Introdução. A fertilização in vitro (FIV) é uma opção de tratamento para pacientes inférteis com endometriose (EDT), e a escolha dos embriões a serem transferidos é um passo fundamental para o sucesso do processo. A endometriose é capaz de interferir em todas as etapas do processo de FIV, a qual pode estar associada à foliculogênese anormal, a defeitos de implantação e à qualidade oocitária inferior, bem como a alterações na receptividade endometrial. Em estágios mais severos da doença, pode-se observar um importante substrato anatômico associado. O Escore de Graduação Embrionário (GES) é uma classificação baseada numa combinação da morfologia pronuclear, clivagem precoce e morfologia no terceiro dia após a fertilização que permite avaliar quais embriões terão maiores chances de implantação/gestação. Ainda não há, na literatura, relatos de comparação do GES com pacientes portadoras de endometriose. Objetivo. Determinar o escore de graduação embrionário médio entre as pacientes inférteis com endometriose submetidas à fertilização in vitro e comparar com pacientes inférteis sem endometriose. Métodos. Foram comparados, através de uma coorte prospectiva, 706 embriões (162 pacientes), sendo 472 embriões pertencentes às pacientes do grupo controle sem endometriose (n=109; infertilidade tubária ou por fator masculino) e 234 embriões de pacientes com endometriose (n=53). Todas as pacientes foram submetidas à fertilização in vitro, utilizando o protocolo de indução da ovulação com antagonista do GnRH. Todos os embriões foram transferidos no dia 3 após fertilização. O escore de graduação embrionário (GES) foi realizado através de avaliação de todos os embriões por 3 vezes, nos tempos: 16 – 18 horas, 25 – 27 horas e 64 – 67 horas, sempre pelo mesmo embriologista. Para pontuação do GES, avaliaram-se: citoplasma, morfologia pronuclear, fragmentação, alinhamento nucleolar, posição do corpo polar, número de blastômeros/morfologia e simetria. Consideramos escore médio a soma dos escores embrionários dividida pelo número de embriões obtidos; e o escore embrionário médio transferido como a soma dos GES dividido pelo número de embriões transferidos. Foi considerado estatisticamente significativo quando P < 0,05 utilizando-se os testes t Student e qui-quadrado. Resultados. Os grupos foram comparados quanto à idade, índice de massa corporal, perfil obstétrico (infertilidade primária, secundária, paridade, abortamentos) e perfil hormonal, não sendo encontrada diferença estatisticamente significativa. Apesar de o número de embriões transferidos ser maior em pacientes com endometriose quando comparadas ao grupo controle (2,38 ± 0,66 versus 2,15 ± 0,54, respectivamente; P=0,001), não se observou diferença no escore de graduação embrionário médio entre os grupos de estudo e controle (71 ± 19,8 versus 71,9 ± 23,5, respectivamente; P=0,881). Quanto às taxas de fertilização, houve semelhança entre os grupos, correspondendo a 61% nas pacientes com endometriose e 59% naquelas do grupo controle (P=0,511). Também não houve diferença estatisticamente significativa quando os grupos foram comparados quanto às taxas de implantação (21% versus 22%, respectivamente; P=0,989) e gestação (26,4% versus 28,4%, respectivamente; P=0,989). Para estes parâmetros de avaliação, os diferentes graus de severidade da endometriose não interferiram significativamente nos resultados. Conclusão. A presença de endometriose não impactou significativamente na qualidade embrionária. Da mesma forma, não foram observadas diferenças nos desfechos reprodutivos avaliados entre pacientes inférteis com e sem endometriose. / Objective. To determine embryo quality (Mean Graduated Embryo Score, MGES) in infertile patients with endometriosis submitted to in vitro fertilization (IVF-ET). Design. Prospective cohort. Setting. Private Human Reproduction Center. Patients. We compared 706 embryos (162 patients) divided in two groups: 472 embryos derived from patients without endometriosis (n=109, infertile patients with tubal or male infertility) and 234 embryos from patients in study group (n=53, infertile patients with endometriosis). Interventions. All patients were submitted to IVF using an