O presente trabalho foi composto de dois estudos distintos. O Estudo I, com um foco conservacionista, teve como objetivo a avaliação e comparação de diferentes métodos de criopreservação de sêmen de bugio-preto (Alouatta caraya). Para tanto, o estudo foi dividido em dois experimentos: Experimento I composto de dois ensaios no primeiro ensaio foram comparados dois diluidores comerciais BotuBOV e Test-yolk buffer (TYB) e no segundo ensaio foram comparados dois métodos de criopreservação de sêmen congelação lenta e vitrificação; Experimento II avaliação dos efeitos da adição de DHA e de Trolox (análogo da vitamina E) ao diluidor para criopreservação de sêmen. O diluidor TYB apresentou melhores resultados quando comparados ao BotuBOV. A congelação lenta apresentou melhores resultados quando comparada à vitrificação. Não houve diferença entre o diluidor controle e os diluidores com Trolox, DHA ou combinação dos dois (DHAT), com exceção da integridade de acrossoma, que foi significativamente menor para o diluidor DHAT. Conclui-se que são necessários mais estudos, com utilização de outras doses de DHA e Trolox, além de outros antioxidantes. O Estudo II, com foco em pesquisa biomédica, teve como objetivo a criopreservação de sêmen de macaco-rhesus (Macaca mulata) e avaliação da qualidade pós-descongelação por meio de fecundação in vitro (FIV). Para tanto, o estudo foi dividido em três experimentos: Experimento I avaliação e comparação de dois métodos de criopreservação de sêmen congelação lenta e vitrificação; Experimento II avaliação e comparação de quatro métodos de preparação do sêmen pós-descongelação lavagem simples (LS), swim-up (SU), separação por gradiente de densidade (SGD) e filtragem em lã de vidro (FLV); Experimento III avaliação da qualidade seminal pós-descongelação por meio de FIV. A congelação lenta apresentou melhores resultados que a vitrificação (p<0,05). LS apresentou os melhores resultados, seguido por SGD e SU, enquanto FLV apresentou os piores resultados. LS e SGD foram utilizados para avaliação da qualidade seminal por meio de FIV, utilizando sêmen fresco como controle. As taxas de fecundação (média±EPM%) para oócitos MI inseminados com sêmen fresco (43.5±16.4) foram significativamente maiores (p<0,05) que LS (2.0±2.0), mas não diferiram de SGD (25.1±14.2). Não houve diferença na taxa de blastocistos (média±EPM%) entre os tratamentos (variação de 0 a 11.9±7.9). As taxas de fecundação para oócitos MII inseminados com sêmen fresco (41.4±3.6) também foram significativamente maiores que SGD e LS (18.4±6.9 e 12.7±7.7, respectivamente), assim como a taxa de blastocistos (64.7±13.6; 4.7±4.7; 30.9±13.8, respectivamente). Conclui-se que, espermatozoides criopreservados foram capazes de fertilizar oócitos e os embriões atingiram o estágio de blastocisto. A SGD selecionou espermatozoides pós-descongelação de melhor qualidade para FIV em macacos-rhesus, quando comparada à LS / This work was divided in two studies. The objective of Study I was to test and compare different cryopreservation methods for sperm from black-and-gold howler monkeys (Alouatta caraya), with a focus on species conservation. The study was divided in two experiments. Experiment I composed of two trials the first trial compared two commercial extenders BotuBOV and Test-yolk buffer (TYB), and the second trial compared two cryopreservation methods slow freezing and vitrification; Experiment II evaluation of the effects of DHA and Trolox (vitamin E analog) as additives to the freezing extender. TYB had better results when compared to BotuBOV. Slow freezing had better results when compared to vitrification. There was no difference between control extender (TYB) and extender containing Trolox, DHA or a combination of both (DHAT), except for acrosome integrity, which was significantly lower for DHAT. In conclusion, more studies are necessary, using other doses of DHA and Trolox, as well as other antioxidants. The objective of Study II was to assess the quality of frozen-thawed sperm from rhesus macaques (Macaca mulatta) by in vitro fertilization (IVF). The study was divided in three experiments. Experiment I evaluation and comparison of two cryopreservation