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.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

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.

Made available in DSpace on 2014-06-11T19:32:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-02Bitstream added on 2014-06-13T20:48:03Z : No. of bitstreams: 1 martinhago_cd_dr_botfm_prot.pdf: 1810275 bytes, checksum: 5592d92db2f6fd8dea862970f2f6df15 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Cpdp - Centro Paulista de Pesquisa e Diagnostico / O diagnóstico genético pré-implantacional (PGD) é um procedimento o qual permite que embriões sejam testados perante uma doença genética antes de sua transferência para o útero materno, ou seja, antes... / Preimplantation genetic diagnosis (PGD) is a procedure that permits embryos to be tested for a possible genetic diseade before being transferred to the maternal uterus, i.e., before the beginning of pregnancy... (Complete abstract click electronic access below)

O arsênio inorgânico, um contaminante ambiental, produz uma série de respostas de estresse em células de mamíferos, incluindo o comprometimento da função mitocondrial, acompanhado por inibição do crescimento celular e carcinogênese. Como previamente identificamos efeitos deletérios do comprometimento da função mitocondrial e dos radicais livres do oxigênio na oogênese, investigamos os efeitos do arsenito na meiose, no desenvolvimento embrionário pré-implantação e na apoptose embrionária em camundongos. Camundongas com 6 semanas de idade foram tratadas com baixa (0,16 mg) ou média dose de arsenito (0,32 mg), por meio de 7 injeções intraperitoneais, 1 a cada 2 dias, durante 14 dias. Os controles foram injetados com solvente. A incidência de anomalias meióticas, caracterizadas por anormalidades do fuso celular e/ou mal alinhamento cromossômico, foi significantemente aumentada tanto nos oócitos in vivo ovulados, como nos in vitro maturados, oriundos dos animais tratados com arsenito. Foram detectadas reduções significativas das taxas de clivagem (24 horas de cultivo), de formação de mórula (72 h) e de desenvolvimento para blastocisto (96 h), nos embriões dos grupos tratados com arsenito. Apesar do número total de núcleos não ter diferido significativamente entre os blastocistos dos grupos controle e de tratamento, a percentagem de núcleos apoptóticos foi significantivamente maior nos blastocistos derivados dos animais tratados com a dose média de arsenito. Estes dados sugerem que o arsenito causa aberrações meióticas, que podem contribuir tanto para o comprometimento do desenvolvimento embrionário pré-implantação, como para a apoptose embrionária. / Inorganic arsenic, an environmental contaminant, produces a variety of stress responses in mammalian cells, including mitochondrial uncoupling accompanied by growth inhibition and carcinogenesis. Because previously we identified detrimental effects of mitochondrial uncoupling, and reactive oxygen species (ROS) on oogenesis, we investigated effects of arsenite on meiosis, early embryo development, and apoptosis in mice. Six-week-old CD-1 mice were treated with either low (0.16mg) or medium (0.32mg) doses of arsenite every two days by 7 intraperitoneal injections for 14 days, and controls were injected with solvent. The incidence of meiotic anomalies, characterized by spindle disruption and/or chromosomal misalignment or spreading, was significantly increased in both in vivo and in vitro treated oocytes. Further, we found a significant decrease in cleavage rates at 24h, morula formation at 72h, and development to blastocyst at 96h in treated groups. Although the total number of nuclei in developed blastocysts did not significantly differ between the treated and control groups, the percentage of apoptotic nuclei was significantly increased in blastocysts derived from the medium dose treated group. These data suggest that arsenite causes meiotic aberrations, which may contribute to decreased cleavage and preimplantation development, as well as increased apoptosis.

