Female gametes, or oocytes grow and mature in a niche environment maintained by the somatic cells of the ovarian follicle. At ovulation ovarian follicle cells respond to the luteinising hormone (LH) surge coordinating the final maturation, meiotic resumption and release of oocytes. Simultaneously, production of a unique “mucified” extracellular matrix surrounding the oocyte through synthesis of Hyaluronan (HA) and HA cross-linking proteins produces an “expanded” and stabilised cumulus oocyte matrix with a specific composition, structure and function. In vitro maturation (IVM) of oocytes is a procedure by which cumulus oocyte complexes (COCs) are stimulated to produce cumulus matrix and undergo oocyte maturation ex vivo. In vitro maturation is a useful procedure for studying oocyte competence as well as offering health benefits for patients undergoing assisted reproduction. Oocytes derived from IVM have much lower developmental competence than in vivo matured oocytes, likely as a result of altered environmental conditions and gene expression leading to suboptimal maturation and/or inappropriate metabolic control in oocytes. Cumulus matrix expansion is widely used as an indicator of good oocyte developmental potential, however, the mechanism(s) that endow oocyte quality and how these may be influenced by the cumulus matrix are poorly understood. To better understand the process by which cumulus matrix is linked to the final stages of oocyte maturation, I undertook investigation of mouse COC matrix composition and function after in vivo maturation in comparison to IVM. The gene responsible for Hyaluronan synthesis, Has2, was not impaired under IVM conditions. In contrast, two key extracellular matrix proteins; Versican and Adamts1, which are normally selectively incorporated into periovulatory COCs in vivo, were greater than 10-fold reduced in IVM whether stimulated with Egf and/or FSH. This work is the first to show that commonly used IVM conditions result in altered gene expression in cumulus cells. Furthermore, the absence of Adamts1 and Versican suggest that COC matrix may be functionally insufficient. Although associated with good developmental potential, the function of the COC matrix in oocyte maturation is unknown. I assessed the properties of COC matrix that control metabolite supply to oocytes by examining transport of fluorescently labelled glucose and cholesterol across mouse COCs. Profound differences in the control of metabolite supply to oocytes in IVM were observed. In vivo matured complexes were capable of excluding glucose from the entire COC and cholesterol was excluded from oocytes. Conversely IVM COCs were more permissive to rapid equilibration of glucose and cholesterol concentrations across the complex and in oocytes. In fact both metabolites accumulated rapidly in IVM oocytes resulting in inverse gradient patterns of glucose and cholesterol abundance with highest concentrations accumulating in the oocyte after IVM vs highest concentrations surrounding the COC after in vivo maturation conditions. As oocytes are highly sensitive to high glucose my results indicate that metabolic balance in IVM may be disrupted due to impaired molecular filtration properties of the mucified COC matrix that controls supply of hydrophilic and lipophylic substrates. Importantly these novel findings can explain the glucose sensitivity of IVM oocytes and identifies a mechanism by which IVM may lead to poorer oocyte developmental competence. To translate these findings into the improvement of IVM I generated recombinant expression plasmid constructs for several Adamts1 and Versican functional domains. The efficacy of Versican as an IVM supplement that activates cumulus cell signal transduction was proved in principle, by showing enhanced COC matrix expansion when added to mouse IVM cultures. Similar mechanisms are likely to be functional in human COCs since I demonstrated VERSICAN and ADAMTS1 expression in human in vivo matured cumulus and granulosa cells. This work has advanced our understanding of oocyte maturation and will lead to improvements in IVM and healthier outcomes from reproductive therapies. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1342419 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