methods slow freezing and vitrification; Experiment II evaluation and comparison of four preparation methods for frozen-thawed sperm simple wash (SW), swim-up (SU), density gradient centrifugation (DGC), and glass wool filtration (GWF); and Experiment III evaluation of frozen-thawed sperm quality by IVF. Slow freezing had better results when compared to vitrification (p<0,05). SW had better results, followed by DGC and SU, while GWF had the worse results. SW and DGC were further evaluated by IVF. Fertilization rates (mean±SEM%) with MI oocytes using fresh sperm were significantly higher (43.5±16.4) than with SW (2.0±2.0) and did not differ from DGC (25.1±14.2). There was no difference in blastocyst rates between treatments (range 0 to 11.9±7.9). Fertilization rates with MII ova were also significantly higher with fresh sperm (41.4±3.6) than DGC and SW (18.4±6.9 and 12.7±7.7, respectively), and more blastocysts developed from MIIs fertilized with fresh sperm (64.7±13.6) than SW and DGC (4.7±4.7 and 30.9±13.8, respectively). In conclusion, frozen-thawed sperm were able to fertilize oocytes and embryos reached the blastocyst stage. DGC yielded better frozen-thawed sperm for IVF in rhesus macaques, when compared with SW

Submitted by Sandro Camargo (sandro.camargo@unipampa.edu.br) on 2015-03-08T18:29:37Z No. of bitstreams: 1 117110033.pdf: 882213 bytes, checksum: 12c52b9fad305c8e13e54eeab7fca3ac (MD5) / Made available in DSpace on 2015-03-08T18:29:38Z (GMT). No. of bitstreams: 1 117110033.pdf: 882213 bytes, checksum: 12c52b9fad305c8e13e54eeab7fca3ac (MD5) Previous issue date: 2013-04-26 / A centrifugação por gradiente de Percoll ® é o método mais utilizado para a seleção de sêmen bovino para a fecundação in vitro (FIV), no entanto, até o momento não existe nenhum estudo que indique uma força de centrifugação capaz de remover com sucesso o plasma seminal e outros constituintes, sem causar perdas na recuperação espermática ou danos funcionais aos espermatozoides obtidos no pellet. O presente estudo foi desenvolvido para verificar o efeito de diferentes forças de centrifugação na seleção espermática por gradientes de Percoll ® , através de avaliação de parâmetros de qualidade espermática e posterior desenvolvimento e qualidade de embriões bovinos produzidos in vitro (PIV). Parâmetros de estresse oxidativo também foram avaliados para verificar uma possível relação com os achados na avaliação de esperma. No experimento 1, amostras de sêmen de quatro touros foram homogeneizadas e submetidas a centrifugação em gradientes descontínuo de Percoll ® (30,60 e 90%) em diferentes forças: F1 (9000 X g), F2 (6500 X g), F3 (4500 X g) e F4 (2200 X g). No Experimento 2, as amostras de sêmen de cada touro foram processadas individualmente e centrifugadas em: F1 (9000 X g) e F4 (2200 X g). Todas as amostras espermáticas foram avaliadas quanto a concentração, motilidade, vigor, morfologia, espécies reativas de oxigênio (EROs) e integridade da membrana plasmática, sendo no experimento 2 avaliados ainda a peroxidação lipídica, defesas antioxidantes e desenvolvimento embrionário. Não foi observada diferença na concentração espermática nos sêmens submetidos a diferentes forças centrifugação. No Experimento 1, a percentagem de espermatozoides móveis foi superior (p <0,05) após centrifugação em F3 e F4 e a produção de EROs de F1 foi superior (p <0,05) em comparação com outras forças. No Experimento 2, quando o sêmen de cada touro foi processado individualmente, não foram observadas diferenças significativas nos parâmetros de qualidade espermática, peroxidação lipídica, defesas antioxidantes, taxa de clivagem e tempo médio da primeira clivagem entre F1 e F4. No entanto, o aumento da força de centrifugação reduziu a taxa de penetração e a fertilização normal (P<0,05). Este trabalho demonstrou pela primeira vez que a força de centrifugação de 2200 X g aumentou a penetração e a taxa de fecundação em relação à força de centrifugação habitual (9000 X g) utilizadas na separação de espermatozoides por gradientes descontínuos Percoll ® em bovinos. Estes resultados sugerem que esta força de centrifugação pode ser utilizada com sucesso na PIV de embriões bovinos, uma