The purpose of this thesis is to query the legitimacy of offering preimplantation genetic diagnostic (PGD) testing against Down's syndrome on the basis of United Kingdom (UK) law and policies. I will argue that extending PGD testing for Down’s syndrome as a permissible use of this technology does not (straightforwardly) adhere with the Human Fertilisation and Embryology Authority (HFEA) Code of Practice's stated factors which are to be considered when assessing the appropriateness of PGD applications. Indeed, due consideration of the evidence given in the relevant literature about the capacities and quality of life possible for persons living with Down's syndrome would seriously call into question the validity of a positive judgment recommending PGD as a treatment service for Down's syndrome according to the current UK regulatory instruments. I end the thesis by considering why the HFEA's relatively recent decision to limit client access according to an exclusive list of "serious" and therefore "in principle" test-worthy genetic conditions—understood as legitimate applications for PGD—stands to entrench prejudice, stigma, social bias, and unfair discrimination against the disadvantaged social group of persons living with Down's syndrome.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: With the development and continued developing of medical technology, treatments become available without the time to reflect ethically on them. Given how fast things change in medical technology, it is important to constantly reflect anew. Ethical reflection, however, seems to be lagging far behind bio-technological developments. Pre-implantation Genetic Diagnosis (PGD) and Human Genetic Manipulation (GM) is fast becoming an everyday reality and must therefore be reflected upon. Few Christian bioethical studies have been done on the impact that this could have on the larger populace, especially the local population in South Africa, where only a small percentage would be able to access these possible treatments. This study is motivated by the quest of ethicists in general and Christian ethicists in particular, to respond adequately and appropriately to the challenges posed by bio-technological developments. The study will outline and discuss the various Christian perspectives on PGD and GM. It will be shown that most Christian responses to bio-technological matters are done from within the framework of the doctrine of creation. In response, this study will then discuss a trinitarian perspective on the confession of God as creator and investigate whether this perspective might advance and enrich, and even amend, the quests of Christians to formulate ethical responses to the challenges posed by PGD and GM. I have made the decision to focus, for the most part, only on the work of one theologian, and will therefore be applying the trinitarian doctrine of creation as found in the work of Jürgen Moltmann to the development of a Christian bioethical perspective. Seeing that Christian ethics in general is concerned with human dignity, social justice and wellbeing, as well as moral upliftment, the ethical implications of this type of medical technology in the South African context, with its uneven distribution of wealth and access to medical care, must also be addressed from the perspective of this study. In this regard, the concept of human beings created imago Dei (in the image of God), with inherent human dignity, is of particular importance. / AFRIKAANSE OPSOMMING: Met die ontwikkeling en voortdurende ontwikkeling van mediese tegnologie word behandelinge beskikbaar sonder dat daar tyd is om eties daaroor te reflekteer. Gegewe hoe vinnig dinge verander in mediese tegnologie is dit belangrik om voortdurend nuut te reflekteer. Pre-implantasie Genetiese Diagnose (PGD) en Menslike Genetiese Manipulasie (GM) is vinnig beter om ‘n alledaagse realiteit te word en daarom moet daar daaroor reflekteer word. Daar is min Christelike bio-etiese studies gedoen oor die impak wat dit op die groter samelewing kan hê, veral in die plaaslike bevolking van Suid-Afrika, waar slegs ‘n klein persentasie toegang tot hierdie moontlike behandelinge sal hê. Hierdie studie word gemotiveer deur die poging van etici in die algemeen en Christelike etici spesifiek, om behoorlik en toepaslik te reageer op die uitdagings wat bio-tegnologiese ontwikkelinge bied. Die studie sal die verskillende Christelike perspektiewe op PGD en GM uiteensit en bespreek. Daar sal aangedui word dat die meeste Christelike antwoorde op die bio-tegnologiese kwessies gedoen word binne die raamwerk van die skeppingsleer. In reaksie hierop sal hierdie studie dan 'n trinitariese perspektief op die belydenis van God as Skepper bespreek en ondersoek of hierdie perspektief die poging om ‘n Christelike etiese antword te formuleer op die uitdagings wat PGD en GM bied kan bevorder en verryk, en moontlik selfs wysig. Ek het die besluit geneem om hoofsaaklik net op die werk van een teoloog te fokus, en sal dus die trinitariese skeppingsleer soos gevind in die werk van Jürgen Moltmann toepas tot die ontwikkeling van 'n Christelike bio-etiese perspektief. Aangesien die Christelike etiek in die algemeen gemoeid is met menswaardigheid, maatskaplike geregtigheid en welstand, asook morele opheffing, moet die etiese implikasies van hierdie tipe mediese tegnologie in die Suid-Afrikaanse konteks, met sy ongelyke verspreiding van rykdom en toegang tot mediese sorg, ook aangespreek. In hierdie verband is die konsep van die mens geskep Imago Dei (na die beeld van God), met inherente menswaardigheid, van besondere belang.

La chromatine embryonnaire subit des changements nécessaires pour l’établissement d’un nouveau programme développemental. Ce travail a étudié l’organisation de l’hétérochromatine au cours du développement sous trois facettes. La première étant celle de d’hétérochromatine constitutive, à travers, l’établissement forcé de la marque H4K20me3 qui provoque un arrêt du développement préimplantatoire. Ce phénotype dépend spécifiquement de l’activité de la methyltransferase SUV4-20h2 et induit l’activation de la voie de signalisation ATR qui bloque la phase de réplication. En deuxième partie, l’hétérochromatine facultative a été le sujet d’une analyse de l’expression des protéines du complexe non-canonique PRC1 et de la modification H2AK119ub qui en résulte. Finalement, une analyse de la chromatine embryonnaire a été mise en place et a permis la détection des changements de niveau de compaction au cours du développement préimplantatoire. / Embryonic chromatin undergoes necessary changes to establish a new developmental program. This work has addressed the organization of heterochromatin in preimplantation embryos from three angles. The first part probed the absence of constitutive heterochromatin by forcing the establishment of the H4K20me3 mark which results in an embryonic arrest prior to the 2-cell stage. This phenotype is due to the specific histone methyl-transferase activity of SUV4-20h2 and is induced by ATR activation which blocks replication. In the second part, facultative heterochromatin was studied by analyzing the levels of several members of the non-canonical PRC1 complex as well as the resultant modification H2AK119ub. Finally, an analysis of the embryonic chromatin was set up and allowed for the measurement of changes in the chromatin openness during preimplantation development.

In vitro fertilization (IVF) is used to help infertile couples achieve a live birth. Clinical studies have suggested that multiple, consecutive cycles of IVF can increase live birth rate significantly. Others have documented improved live birth rates from the use of new laboratory techniques for preimplantation genetic screening (PGS). This genetic screening technique seeks to determine the ploidy of the embryo prior to implantation into the woman. To date, no study has examined the cost-effectiveness of using IVF in conjunction with PGS compared to that of IVF alone for 3 consecutive cycles in achieving a live birth. This study compared the incremental cost-effectiveness ratios (ICER) from each intervention arm based on the clinical probabilities for each outcome and this study was grounded in the protection motivation theory. Costs were obtained from secondary sources, such as the literature and government databases. The model was constructed using a decision-analytical approach that allowed for z test statistical analysis of the outcomes, where the ICER is the dependent variable and the independent variables are the 2 interventions. The robustness of the model was tested through univariate and probabilistic sensitivity analysis and stratified by age groups. The results showed that PGS with IVF was cost-effective for women aged under 40 and women aged 40-42, but not for women over 42. Based on a willingness-to-pay threshold of $100,000, IVF with PGS was the most cost-effective strategy in all age groups. The positive social change implication of this study is such that understanding the costs associated with a new technology to achieve a live birth is significant for society to help guide clinical treatment of these patients.