Female gametes, or oocytes grow and mature in a niche environment maintained by the somatic cells of the ovarian follicle. At ovulation ovarian follicle cells respond to the luteinising hormone (LH) surge coordinating the final maturation, meiotic resumption and release of oocytes. Simultaneously, production of a unique “mucified” extracellular matrix surrounding the oocyte through synthesis of Hyaluronan (HA) and HA cross-linking proteins produces an “expanded” and stabilised cumulus oocyte matrix with a specific composition, structure and function. In vitro maturation (IVM) of oocytes is a procedure by which cumulus oocyte complexes (COCs) are stimulated to produce cumulus matrix and undergo oocyte maturation ex vivo. In vitro maturation is a useful procedure for studying oocyte competence as well as offering health benefits for patients undergoing assisted reproduction. Oocytes derived from IVM have much lower developmental competence than in vivo matured oocytes, likely as a result of altered environmental conditions and gene expression leading to suboptimal maturation and/or inappropriate metabolic control in oocytes. Cumulus matrix expansion is widely used as an indicator of good oocyte developmental potential, however, the mechanism(s) that endow oocyte quality and how these may be influenced by the cumulus matrix are poorly understood. To better understand the process by which cumulus matrix is linked to the final stages of oocyte maturation, I undertook investigation of mouse COC matrix composition and function after in vivo maturation in comparison to IVM. The gene responsible for Hyaluronan synthesis, Has2, was not impaired under IVM conditions. In contrast, two key extracellular matrix proteins; Versican and Adamts1, which are normally selectively incorporated into periovulatory COCs in vivo, were greater than 10-fold reduced in IVM whether stimulated with Egf and/or FSH. This work is the first to show that commonly used IVM conditions result in altered gene expression in cumulus cells. Furthermore, the absence of Adamts1 and Versican suggest that COC matrix may be functionally insufficient. Although associated with good developmental potential, the function of the COC matrix in oocyte maturation is unknown. I assessed the properties of COC matrix that control metabolite supply to oocytes by examining transport of fluorescently labelled glucose and cholesterol across mouse COCs. Profound differences in the control of metabolite supply to oocytes in IVM were observed. In vivo matured complexes were capable of excluding glucose from the entire COC and cholesterol was excluded from oocytes. Conversely IVM COCs were more permissive to rapid equilibration of glucose and cholesterol concentrations across the complex and in oocytes. In fact both metabolites accumulated rapidly in IVM oocytes resulting in inverse gradient patterns of glucose and cholesterol abundance with highest concentrations accumulating in the oocyte after IVM vs highest concentrations surrounding the COC after in vivo maturation conditions. As oocytes are highly sensitive to high glucose my results indicate that metabolic balance in IVM may be disrupted due to impaired molecular filtration properties of the mucified COC matrix that controls supply of hydrophilic and lipophylic substrates. Importantly these novel findings can explain the glucose sensitivity of IVM oocytes and identifies a mechanism by which IVM may lead to poorer oocyte developmental competence. To translate these findings into the improvement of IVM I generated recombinant expression plasmid constructs for several Adamts1 and Versican functional domains. The efficacy of Versican as an IVM supplement that activates cumulus cell signal transduction was proved in principle, by showing enhanced COC matrix expansion when added to mouse IVM cultures. Similar mechanisms are likely to be functional in human COCs since I demonstrated VERSICAN and ADAMTS1 expression in human in vivo matured cumulus and granulosa cells. This work has advanced our understanding of oocyte maturation and will lead to improvements in IVM and healthier outcomes from reproductive therapies. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1342419 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