estradiol-antagonist-recombinant FSH protocol for ovarian stimulation. MGES was performed evaluating all embryos three times: 16 – 18 hours, 25 – 27 hours and 64 – 67 hours, by the same embryologist. Embryo evaluation was performed according to the following parameters: fragmentation, nucleolar alignment, polar body apposition, blastomere number/morphology and symmetry. Main Outcome Measures. MGES scores, fertilization, implantation and pregnancy rates. Results. Groups were compared for age, body mass index (BMI), past ob/gyn characteristics (primary or secondary infertility, parity, abortions) and hormonal profile. Although the number of embryos transferred was greater in patients with endometriosis than in the control group (2.38 ± 0.66 versus 2.15 ± 0.54, respectively; P = 0.001), the MGES was similar in both groups (71 ± 19.8 versus 71.9 ± 23.5, respectively; P = 0.881). Likewise, fertilization rate was similar in groups, 61% in patients with endometriosis and 59% in control group (P=0,511). Moreover, no significant differences were found in implantation rates (21% versus 22%, respectively [P=0,989]) and in pregnancy rates (26,4% versus 28,4%, respectively [P=0,989]). The severity of endometriosis did not influence the results. Conclusions. Embryo quality measured by MGES was not influenced by endometriosis. Likewise, the reproductive outcomes evaluated were similar between infertile patients with and without endometriosis.

Submitted by (edna.saturno@ufrpe.br) on 2016-08-17T16:44:17Z No. of bitstreams: 1 Christiane Medeiros Florentino.pdf: 580695 bytes, checksum: fa516dd272059d73278f42fc85ddecd9 (MD5) / Made available in DSpace on 2016-08-17T16:44:17Z (GMT). No. of bitstreams: 1 Christiane Medeiros Florentino.pdf: 580695 bytes, checksum: fa516dd272059d73278f42fc85ddecd9 (MD5) Previous issue date: 2011-02-17 / The aim of the present study was to evaluate transfer efficiency in bovine embryos in vitro produced (IVP) in the Legally-defined Brazilian Amazon. In order to do so, two experiments were conducted. In Experiment I, embryos at different stages of development were inovulated in cows with different corpus luteum types, either in dry or rainy season. 5403 embryos transfers were evaluated, with IVP embryos at different stages of development. The oocytes were obtained from zebu females through vaginal aspiration guided by sonogram, matured, fertilized, and cultured in vitro. The embryos were inovulated in the stages of morula (MO), early blastocyst (BI), blastocyst (BL), expanded blastocyst (BX), blastocyst hatching (BN) and hatched blastocyst (EB) in cyclic crossbred cows. Corpus lutea were classified by ultrasonography in protruded (CL1:> 200mm, CL2: 160 to 200mm, CL3: 100 to 159mm) and included (CL <150 mm). 34.30% (1855) of the animals conceived, with significantly higher pregnancy rate of stage BX embryos (38.2%). The result of BX only did not differ significantly from that obtained in females inovuladas with embryos stage BN (35.9%) and BN did not differ MO, BI and BL (P>0.05). The analyses showed no correlation between corpus luteum size or type and the development of IVP embryos inovulated at different stages. Pregnancy rates did not differ for inovulations performed in dry and rain season, with 33.7% and 35.7% mean respectively (P>0.05). Experiment II aimed to evaluate optimal time for inovulation of bovine females with IVP embryos after seven days, as a function of estrus manifestation, either in dry or rainy season. Among 2234 inovulated receptors, Estrus manifestation was observed two days before (D-2) in 31 of the recipients, one day before (D-1) in 473, on the exact day (D-0) in 1.247, one day after (D+1) in vitro fertilization, in 472, respectively, and 11 of the animals revealed estrus behavior two days (D+2) after in vitro fertilization. 