vez que não reduz a recuperação de espermatozoides e aumenta a taxa de fecundação. / Centrifugation by Percoll® gradient is the most widely used method in the preparation of bull sperm for the purpose of in vitro fertilization, however at the moment, no scientific study determined the best centrifugation speed to remove seminal plasma and other constituents, i.e. the speed at which the loss of sperm cells is minimized and where the spermatozoa in the pellet still remain functional. The present study was designed to examine the efficiency of different centrifugation forces in sperm separation Percoll by methods evaluating sperm quality parameters and subsequent development and quality of bovine in vitro production (IVP) embryos. Additionally, we evaluated oxidative stress parameters to verify a possible relationship with the findings in the sperm evaluation. In Experiment 1, the semen samples from each bull were pooled and submitted to centrifugation in discontinuous gradients Percoll (30,60 and 90%) at different forces: F1 (9000 X g), F2 (6500 X g), F3 (4500 X g) and F4 (2200 X g). In Experiment 2, the semen samples from each bull were done separately and submitted to: F1 (9000 X g) and F4 (2200 X g). All sperm samples were evaluated to the concentration, motility, vigor, morphology, reactive oxygen species (ROS) and integrity of the plasma membrane, and in experiment 2 also were evaluated lipid peroxidation, antioxidants assays and embryo development. No difference was observed in the concentration of sperm submitted to different centrifugation forces. In Experiment 1, the total percentage of motile sperm was increased (p < 0.05) after centrifugation in F3 and F4 and the ROS production to F1 was superior (p < 0.05) compared to other forces. In Experiment 2, when the bull semen was processed individually, no significant difference was observed in the sperm quality parameters assessment, lipid peroxidation, antioxidants assays, cleavage rate and average time of the first cleavage between F1 and F4, sires or interaction between them after Percoll. However, the increased force centrifugation reduced the rate of penetration and normal fertilization (P < 0.05). This work demonstrated for the first time that 2200 X g centrifugation force enhanced the penetration and fertilization rates in relation to usual centrifugation force (9000 X g) using sperm separation by discontinuous Percoll gradients in bovine. These findings suggest that this centrifugation force could be used with successful in the bovine IVP embryo since it does not reduce sperm recuperation and increases the fecundation rate.

One out of every 8 couples experiences infertility. Few publications exist examining the association between the addition of adjunctive therapies when applied to the in vitro fertilization (IVF) procedure and pregnancy outcome. The purpose of this study was to compare the effectiveness of 2 types of adjunct acupuncture exposures on IVF outcomes by applying the concept of the epidemiology triad. The association between the type of adjunct acupuncture exposure received (the independent variable) and pregnancy (the outcome) was determined via binary logistic regression using SPSS software. Medical records of women having received the IVF procedure along with adjunct acupuncture were eligible for analysis. A total of 444 qualifying patient records were collected from participating acupuncture clinics. Analysis of the data showed there was no significant difference in pregnancy outcomes between the two types of adjunct acupuncture reviewed. However, an increase in pregnancy outcomes was noted in the adjunct acupuncture groups when compared with pregnancy outcomes in IVF protocols without the addition of adjunct acupuncture. Additionally, there was no significant difference between pregnancy outcomes when accounting for various race or age groups. This study has implications for positive social change in the form of both providing data to women undergoing the IVF procedure to allow for more informed decision making and ultimately with hopes of improving the odds of success to the nearly 165,000 IVF procedures undergone in America each year.