Master of Science in Medicine (by research) / Assisted reproductive technologies (ARTs) are very efficient in producing embryos, however many of these embryos have poor viability. No more than 50% of IVF embryos complete preimplantation development (Hardy et al. 2001). The poor viability is manifested as a reduced rate of cell proliferation and increased rates of apoptosis in the early embryo, resulting in high rates of embryo mortality (Hardy et al. 2001). The reduced viability occurs as a response to a range of cellular stressors that are a consequence of embryo culture (Hardy et al. 2001). The stress of culture disrupts some survival signalling pathways, metabolism of substrates and induces redox stress (Hardy et al. 2001). The cellular stress sensor p53 is expressed in the early embryo but is normally kept at very low levels (Li et al. 2005). This latency may be breached in IVF embryos following culture of zygotes in vitro for 96 hours, resulting in the up-regulation and nuclear accumulation of p53 (Li et al. 2005). Activation of the p53 stress-sensing pathway in the early mouse embryo by culture in vitro causes a marked loss of their developmental competence (Li et al. 2005). This study aimed to establish whether benefits could be obtained by culturing mice IVF embryos in the presence of p53 protein inhibitors. IVF zygotes were cultured individually in 10µl drops of 1.25, 2.5, 5 or 10µM Pifithrin-a (PFTa) in 0.05% DMSO for 96 hours. On day 5 the development stage was assessed. Embryos reaching the blastocyst stage were fixed and stained with Hoechst 33342 for total cell count and the proportion of nuclei with normal and abnormal morphology. There was an increase in the blastocyst rate, total cell count and the proportion of nuclei in a blastocyst with normal nuclei in 10µM-treated embryos. This study also aimed to determine whether benefits could be obtained by incubating mouse IVF sperm with p53 protein inhibitors during IVF. IVF sperm was treated with 1.25, 2.5, 5 or 10µM of PFTa in 0.05% DMSO during incubation with oocytes for 6 hours. Resulting zygotes were cultured for 96 hours individually in 10µl drops of MODHTFM. On day 5 the development stage was assessed. Embryos reaching the blastocyst stage were fixed and stained with Hoechst 33342 for total cell count and the proportion of nuclei with normal and abnormal morphology. There was a reduction in the proportion of fragmented nuclei in blastocysts derived from 1.25 and 10µM-treated sperm. 10µM treated sperm increased the total cell count, the proportion of normal nuclei in a blastocyst and the blastocyst development rate. IVF sperm incubated with 1.25µM PFTa during insemination of oocytes increased the fertilisation rate. Another aim of this study was to establish whether p53 siRNA could inhibit p53 mRNA in mice IVF embryos and if so, whether this would improve embryo viability in culture. IVF zygotes were transfected with 15nM p53 small inhibiting RNA (siRNA) and 0.8% Oligofectamine Reagent immediately, 24 h, 48 h and 72 h after IVF then cultured individually in 10µl drops of MOD-HTFM for a total of 96 hours. On day 5 the blastocyst rate was assessed and immunofluorescence performed probing for p53. There was no significant reduction in p53 expression and no improvement in blastocyst rate at any of the transfection times. However, there was a decrease in the proportion of nuclei which expressed p53 when p53 siRNA was transfected 72 hours after IVF. Also, it was determined that siRNA was efficiently being delivered into the preimplantation embryo with Oligofectamine Reagent. Lastly, this study aimed to determine whether mice sperm with p53 gene deletions have a selective advantage in fertilising the oocyte compared to their wild-type counterparts. p53+/- males were mated with p53+/+ females and the resulting zygotes genotyped after 24 hours of culture. More than 50% of offspring had a p53+/+ genotype. There was no selective advantage for p53 null sperm to fertilise the oocyte, there was actually a disadvantage. The selective disadvantage for p53 null sperm to fertilise the F1 hybrid oocyte in IVF compared to its wild-type counterparts may imply that p53 null sperm are not as viable and may have a survival disadvantage. The reduction in fertility of p53 null sperm in vitro infers that p53 function may be important for the fertility of the mouse sperm in vitro. The results of this thesis could establish means of improving human embryo viability in ART, some examples being P53 protein inhibition in preimplantation embryos during culture prior to transfer to the uterus, or P53 protein inhibition in IVF sperm. The use of the new technology, p53 siRNA was not effective in inhibiting p53 expression, although the build-up experiments determined that siRNA is efficiently delivered into the preimplantation embryo with Oligofectamine Reagent. The demonstration that p53 null sperm has a selective disadvantage in fertilising the oocyte compared to their wild-type counterparts does not indicate a positive selection pressure for naturally occurring mutations to this gene. And so, there is no concern regarding the genetic and epigenetic risks to progeny arising from assisted reproductive technologies with respect to sperm.