35.27% of all inoculated animals conceived. The pregnancy rate for D0 was 57.90%, , while for D-2, D-1, D+1 and D+2, such percentages were 0.37%, 22.34%, 19.02 and 0.37%, respectively. Considering pregnancy rates, no significant difference (P>0.05) was observed between D-1, D-0 and D+1, which also displayed no significant difference for synchronization to D-1, D-0 and D+1 in either seasons (P>0.05). The results obtained after embryo transfer with IVP from Amazon Region were similar to studies in other regions and not influenced by the season. These results show the need for continuous research in order to address oocyte development problems for the production of quality embryos, augmenting the knowledge in the area and optimizing the use of IVP, which will ultimately enable the dissemination of this technology, and increase livestock production efficiency. Therefore, it can be concluded that a synchronism of ± 1 day interval from estrus manifestation is optimal for the achievement of pregnancy through in vitro fertilization, and that in the Legally-defined Brazilian Amazon, the season is not a limiting factor for the maintenance of successful pregnancies. / om o objetivo de avaliar a eficiência da transferência de embriões bovinos produzidos in vitro (PIV) em fêmeas bovinas na região da Amazônia Legal, foram realizados dois experimentos. No experimento I, embriões em diferentes estádios de desenvolvimento foram inovulados em receptoras com corpos lúteos de diferentes tipos, nos períodos chuvoso e seco. Foram avaliadas 5403 transferências com embriões PIV em seis estádios de desenvolvimento. Os oócitos foram obtidos de fêmeas zebuínas, por aspiração vaginal guiada por ultrassonografia, maturados, fecundados e cultivados in vitro. Os embriões foram inovulados nos estádios de mórula (MO), blastocisto inicial (BI), blastocisto (BL), blastocisto expandido (BX), blastocisto em eclosão (BN) e blastocisto eclodido (BE), em receptoras mestiças cíclicas. Os corpos lúteos das receptoras foram classificados em protusos (CL1: >200mm, CL2: entre 160 a 200mm, CL3: entre 100 a 159mm) e inclusos (CL<150mm), por meio de ultrassonografia. Obteve-se 34,30% (1.855) de receptoras prenhes, com taxa de prenhez significativamente maior nos embriões em estádio de BX (38,2%). O resultado de BX apenas não diferiu significativamente do obtido em fêmeas inovuladas com embriões em estádio BN (35,9%) e BN não diferiu de MO, BI e BL (P>0,05). As análises demonstraram ausência de correlação entre o tamanho/tipo do corpo lúteo com desenvolvimento de embriões PIV inovulados nos diferentes estádios. Não foi evidenciada diferença estatística (P>0,05) entre períodos seco e chuvoso, com taxas de prenhez de 33,7% e 35,7%, respectivamente. No Experimento II, foi avaliado o tempo ótimo para inovulação de embriões com sete dias após PIV, em fêmeas bovinas, em função do momento do estro das receptoras, em períodos chuvoso e seco. Dentre as 2.234 receptoras bovinas inovuladas, 31 apresentaram estro dois dias antes (D-2) da fertilização in vitro, 473, um dia antes (D-1), 1.247, no mesmo dia (D0), 472 um dia após (D+1) e 11 receptoras apresentaram estro dois dias após (D+2) a fertilização in vitro. Das receptoras inovuladas, 35,27% apresentaram-se prenhes. Destas, 57,90% foram inovuladas no D0, enquanto que para os dias D-2, D-1, D+1 e D+2, os percentuais de fêmeas prenhes foram de 0,37%, 22,34%, 19,02% e 0,37%,respectivamente. Não foram evidenciadas diferenças estatisticamente significativas (P>0,05) entre as taxas de prenhez obtidas nos grupos de sincronização D-1, D-0 e D+1. Também não foram evidenciadas diferenças estatísticas (P>0,05) quanto à estação do ano em que foram realizadas as inovulações para sincronização D-1, D-0 e D+1. Os resultados de prenhez obtidos após inovulação com PIV na região da Amazônia Legal foram semelhantes aos estudos realizados em outras regiões, os quais demonstraram não haver influencia do período do ano sobre as inovulações. Estes resultados reforçam a necessidade de se continuar a pesquisa em busca da solução dos problemas acerca do desenvolvimento do oócito para produção de embriões de qualidade, ampliando o conhecimento e dessa forma otimizando a utilização da PIV, o que possibilitará a difusão desta tecnologia visando o aumento da produtividade da pecuária nacional. Conclui-se que a sincronia de até ± 1 dia é melhor para o estabelecimento da prenhez obtida a partir de embriões PIV e que na Amazônia Legal a estação do ano não é fator limitante para a manutenção da gestação destes embriões.

