol, paraformaldehyde - PFA 1% or 4%) and bisbenzimide. To evaluate the staining quality and count the nuclei differentially, blastocysts were mounted and viewed using epifluorescence microscopy. Staining quality scores were significantly different (P < 0.05) among all fixative solutions with the highest for ethanol followed by methanol, PFA1%, and PFA4%. Changes in PI concentration and use of different fixative solutions revealed significant effects on inner cell mass (ICM) cell count and ICM/trophectoderm (TE) ratio. Different concentrations of the detergent used for cell permeabilization showed significant effects on TE cell counts and ICM/TE ratio. I concluded that the protocol using 1% TX-100 for cell permeabilization, 50 g/mL of PI for TE cell staining, and ethanol as a fixative solution is the most efficient method for cell lineage differential staining and counting at the blastocyst stage. In the second project the objective was to evaluate the effects of preand/ or postnatal exposure to ambient air on fertilization, embryo development, and cell lineage segregation in preimplantation blastocysts using the IVF mouse model. Six-week old superovulated mice were preand/ or postnatally exposed to filtered air (FA-FA), filtered-ambient air (FAAA), or ambient air (AA-AA) in exposure chambers 24/7 for nine weeks. Reproductive endpoints evaluated included gestation length, litter size, litter birth weight, live birth index, sex ratio, ovarian response to superovulation, fertilization rate, embryo development, blastocyst and hatching rates, total cell count, and proportion of cell allocation to ICM and TE. Gestation length, litter size, litter birth weight, live birth index, and sex ratio were similar among exposure groups. Ovarian response was not affected by the exposure protocol. A multivariate effect for pre- and/or postnatal exposure to ambient fine particulate matter on IVF, embryo development, and blastocyst differential staining was found. Cell counts in ICM and ICM/TE ratios in blastocysts produced in the FA-FA protocol were significantly higher than in blastocysts produced in FA-AA and AA-AA protocols. No difference in the total cell count was observed. Based on these observations the study suggests that exposure to ambient fine particulate matter in a large urban center may negatively affect female reproductive health by disrupting the lineage specification at the blastocyst stage. Finally, the purpose of the third project was to evaluate the effects of pre- and/or postnatal exposure to particulate air pollution on fertilization, embryo development, and cell lineage segregation in preimplantation blastocysts during the late-life reproductive period using the IVF mouse model. Five-month-old superovulated mice were pre- and/or postnatally exposed to filtered air (FA-FA), filtered-ambient air (FA-AA), or ambient air (AA-AA) in exposure chambers 24/7 during six months. Reproductive endpoints were the same as the ones selected for the second project. Gestation length, litter size, litter birth weight, live birth index, and sex ratio were similar among exposure groups. Ovarian response was not affected by the exposure protocol. A multivariate effect for pre- and/or postnatal exposure to ambient air on blastocyst differential staining but not on IVF and embryo development was found. Cell counts in ICM and ICM/TE ratios in blastocysts produced in the FA-FA protocol were significantly higher than in blastocysts produced in FA-AA and AA-AA protocols. Cell counts in TE cells in blastocysts produced in the FA-FA protocol were significantly lower than in blastocysts produced in FA-AA and AA-AA protocols. The total cell count was similar among groups. This study suggests that exposure to particulate air pollution in a large urban center has no effect on ovarian function but may negatively affect female reproductive health in the late-life period by disrupting the lineage specification at the blastocyst stage.