Despite the many advances of medical technology to help treat infertility, approximately half of women seeking fertility treatment will never give birth to a child. Women coping with treatment failure face many challenges, including deciding when to abandon treatment and how to let go of their dreams of having a baby to focus on other pursuits. In order to better understand how women cope with these challenges, in depth interviews and a focus group were carried out with 12 women for whom fertility treatment had not been successful. Data was gathered and analysed using Grounded Theory, and a model of the process of adjustment from pursuing treatment to coming to terms with involuntary childlessness was co-constructed from the data. The model conceptualizes women's journey as moving through three main phases; 'living in limbo' in which women are still undergoing treatment, 'leaving treatment' in which women decide to terminate treatment and abandon the search for a resolution to their infertility, and finally 'learning to live with involuntary childlessness' in which women start the 'work' of grappling with the questions that childlessness seems to raise about the meaning of their lives, their identity and self image, and their sense of social belonging. The model goes on to highlight the factors which seem to aid women in resolving these challenges. The findings of this study suggest that the emotional challenges of coping with unsuccessful fertility treatment extend well beyond the end of treatment, highlighting the need for good access to therapeutic support for women coping with involuntary childlessness longer term. Results also point to certain sources and types of support which may be particularly helpful, including peer support from other childless women, and therapeutic interventions which help women develop more positive perspectives on childlessness and to identify alternative sources of fulfillment. The results of this study also point to the need for social action which works to challenge the misconceptions and stigma surrounding infertility and childlessness which add a further challenge to the lives of women who are involuntarily childless.

Assisted reproductive technology procedures has become a more complex treatment over the years after implementation of preimplantation genetic diagnostics and cryopreservation methods such as slow freeze and vitrification. When embryos undergo these methods they are exposed to external damage that threaten to affect their quality and thereby lead to lower survival rates and lower pregnancy rates. The aim of this study was to document blastocysts quality after vitrification, re-vitrification and preimplantation genetic diagnosis with subsequent vitrification. A total of 126 blastocysts were collected, of which 119 blastocysts were documented with the help of an experienced embryologists and the remaining seven blastocysts were from a new series of re-vitrified embryos. The 126 collected blastocyst were allocated into groups depending on their degree of preimplantation genetic diagnosis and vitrification. The gathered data was scoring according to morphology, expansion and proportion of necrotic cells at 2 and 4 hours of the expansion phase. Fisher exact test was used for statistical evaluation. There were no significant difference when comparing data before and after vitrification and preimplantation diagnosis, which indicates that these methods do not cause morphological damage to the blastocyst.

Assisted reproductive technology (ART) has been rapidly expanding since the birth of Louise Brown, the first test tube baby, in 1978. Although an increasingly complex array of laboratory skills and procedures have been developed for infertility treatments, the success rate of ART remains low. In an attempt to make ART safer and more efficient, international medical practice is trending towards single embryo transfers and the use of innovative, sophisticated technologies to identify promising gametes and embryos with the highest potential to generate a pregnancy. Laser technology is increasingly being used to accomplish these aims. The application of lasers for ART has been successfully employed in clinical practice for some time now and is continually the subject of investigative research in order to generate new methods to improve operations. Moreover, lasers serve as a powerful tool at the forefront of investigative research in the reproductive sciences, assisting in broadening our understanding of reproductive and developmental biology. Nevertheless, there is a paucity of literature pertaining to the safe standardisation of such laser procedures with evidence at the molecular level. The primary aim of this thesis was to optimise applications of laser technology for clinical ART and research applications in the reproductive sciences. This thesis utilised the mouse embryo model to investigate potential deleterious effects of different laser treatment applications, both by the operator and hardware manufacturer. Safe and unsafe laser operator parameters were elucidated by assessing deleterious effects to the plasma membrane integrity, blastocyst survival rate, DNA fragmentation levels, and changes in gene expression of key developmental genes. The effect of altering the laser hardware to lower the power output was evaluated and it was determined that if a lower power laser is used to deliver a set amount of energy over a longer period of time, a smaller amount of damage is incurred. Work in this thesis also established a new method in which laser technology can be used as a research tool for the reproductive sciences, by creating a novel stimuli-responsive laser-activated nanoparticle delivery system with spatial control and increased efficiency in a mammalian cell model. The field of reproductive science continues to benefit greatly from laser application clinically to improve infertility treatments, and in research, to elucidate mechanisms underlying infertility, with a hope of increasing our understanding and eventually developing new treatment options.