A maturação in vitro do oócito é um dos fatores limitantes na produção in vitro de embriões. In vivo, esta maturação é um processo altamente orquestrado no qual a meiose é retomada pela onda de gonadotrofina que antecede a ovulação e que induz à queda dos níveis de AMPc no oócito. No entanto, os oócitos aspirados ao serem retirados dos folículos ovarianos retomam espontaneamente a maturação comprometendo a competência de seu desenvolvimento. O AMPc é sintetizado pela adenilato ciclase (AC) e degradado pelas fosfodiesterases (PDE), existindo algumas relacionadas à degradação do AMPc e outras do GMPc. Sendo assim, a proposição deste trabalho foi averiguar a contribuição de diferentes isoformas de fosfodiesterases na retomada da meiose e nos níveis de GMPc, AMPc e ainda, determinar quando há manutenção de AMPc em níveis elevados observando sua influência na competência oocitária e ativação da MAPK. Para isso, os complexos cumulus-oócito (CCOs) foram maturados in vitro na ausência, presença ou associação de inibidores de PDEs-AMPc e GMPc específicas e FSHr. As amostras foram avaliadas em relação a: 1) taxa de maturação; 2) níveis intracelulares de AMPc e GMPc nos CCOs; 3) taxa de desenvolvimento de blastocistos ; 4) ativação da MAPK em oócitos e células do cumulus. Os resultados obtidos no primeiro experimento indiaram que o inibidor da PDE3 foi o mais eficaz (p&lt;0,05) em atrasar a retomada da meiose, às nove horas de maturação, porém, isolado ou em associação com o inibidor da PDE8, não foi capaz de alterar (p&gt;0,05) os níveis de AMPc. No experimento dois, o inibidor da PDE5 isolado não influenciou a retomada da meiose (p&gt;0,05), porém, quando associado aos inibidores da PDE3 e 8 houve atraso na retomada (p&lt;0,05) e ainda alteraram os níveis de GMPc e AMPc (p&lt;0,05) nas primeiras horas de maturação. O experimento três mostrou a influencia do FSHr durante a MIV, o qual estimulou a retomada da meiose, mas em associação com inibidores da PDE5 e 8 atrasa a retomada (p&lt;0,05). Além disso, o FSHr provoca aumento do nível de AMPc e sua associação com inibidores de PDE5 e PDE8 ocasionou elevação adicional (p&lt;0,05). As condições de cultivo estudadas no experimento quatro mostraram que a maturação induzida (pré-MIV de duas horas com agentes para elevar AMPc seguindo de 22 horas de MIV com FSH associado a inibidores de PDEs) atrasaram a retomada da meiose às nove horas de maturação, mas não afetaram progressão da meiose às 24, 28 e 30 horas. Os tratamentos, porém, não melhoraram a competência oocitária após a fertilização in vitro e ocasionaram pequenas variações na ativação da MAPK em oócitos e células do cumulus. / In vitro maturation of oocytes is a limiting factor in the in vitro production of bovine embryos. In vivo, this maturation is a highly orchestrated process in which meiosis resumption by the gonadotropin surge that precedes ovulation induces the decrease in cAMP levels in the oocyte. However, when oocytes are removed from follicles, they spontaneously resume maturation compromising the competence for its development. cAMP is synthesized by adenylyl cyclase (AC) and degraded by phosphodiesterases (PDE), and there are some PDEs related to degradation of cAMP and/or cGMP. Thus, the purpose of this work was to investigate the contribution of different isoforms of phosphodiesterases in the resumption of meiosis and levels of cAMP and, also, to determine differences in signaling pathways when maintaining high levels of cAMP and its influence on oocyte competence. For this purpose, cumulus-oocyte complexes (COC) were matured in vitro in the presence, absence or combination of inhibitors of cAMP- and cGMP-specific PDEs and FSH. Samples be were evaluated in relation to: 1) maturation rate, 2) intracellular levels of cAMP and cGMP in COCs, 3) rate of blastocyst development and 4) activation of MAPK in oocytes and cumulus cells. The results of the first experiment showed that the PDE3 inhibitor is more effective (p &lt;0.05) in delaying meiosis resumption, at nine hours of maturation, but was not capable of altering cAMP levels (p&gt; 0.05) either alone or in combination with the PDE8 inhibitor. In experiment two, the PDE5 inhibitor alone did not affect the meiosis resumption (p&gt; 0.05), however, when associated with PDE3 and PDE8 inhibitors it delayed their resumption (p &lt;0.05) and also altered cGMP and cAMP levels of (p &lt;0.05) in