The psychological impacts of infertility have been well documented in the literature, providing evidence to support that at least some women who confront infertility are at risk for heightened distress and depressive symptoms. In response to this accumulated evidence, it has been argued that psychoeducational interventions may provide an important component to the treatment of infertility. While several theoretical models postulate the effects of stress on infertility treatment outcomes, results of these investigations have led to conflicting conclusions. However, a synthesis of the accumulated data examining the effects of stress on ART treatment outcomes was nonexistent until the conduct of this study. Therefore, the purpose of this study was to investigate the impact of stress on ART treatment outcomes and to determine whether psychoeducational interventions mitigate the impact of stress experienced by women during an ART treatment program. Two hypotheses were tested: 1. Increased levels of stress will be associated with a lower likelihood of Assisted Reproductive Technology (ART) treatment success, and 2. Psychoeducational interventions will mitigate the effects of stress experienced during Assisted Reproductive Technology (ART) treatment. A meta-analysis analyzing the results for each hypothesis was tested through a hierarchical linear regression model. A total of 13 studies, representing 43 effect sizes, were included in the analysis investigating the relationship between stress and ART treatment outcomes. Results of the HLM regression model suggest that stress has a small negative association with ART treatment outcomes (d=0.2012, p< .05). The analysis investigating the relationship between psychoeducational interventions and stress included a total of 4 studies, representing 12 effect sizes. Empirical evidence gathered through this analysis revealed that the effect of psychoeducational interventions on the stress experienced by women participating in an ART treatment program were not statistically significant (d=0.3071, p>.05). However, because this analysis was based on such a small sample of studies, generalizations regarding the efficacy of psychoeducational interventions cannot be made. Therefore, research aimed at investigating the impacts of a variety of programs should continue in an effort to provide more conclusive information.

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.

Pre-pubertal pig oocytes possess lower developmental competence than those from adult pigs following in vitro maturation (IVM). Previous studies have demonstrated that exposure of pre-pubertal oocytes to 1 mM dibutyryl cAMP (dbcAMP), a membrane permeable cyclic adenosine monophosphate (cAMP) analogue, for the first 20 h of IVM improves the rate of blastocyst development. Developmental competence of in vitro matured pig oocytes has been reported to increase with increasing follicle size. In this thesis, experiments were carried out using pre-pubertal and adult pig oocytes to investigate the relationship between donor age, intra-oocyte cAMP level and follicle size in terms of oocyte maturation and developmental competence. These experiments demonstrated that, while ovarian, follicular and oocyte morphology are immediately altered with the onset of puberty, pre-pubertal oocytes must be exposed to more than the first oestrous cycle to achieve improved developmental competence in vitro. Later experiments demonstrated that pre-pubertal oocytes accumulate less cAMP during IVM, undergo more rapid meiotic progression and display reduced rates of blastocyst development compared to in vitro matured adult oocytes. Treatment with dbcAMP for 22 h IVM increased the cAMP content of pre-pubertal oocytes, slowed meiotic progression during IVM and improved the rate of blastocyst formation. While the cAMP concentration of pre-pubertal oocytes was increased to levels similar to that of adult oocytes, rates of blastocyst formation remained lower, suggesting that additional factor(s) are required for oocyte maturation. This thesis also examined the follicle size cohorts that make up the 3-8 mm aspiration range on pig ovaries. The surface of pre-pubertal ovaries contained around double the number of 3 mm follicles compared with adult ovaries. Blastocyst development of pre-pubertal oocytes increased with increasing follicle size and was highest using oocytes from 5-8 mm follicles, while adult oocytes from all follicle size cohorts displayed similar high rates of blastocyst formation. The interaction between follicle size and cAMP content in pre-pubertal oocytes was examined next. Cumulus-oocyte complexes (COCs) from 3 mm follicles accumulated less intra-oocyte and inter-COC cAMP and displayed reduced cumulus expansion compared with COCs from 5-8 mm follicles. While dbcAMP treatment increased the cAMP content of oocytes from 3 mm follicles, it had no effect on the cAMP content of the whole COC. These findings suggest that inadequate levels of intra-oocyte cAMP during IVM contribute to the low developmental competence of pre-pubertal oocytes from 3 mm follicles, suggesting that cAMP transfer, production or degradation processes are incomplete. Analysis of steroid content from different follicle size cohorts revealed that