AN ABSTRACT OF THE DISSERTATION OF AKILAH MORRIS SMITH, for the Doctor of Philosophy degree in Public Health, presented on April 11th 2018, at Southern Illinois University Carbondale. TITLE: KNOWLEDGE, INTENTIONS, AND BELIEFS ABOUT FERTILITY AND ASSISTED REPRODUCTIVE TECHNOLOGY AMONG ILLINOIS COLLEGE STUDENTS MAJOR PROFESSOR: Roberta Ogletree H.S.D and Juliane P. Wallace PhD The purpose of this quantitative cross sectional study was to examine knowledge, beliefs, and intentions about fertility and assisted reproductive technology among college students. This study differs from previous studies in that it examines knowledge, beliefs, and intentions about fertility and assisted reproductive technology among Illinois college students. Five hundred thirty six undergraduate students from six Illinois universities taking foundational health courses participated in this convenience sample study. Participants included three hundred and five females, 225 males, and five transgender students. The age ranges from 18-60. Five hundred and twenty students were childless. Three hundred and eleven students were single, 195 were in a committed relationship, and 16 were married. An Analysis of Variance (ANOVA) detected the differences among college students’ knowledge, beliefs, and intentions, based on race, sexual orientation, age, parental status, relationship status, and gender. Additionally Multiple Linear Regression analysis determined variations among race, sexual orientation, age, parental status, relationship status, and gender based on intentions, beliefs, and knowledge of fertility and ART treatment options. The first findings indicate that age, race and relationship status variables strongly impacted fertility intentions. The second findings reveal that gender and race impact beliefs influencing fertility and ART treatment options. None of the six variables significantly affected knowledge, which does not correlate with the literature. The students replied that they were not informed about women’s fertility as well as ART treatment options. Caucasians and older students’ intended on delaying parenthood supports the current literatures. According to Martinez, Daniels, and Chandra (2012), Caucasians are more likely to delay parenthood, which this research study supports. Secondly, Caucasians and males students had beliefs that supported the delaying of parenthood. Amongst the six groups, none of the groups affected knowledge. Daniluk and Koert (2012) show that while college student’s lack knowledge researchers are not sure what strongly predict their fertility and ART knowledge treatments.