the early hours of maturation. The third experiments showed the influence of FSHr during IVM, which stimulated the resumption of meiosis, but in combination with PDE5 and PDE8 inhibitors meiosis was delayed (p &lt;0.05). Furthermore, FSHr causes increased levels of cAMP and its association with PDE5 and PDE8 inhibitors caused an additional increase (p &lt;0.05). Culture conditions studied in experiment four showed that induced maturation (pre-IVM for two hours with agents to elevate cAMP followed by 22 hours IVM with FSH associated with PDE inhibitors) delayed the resumption of meiotic maturation at nine hours, but has no effect on meiosis progression at 24, 28 and 30 hours. The treatments, however, did not improve oocyte competence after in vitro fertilization and caused minor variations in the activation of MAPK in oocytes and cumulus cells.

A integridade da cromatina espermática é fundamental para a transmissão das informações genéticas paternas, sendo que alterações de DNA podem levar a falhas no processo reprodutivo. Danos na cromatina espermática podem ocorrer por diversos motivos, sendo que os mais importantes são a apoptose, a deficiência de protamina e os danos causados pelas espécies reativas de oxigênio (EROs). Os danos de DNA espermático não impedem que o espermatozóide fecunde o oócito, entretanto podem causar perda embrionária por apoptose. A importância da integridade da cromatina espermática no desenvolvimento embrionário já foi descrita em humanos, sendo que pode ser atribuída como uma das causas de infertilidade masculina, quando está alterada. Contudo, as informações sobre a influência da integridade do DNA espermático no processo de fecundação e do desenvolvimento embrionário na espécie bovina são muito escassas. Devido à falta de informação sobre a relação da fragmentação de DNA e conseqüente alteração nos índices de desenvolvimento embrionário nessa espécie, este trabalho teve como objetivo avaliar o índice de fragmentação de DNA espermático em uma população de touros e verificar a influência dos possíveis danos de DNA espermático na produção in vitro (PIV) de embriões. Para isto, o sêmen congelado de 221 touros foi avaliado pelo ensaio de estrutura de cromatina espermática (SCSA). Após o ensaio, os animais foram divididos em seis grupos experimentais de acordo com o índice de fragmentação de DNA apresentado, sendo sete animais de cada grupo, escolhidos aleatoriamente para as avaliações espermáticas: motilidade, fragmentação de DNA espermático pelos ensaios SCSA e Cometa Alcalino e estresse oxidativo pelo ensaio de substâncias reativas ao ácido tiobarbitúrico (TBARS). Posteriormente foi realizada a PIV de embriões, sendo avaliados os índices de clivagem e de blastocisto e realizado o ensaio de TUNEL para a avaliação da apoptose dos blastocistos produzidos. Em relação à motilidade espermática, o grupo 1 apresentou menor motilidade em relação ao grupo 4 (p<0,05). Não foi verificada diferença significativa da motilidade entre os outros grupos experimentais. O ensaio SCSA revelou que o grupo 1 apresentou menor susceptibilidade à fragmentação de DNA espermático quando comparado com o grupo 5 (p< 0,05). O grupo 2 apresentou susceptibilidade à fragmentação de DNA espermático inferior, quando comparado aos grupos 3, 4, 5 e 6 (p<0,05). Em relação ao ensaio Cometa Alcalino, a avaliação do estresse oxidativo e a PIV de embriões, não houve diferença significativa entre os grupos experimentais. Além disso, foi observada correlação negativa entre a intensidade média do Cometa e o índice de blastocisto produzido in vitro. O índice de blastocisto também apresentou correlação negativa com a intensidade média da cabeça do Cometa e ao ensaio TBARS. O ensaio TBARS apresentou correlação negativa com o índice de clivagem dos embriões PIV. Por outro lado, o ensaio TBARS apresentou correlação positiva com os ensaios SCSA e TUNEL. Considerando as condições experimentais, a fragmentação de DNA espermático não influenciou a PIV de embriões bovinos. / Sperm chromatin integrity is essential for the transmission of paternal genetic information. Changes in sperm DNA can lead to failures in the reproductive process. Loss of chromatin integrity may occur for several reasons, and the most important are apoptosis, protamine deficiency and damages caused by reactive oxygen species (ROS). The sperm DNA damage does not prevent the sperm to fertilize the