the progesterone content of prepubertal follicular fluid (FF) increased with increasing follicle size, yet overall was lower than that of adults. This suggests that differences may exist in the gonadotropinstimulated steroidogenic activity of granulosa cells of pre-pubertal COCs from different follicle sizes. Since progesterone secretion did not differ between pre-pubertal and adult COCs, it appears that the downstream pathway from the granulosa cell response rather than the actual quantity of progesterone is important for subsequent maturation processes. These studies then examined gap junction communication (GJC) within the pre-pubertal COC during IVM to examine whether the positive effects of increasing follicle size and dbcAMP on intra-oocyte cAMP levels relates to improved cAMP transfer between the cumulus cell layer and oocyte. Cumulus cell-oocyte GJC during IVM was maintained for a longer period in pre-pubertal COCs from 3 mm follicles than in those from 5-8 mm follicles. Treatment with dbcAMP had minimal effect on GJC in either COC type, thus the dbcAMP-induced increase in intra-oocyte cAMP levels appears independent of GJC. Differences in GJC during IVM together with the COCs ability to increase intraoocyte cAMP levels during IVM, suggests that differences may exist in the quantity of gonadotropin receptors, which are responsible for cAMP production, within the cumulus layer of COCs from 3 mm compared with 5-8 mm follicles. In conclusion, this thesis has demonstrated that an increase in intra-oocyte cAMP is necessary during maturation for completion and synchronisation of maturation and high developmental competence of the pig oocyte. Comparison of 3, 4 and 5-8 mm follicle sizes in the pre-pubertal pig, as described here, provides an excellent model for further investigation into the role of cAMP and the other factors required for co-ordination of oocyte nuclear and cytoplasmic maturation and subsequent embryo production. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1297309 / Thesis (Ph.D.) -- School of Paediatrics and Reproductive Health, 2007

Research on in vitro embryo production (IVP) in the equine is impeded by the limited availability of mature oocytes as the mare is mono ovulating and superovulation is still difficult (Dippert and Squires, 1994; Bezard et al., 1995; Alvarenga et al., 2001b). Despite recent improvement in IVM of equine oocytes, success rates of IVM in that species remain low in all culture media tested compared to other species (Goudet et al., 2000b). However, most studies have focused on the percentage of oocytes reaching the metaphase II stage (nuclear maturation) but few concentrated on the final oocyte competence as measured by its ability to develop into a blastocyst and further establish a pregnancy. Blastocyst production rate is influenced not only by culture environment but also by oocyte maturation conditions. Under in vitro culture conditions, oxidative modifications of cell components via increased ROS represent a major culture induced stress (Johnson and Nasr-Esfahani, 1994). Anti-oxidant systems can attenuate the deleterious effects of oxidative stress by scavenging ROS (Del Corso et al., 1994). Glutathione, a tripeptide thiol, is the major non-protein sulfydryl compound in mammalian cells that plays an important role in protecting the cell from oxidative damage (Meister and Tate, 1976; Meister and Anderson, 1983). It has been suggested that GSH content in oocytes may serve as a reservoir protecting the zygote and the early embryos from oxidative damage before genomic activation and de novo GSH synthesis occur (Furnus et al., 1998; de Matos and Furnus, 2000). The addition of GSH synthesis precursors, such as cysteamine, a thiol compound, to IVM media has been shown to improve IVP in various species (Takahashi et al., 1993; de Matos et al., 1995; Grupen et al., 1995; de Matos et al., 2002a; de Matos et al., 2002b; de Matos et al., 2003; Gasparrini et al., 2003; Oyamada and Fukui, 2004; Balasubramanian and Rho, 2007; Anand et al., 2008; Singhal et al., 2008; Zhou et al., 2008). Very little information on the use of thiol compounds in the equine is available. Conventional in vitro fertilization (IVF) has not been successful in the mare, and a repeatable IVF technique has not yet been developed (Alm et al., 2001). To overcome the limitation of conventional IVF procedures, other methods to produce embryos from oocytes, either in vivo or in vitro, have been investigated. Among these, intra cytoplasmic sperm injection (ICSI) has permitted efficient equine in vitro blastocyst production (Galli et al., 2002; Lazzari et al., 2002; Choi et al., 2006a; Choi et al., 2006c). However, ICSI requires specific equipment and skills. Transfer of an immature oocyte into the preovulatory follicle of an inseminated recipient mare (Intra-Follicular Oocyte Transfer, IFOT) has produced embryos but the success rate was low (Hinrichs and Digiorgio, 1991). Similarly, oocyte transfer (OT) into the oviduct of an inseminated recipient mare was investigated (McKinnon et al., 1988; Carnevale, 1996; Hinrichs et al., 1997; Carnevale et al., 2001; Carnevale et al., 2003; Carnevale, 2004), and commercial programs using OT for mares with reproductive abnormalities are now available (Carnevale et al., 2001). Unfortunately, IFOT is poorly documented in the literature and reports of OT have been published by various laboratories and under various conditions, making comparisons between results and choosing among these as substitutive techniques to ICSI or embryo transfer difficult. The first aim of the present work was to investigate if there is an influence of supplementation with 100 µM of cysteamine on conventional IVF success rate. Cumulus oocytes complexes (COCs) retrieved by transvaginal ultrasound guided aspiration were matured in vitro with or without cysteamine supplementation and were then submitted to conventional IVF using either calcium ionophore or heparin as capacitation treatment for spermatozoa. A total of 131 oocytes were evaluated for evidence of sperm penetration. Both techniques (ionophore or heparin) yielded 6% of IVF and results were similar both for the cysteamine and the control group. This success rate of IVF is low compared to some published data (Palmer et al., 1991; Dell'Aquila et al., 1996; McPartlin et al., 2009) but similar to what others reported in the literature (Choi et al., 1994; Dell'Aquila et al., 1997a). Although, it seems likely that cysteamine did not significantly improve IVF rates under our conditions, our general success rates for IVF procedures may be too low for us to conclude definitely about the effect of cysteamine. As ICSI was not available to us, the second aim of this work was to determine what in vivo technique could best bypass the lack of an efficient conventional IVF procedure. We compared embryo production following transfer of in vivo recovered oocytes (1) into a recipients oviduct or (2) into her preovulatory follicle either immediately after ovum pick up or (3) after in vitro maturation. Recipients were inseminated with fresh semen of a stallion with a known normal fertility. Ten days after transfer, rates of embryos collected in excess to the number of ovulations were calculated and compared for each group. Embryo collection rates were 32.5% (13/40), 5.5% (3/55) and 12.8% (6/47) for OT, post-IVM and immediate IFOT respectively. OT significantly yielded more embryos than immediate and post-IVM IFOT did. These results show that, when ICSI is not an option, intra-oviductal oocyte transfer is to be preferred to IFOT, as an in vivo alternative, to bypass the inadequacy of conventional in vitro fertilization and to assess oocyte developmental competence. After it was established that in comparison to IFOT, OT is the most reliable in vivo alternative to in vitro fertilization where ICSI technology is not available, this technique was used to assess the effect of cysteamine supplementation on nuclear maturation and oocyte competence. The third aim of this work was to investigate the influence of supplementation with 100 µM of cysteamine on in vitro nuclear and cytoplasmic maturation by specific DNA staining and the ability of oocytes to undergo in vivo fertilization after OT. Oocytes were collected by transvaginal ultrasound guided aspiration and matured in vitro with (cysteamine group) or without (control group) cysteamine. The nuclear stage after DNA Hoechst staining and the embryo yield following OT were used as a criterion for assessing nuclear and cytoplasmic maturation, respectively. Overall maturation rate was 52%, which is rates reported in the literature ranging from 40 to 70% in the equine (Goudet et al., 1997a; Bogh et al., 2002; Hinrichs et al., 2005; Galli et al., 2007). Nuclear maturation was not statistically different (p>0.05) between oocytes cultured with or without cysteamine (55% and 47% respectively). From 57 oocytes transferred to the oviduct in each group, the number of embryos collected was 10 (17%) in the control group and 5 in the cysteamine group (9%). Those two percentages were not statistically different (p>0.05). Contrary to the data described in other domestic species, there was no effect of cysteamine on in vitro nuclear maturation, or in vivo embryonic development under our conditions. Under our conditions, the addition of 100 µM of cysteamine to a classic culture medium does not improve equine oocyte maturation or embryonic development after OT. The same dose failed to increase GSH content in the equine (Luciano et al., 2006). However, the effect of cysteamine supplementation is highly species and concentration dependant. The inadequacy of the chosen concentration may explain that equine embryo production has not been increased by the cysteamine under our conditions as opposed to what has been observed in many other species. Alternatively, we can hypothesize that some substances present in the IVM medium can interfere with GSH synthesis. This has been suggested for FSH and estradiol (Bing et al., 2001) and, although our maturation medium is not supplemented with gonadotropins or estradiol, factors contained in fetal calf serum or EGF might also have an effect on GSH synthesis. Considering its beneficial effects in many other species, supplementation with cysteamine to different IVM media should be further investigated in the equine. Ideally combining different concentrations and ICSI or OT in order to determine an optimal concentration and its effects on oocyte developmental competence.