La mobilité est une fonction clé de la qualité fécondante et de sélection des spermatozoïdes des techniques d’assistance médicale à la procréation (AMP). Nous manquons cependant d'information des mécanismes moléculaires contrôlant cette mobilité. Ce travail de thèse est articulé autour de 2 protéines d’intérêts impliquées dans la mobilité spermatique: la protéine Tau (Tubule-associated unit) associée à la polymérisation des microtubules dans le neurone, et la protéine d’ancrage aux kinases A4 (AKAP4), protéine majeure de la gaine fibreuse du flagelle spermatique, connue pour son implication dans la mobilité spermatique.Très peu d’études traitent de la protéine Tau dans l’appareil génital masculin, et aucune chez l’homme. Dans une première partie de ce travail, nous avons analysé l’expression de la protéine Tau dans le spermatozoïde et le testicule humain par une approche immuno-histo-chimique (article 1). La protéine Tau est localisée dans la pièce intermédiaire du flagelle du spermatozoïde, et dans le spermatocyte et la spermatide dans le testicule. Les rôles potentiels de la protéine Tau durant la spermatogenèse sont discutés dans une revue de la littérature (article 2).Les avancées dans le domaine de la protéomique permettent aujourd’hui d’étudier le protéome du spermatozoïde et de ses compartiments cellulaires de façon hautement résolutive. Le protéome global du spermatozoïde a été étudié chez des hommes consultant le centre de procréation assistée du CHRU de Lille. Il a permis de définir un groupe de spermatozoïdes présentant majoritairement des protéines de haut poids moléculaire et un groupe présentant une protéolyse aux dépends des protéines de haut poids moléculaire. L’AKAP4 a été identifiée parmi les protéines de hauts poids moléculaire. Nous avons étudié, quantifié le profil d’expression de l’AKAP4 en Western Blot dans le sperme d’hommes venant effectuer un spermogramme et corrélé les données biochimiques du protéome et de l’AKAP4 au spermogramme (article en soumission 3). Le rôle de l’AKAP4 comme marqueur prédictif en AMP est ensuite abordé. Les données clinico-biologiques (logiciel INFOFIV) des tentatives d’AMP au CHRU de Lille ont été confrontées aux données quantitatives du protéome global et de l’AKAP4 (article en préparation 4).Les protéines Tau et AKAP4 sont des protéines d’intérêts dans la prise en charge de l’homme infertile. L’AKAP4 pourrait être proposée comme biomarqueur dans la prise en charge en AMP. / Motility is a key function of the fertilizing quality and the selection of sperm for assisted reproductive technology (ART). However, we lack information on molecular mechanisms controlling this mobility. This thesis is structured around 2 proteins of interest involved in sperm mobility: Tau (tubule-associated unit) associated with microtubule polymerization in the neuron, and the A-kinase anchoring protein 4 (AKAP4), the main protein of the fibrous sheath of the sperm’s flagellum, known for its involvement in sperm motility.Very few studies focus on the Tau protein in the male reproductive system, and none in humans. In the first part of this work, we analysed the expression of the Tau protein in the sperm and human testis through an immuno-histo-chemical approach (Article 1). The Tau protein is localized in the intermediate part of the sperm flagellum and in the spermatocyte and spermatid in the testis. The potential roles of the Tau protein during spermatogenesis are discussed in a review of the literature (Article 2).Advances in the field of proteomics now allow the study the proteome of sperm and its cellular compartments in a highly-resolutive way. The global sperm proteome was studied in men visiting the assisted reproduction center of Lille University Hospital. This has led to a group of sperm with predominantly high molecular weight proteins being identified as well as a group having proteolysis at the expense of high molecular weight proteins. The AKAP4 was identified among the high molecular weight proteins. We studied, quantified the profile of AKAP4 expression by Western Blot in the sperm of men undergoing a semen analysis and correlated the biochemical data of the proteome and AKAP4 to the semen analysis (article 3 in submission). The role of AKAP4 as a predictive marker of ART success is then discussed. Clinical and biological data (INFOFIV software) of ART attempts at the Lille University Hospital were compared with quantitative data of the global proteome and AKAP4 (Article 4 in preparation).Tau protein and AKAP4 are proteins of interest in the management of infertile men. The AKAP4 could be proposed as a biomarker in ART treatments.