oocyte, however it can cause embryonic loss by apoptosis. The importance of sperm chromatin integrity in embryonic development has been described in humans, and can be considered a cause of male infertility when it is disrupted. However, information on the influence of sperm DNA integrity in the process of fertilization and embryonic development in cattle are very scarce. Due to the lack of information on the relationship between DNA fragmentation and its consequence on embryonic development rates in this species, this study aimed to evaluate the sperm DNA fragmentation rate in a population of bulls and the influence of possible sperm DNA damage on in vitro production (IVP). For this, frozen semen from 221 bulls was evaluated by sperm chromatin structure assay (SCSA). After that, animals were divided into six groups, according to the DNA fragmentation rate observed. Then seven animals from each group were randomly chosen for sperm evaluation: motility, sperm DNA fragmentation by the SCSA and Alkaline Comet assay and oxidative stress by tiobarbituric acid reactive substances (TBARS). After sperm evaluations embryo IVP was performed. Cleavage and blastocyst rates were assessed and the obtained blastocysts were submitted to TUNEL assay for apoptosis evaluation. Regarding sperm motility, group 1 had lower motility compared to group 4 (p <0.05). There was no significant difference in motility between the other experimental groups. The SCSA revealed that group 1 showed lower susceptibility to sperm DNA fragmentation when compared to group 5 (p <0.05). Group 2 was less susceptible to sperm DNA fragmentation, when compared to groups 3, 4, 5 and 6 (p <0.05). For the alkaline comet assay, the assessment of oxidative stress and embryo IVP, no significant difference was shown between en experimental groups. Moreover, there was a negative correlation between the comet mean intensity and the blastocyst rate. The blastocyst rate also negatively correlated to the comet head mean intensity and TBARS. The TBARS was negatively correlated to cleavage rate. In addition, TBARS correlated positively to TUNEL assay and SCSA. Considering the experimental conditions, the sperm DNA fragmentation did not influence the IVP of bovine embryos.

A maturação in vitro de oócitos (MIV) é uma importante tecnologia reprodutiva a qual gera oócitos maduros capazes de suportar o desenvolvimento embrionário pré-implantacional e sua completa evolução à termo. Muitos fatores levam ao processo de maturação do oócito, e o AMH (hormônio anti-Mülleriano) tem demonstrado possuir um importante efeito nesta etapa. Neste trabalho nós demonstramos a influência da suplementação de AMH na maturação de complexos cumulus-oócito (COCs). Nossos resultados demonstram que não houve efeito na produção de embriões para COCS grau I. Entretanto, pudemos encontrar diferenças significativas entre os COCs graus II e III maturados na presença de 150ng/ml de AMH. Aqui também demonstramos que não houve diferença significativa na expressão relativa de mRNA para os genes AMHRII e FSHR no oócito, e na expressão relativa de mRNA para os genes AMH, AMHRII e FSHR nas células da granulosa. Nossos resultados corroboram com as importantes funções do AMH na produção de embriões, sugerem que a suplementação do meio de maturação de oócitos com AMH pode ajudar a melhorar a produção de blastocistos. / The in vitro oocyte maturation (MIV) is an important reproductive technology that generates mature oocytes able to support the preimplantation embryonic development and their fully evolution to term. Many factors lead to oocyte maturation process, and the AMH (AntiMüllerian hormone) have demonstrated important effects in the oocyte development. Here, we report the influence of AMH supplementation in the cumulus-oocyte complex (COCs) maturation. We found that AMH had no effect on embryo production of COCs grade I. On the other hand, significant differences between the COCs grade II and COCs grade III matured in AMH 150ng/ml were verified. We have also demonstrated that there were no significant difference in mRNA expression of the genes AMHRII and FSHR in the oocyte, and in mRNA of the genes AMH, AMHRII and FSHR in the granulosa cells. Taken together, the results corroborate the important roles for AMH on embryo production, and suggest that AMH supplementation could achieve successful outcome in the production of blastocysts.