Psychological problems are common in infertile women undergoing in vitro fertilization (IVF) treatment.  The aim of this thesis was to determine the prevalence of psychiatric disorders, such as mood and anxiety disorders, and related risk factors and personality traits in women and men undergoing IVF. Participants were 1090 consecutive women and men, 545 couples, attending a fertility clinic in Sweden during a two-year period. The Primary Care Evaluation of Mental Disorders (PRIME-MD), based on the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV), was used for evaluating mood and anxiety disorders. The participation rate was 862 (79 %) subjects. Any psychiatric disorder was present in 31 % of females and in 10 % of males. Major depression was prevalent in 11 % of females and 5 % of males. Only 21 % of the subjects with a psychiatric disorder had some form of treatment. A negative pregnancy test and obesity (BMI ≥ 30) were risk factors for mood disorders in women and the only risk factor for depression in men was unexplained infertility. Anxiety disorders were less common than in the general population and no IVF-related risk factors were identified. The Swedish universities Scales of Personality (SSP), a self-rating questionnaire, was used for evaluation of personality traits. High scores of personality traits related to neuroticism were associated with mood and/or anxiety disorders among both women and men. Another objective was to explore the experience of childlessness three years after unsuccessful IVF by a qualitative-approach, assessing data by interviews. Failure after IVF was experienced by women in terms of grief, whereas men took upon themselves a supportive role not expressing grief. A need for professional support and counselling in how to handle grief was described. An unstructured end after IVF treatment left unanswered questions. Three years after the end of treatment, men and women were still processing and had not adapted to childlessness, indicating the grieving process was unresolved.

Är det möjligt att välja rätt embryo vid In-vitro fertilisering (IVF)? I sådana fall, vad innebär det att något är det rätta embryot? I denna uppsats diskuteras IVF och de moraliska dilemman som kan uppstå vid val av embryo. Vid IVF är det möjligt att ställa en preimplantorisk genetisk diagnostik (PGD) vilken kan ge information om genetiska sjukdomar och andra anlag, såväl som kön och kromosomfel. Enligt Julian Savulescu, som förespråkar the Principle of Procreative Beneficence, är vi moraliskt skyldiga att välja ett friskt embryo, vilket också anses vara det rätta embryot. I kontrast till detta ställs Christine Overall som menar att Savulescus princip innebär problem för hur vi bör se på barnafödande. Jag diskuterar vidare hur vi utifrån ett socialt och ett samhällsperspektiv kan se det som moraliskt tveksamt att förbjuda att personer med vissa anlag föds, samt försöker visa på Savulescus ignorans för hur IVF fungerar och att detta bidrar till att hans argument fallerar.

Psychological problems are common in infertile women undergoing in vitro fertilization (IVF) treatment.  The aim of this thesis was to determine the prevalence of psychiatric disorders, such as mood and anxiety disorders, and related risk factors and personality traits in women and men undergoing IVF. Participants were 1090 consecutive women and men, 545 couples, attending a fertility clinic in Sweden during a two-year period. The Primary Care Evaluation of Mental Disorders (PRIME-MD), based on the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV), was used for evaluating mood and anxiety disorders. The participation rate was 862 (79 %) subjects. Any psychiatric disorder was present in 31 % of females and in 10 % of males. Major depression was prevalent in 11 % of females and 5 % of males. Only 21 % of the subjects with a psychiatric disorder had some form of treatment. A negative pregnancy test and obesity (BMI ≥ 30) were risk factors for mood disorders in women and the only risk factor for depression in men was unexplained infertility. Anxiety disorders were less common than in the general population and no IVF-related risk factors were identified. The Swedish universities Scales of Personality (SSP), a self-rating questionnaire, was used for evaluation of personality traits. High scores of personality traits related to neuroticism were associated with mood and/or anxiety disorders among both women and men. Another objective was to explore the experience of childlessness three years after unsuccessful IVF by a qualitative-approach, assessing data by interviews. Failure after IVF was experienced by women in terms of grief, whereas men took upon themselves a supportive role not expressing grief. A need for professional support and counselling in how to handle grief was described. An unstructured end after IVF treatment left unanswered questions. Three years after the end of treatment, men and women were still processing and had not adapted to childlessness, indicating the grieving process was unresolved.