Micromotors – untethered, motile, microscopic devices – are implemented in this dissertation for two applications in the field of assisted reproductive technology. First, as synthetic motor units for individual sperm cells, representing a novel approach to counteract sperm immotility (asthenozoospermia), which is one of the most prevalent causes of male infertility. Second, as synthetic carriers of fertilized oocytes (zygotes) towards the realization of non-invasive intrafallopian transfer, representing a novel alternative to the current keyhole surgery (laparoscopy) approach to achieve early embryo transfer after in vitro fertilization. In both applications, magnetically actuated micromotors are utilized to capture, transport, and deliver individual cells in a reproducible, controllable manner. In comparison with established in vitro fertilization routines, the crucial advantage of employing micromotors for the manipulation of gametes, i.e. sperm and (fertilized) oocytes, lies in the potential transfer of decisive steps of the fertilization process back to its natural environment – the fallopian tube of the female patient – taking advantage of the untethered, non-invasive motion and manipulation capabilities of magnetic micromotors. When sperm motility can be restored with magnetic micromotors, sperm can travel to the oocyte under external actuation and control, and the oocyte does not need to be explanted for in vitro fertilization. However, if in vitro fertilization was necessary, fertilized oocytes can be transferred back to the fallopian tube by micromotors in a non-invasive manner, to undergo early embryo development in the natural environment. These novel concepts of micromotor-assisted reproduction are presented and investigated in this thesis, and their potential is analyzed on the basis of proof-of-concept experiments.:1 Introduction 6 1.1 Background and Motivation 6 1.2 Objectives and Structure of this Dissertation 9 2 Fundamentals 11 2.1 Micromotors Definition and Concept 11 2.2 Micromotors for Biomedical Applications 13 2.3 Magnetic Micropropellers 15 2.3.1 Theory 15 2.3.2 Implementation 20 2.4 Microfabrication: Direct Laser Writing 21 2.5 Assisted Reproductive Technology 23 2.5.1 In vitro Fertilization and Intracytoplasmic Sperm Injection 24 2.5.2 Embryo Transfer and Zygote Intrafallopian Transfer 25 2.5.3 The Sperm Cell and the Oocyte 26 2.6 Towards Micromotor-Assisted Reproduction 28 3 Materials and Methods 30 3.1 Fabrication of Microfluidic Channel Platforms 30 3.1.1 Tailored Parafilm Channels 30 3.1.2 Polymer Channels Cast from Micromolds 31 3.1.3 Tubular Channels to Mimic In vivo Ducts 32 3.2 Fabrication of Magnetic Micropropellers 32 3.2.1 Direct Laser Writing of Polymeric Resin 33 3.2.1.1 Design and Programming 33 3.2.1.2 Exposure and Development 35 3.2.1.3 In Situ Direct Laser Writing 35 3.2.2 Critical Point Drying 35 3.2.3 Magnetic Metal Coatings 36 3.2.4 Surface Functionalization 37 3.3 Sample Characterization 38 3.3.1 Optical Microscopy 38 3.3.2 Scanning Electron Microscopy 38 3.4 Cell Culture and Analysis 39 3.4.1 Sperm Cells 39 3.4.2 Oocytes 39 3.4.3 In vitro Fertilization 41 3.4.4 Hypoosmotic Swelling Test 44 3.4.5 Cell Viability Assays 44 3.5 Magnetic Actuation 45 3.5.1 Modified Helmholtz Coil Setup 46 3.5.2 MiniMag Setup 47 3.5.3 Experimental Procedure 48 3.5.3.1 Micromotor Performance Evaluation 48 3.5.3.2 Cell Transport Experiments 49 3.5.3.3 Cell Transfer Experiments 50 4 Micromotor-assisted Sperm Delivery 51 4.1 Micromotor Design and Fabrication 51 4.2 Actuation and Propulsion Performance 53 4.3 Capture, Transport, and Release of Sperm 56 4.4 Delivery to the Oocyte 59 4.5 Sperm Viability and the Ability to Fertilize 61 5 Micromotor-assisted Zygote Transfer 68 5.1 Micromotor Design and Fabrication 68 5.2 Actuation and Propulsion Performance 70 5.3 Capture, Transport, and Release of Zygotes 76 5.4 Transfer between Separate Environments 80 5.5 Zygote Viability and Further Development 82 6 Conclusions and Prospects 85 Appendix 87 Bibliography 93 List of Figures and Tables 108 List of Abbreviations and Terms 109 Theses 111 Selbstständigkeitserklärung 112 Acknowledgments 113 List of Publications 115 Curriculum Vitae 116

Em nossa sociedade, a relação heterossexual ainda parece ser a única possibilidade legitimada para formação de um casal ou até mesmo de uma família. Porém, é cada vez maior o número de pessoas que desafia os discursos normativos presentes e busca a constituição de parcerias afetivo-sexuais com outras de seu próprio sexo, muitas vezes associando essas parcerias à experiência da parentalidade, seja com filhos biológicos ou adotivos. Com as crescentes discussões sobre os direitos sexuais reprodutivos e com o surgimento de novos arranjos familiares, entre eles o formado por casais homossexuais, começa-se a desconstruir o modelo ideal de família nuclear e abre-se caminho para discussão de temas como a maternidade lésbica. Este trabalho pretende contribuir com o debate da homoparentalidade, procurando demonstrar as especificidades existentes entre essas mulheres e suas formas de construir sua cidadania íntima dentro do contexto heteronormativo da sociedade brasileira. Para tanto, foi traçado o seguinte objetivo geral: compreender as concepções sobre a parentalidade de mulheres lésbicas que buscam a gravidez por meio de doadores de sêmen, sejam eles conhecidos ou desconhecidos. O estudo proposto baseia-se nos pressupostos da pesquisa qualitativa, como forma de privilegiar os discursos dos sujeitos como fonte de informação. Doze mulheres lésbicas aceitaram participar do estudo e foram entrevistadas entre os anos de 2009 e 2011. Os dados foram transcritos, organizados e analisados. A partir dos resultados, foi possível perceber que a vivência da maternidade por parte das mulheres lésbicas depende de fatores diversos como o histórico-cultural, o social, o jurídico-legal, o econômico e os relacionados às políticas públicas, além, é claro, da história de vida de cada uma dessas mulheres. Desta forma, para a mulher assumir a homossexualidade em uma sociedade heteronormativa e, ao mesmo tempo, optar pela maternidade, é necessário percorrer um árduo caminho, onde uma das saídas parece ser a luta pela cidadania plena e consolidação dos direitos humanos. Isto aponta para a importância de se abordar o tema em estudos e discussões acadêmicas com outras esferas da política pública e da vida social, incluindo a saúde pública / In our society, the heterosexual relationship still appears to be the only legitimate form to be a couple or to be a family. However, an increasing number of people who challenge the normative discourse are seeking for same-sex partnerships, often associating these partnerships to the experience of parenting, with biological or adoptive children. The increasing discussions about reproductive and sexual rights and the emergence of new family arrangements, including the one formed by homosexual couples, began to deconstruct the ideal model of nuclear family and its opens up the way for new discussions such as lesbian motherhood. This study intend to contribute to the homoparenthood debate, by demonstrating the specificities between these women and their ways to construct an intimate citizenship within the context of Brazilian heternormative society. To do so, the following overall aim was: to comprehend the parenthood concepts of lesbian women who seek pregnancy through known or unknown semen donor. The proposed study is based on the assumptions of qualitative research, which means that the subjects discourse was the source of information. Twelve lesbians were interviewed between the years 2009 and 2011. The data were transcribed, organized and analyzed. From the results, it was revealed that the motherhood experience by lesbians depends on several factors such as historical, cultural, social, juridical, legal, economic, public policies, and, of course, the personal history of each of these women. Thus, for women who come out as a lesbian in a heteronormative society and at the same time, opt for motherhood, they have a hard road to face. The solution seems to be to struggle for citizenship and human rights consolidation. So, its important working up this issue in academic studies and to discuss with other spheres of public policy and social life, including public health