Over three decades ago, W.H.O. declared infertility as a public health issue; due to its impact on millions of people worldwide. While cases of infertility could be multifactorial (affecting both male and female), 50% of cases are due to male factor infertility and this is mostly characterised by reduced sperm motility (asthenozoospermia). Assisted Reproduction Technology (ART) is the only treatment option available for this condition. Over 20 years ago, non-selective phosphodiesterase inhibitors (PDEi), such as pentoxifylline, were shown to enhance motility of human spermatozoa; however, contradictory results and stimulation of premature acrosome reaction has precluded their clinical use. Advancement in our knowledge have now made it clear that human sperm express several different PDEs and these are compartmentalised at different regions of the cells. By using type-specific phosphodiesterase inhibitors, differential modulation of sperm motility can be achieved without affecting other sperm function such as acrosome reaction. Additionally, by enhancing sperm function through PDE inhibition, there is a possibility of increasing IVF rates. The objective of this thesis is to: (1) examine the effect of phosphodiesterase inhibitors on spermatozoa in order to identify compounds that have clinically relevant enhancement of human sperm motility; (2) identify the signalling pathway(s) involved in the motility enhancing effects of identified compounds by targeting the modulator and mediator of cyclic nucleotides; (3) develop an animal IVF model to assess effects of Ibudilast on fertilization; and (4) optimise high performance liquid chromatography (HPLC) techniques for routine detection of cyclic nucleotides in sperm cells. A two phase drug screening approach was used to systematically and comprehensively screen series of compounds in order to identify those that have clinically relevant enhancement of human sperm motility. In phase 1, 6 compounds (out of 43 compounds) were found to have strong effects on poor motility samples, with magnitude of response ≥60% increase in percentage total motility. Additionally, these compounds significantly enhanced sperm penetration into cervical mucus substitute (p≤0.05), and they did not affect sperm acrosomal integrity nor cause externalisation of phosphatidylserine (p=0.6 respectively). 63% of IVF samples treated with compounds #26, #37 and #38 had significant increase in percentage total motility. For ICSI samples, compounds #26, #37 and #38 were the most effective. In respect to total motility, 88%, 81% and 79% of samples treated with these compounds showed significant increases in total motility, and 94%, 93% and 81% of samples showed significant increases in percentage of progressive cells, respectively. Analysis of the signalling pathways, using PKA, sGC and PKG inhibitors, showed that chosen PDE inhibitors were working predominantly through PKA signalling pathways. Additionally, this study revealed that this pathway is needed for the maintenance of basal progressive motility and hyperactivation in human sperm. Animal IVF studies showed that addition of Ibudilast (compound #26) during sperm-oocyte incubation leads to higher IVF rates. Lastly, this study used an HPLC system to detect cAMP in boar sperm. This was done to explore if HPLC system can be used for high throughput detection of cyclic nucleotides in mammalian sperm.

Cryopreservation is used to freeze and store donor’s sperm, from men who are going throughcertain medical treatments such as chemotherapy or radiotherapy and from men withazoopermia or severe oligozoospermia. Stored sperm samples are used in artificialreproductive technologies. Formation of ice crystals is the biggest problem when freezingcells because of the risk severe damage to the cells. To guarantee optimal survival rates infrozen sperm samples, cryoprotectants which protect sperms from ice crystals formations, isadded.The main objective of the study was to compare two different freeze solutions and theirimpact on sperm survival in frozen sperm samples. In addition, an additional aim was todetermine if the amount of motile sperm changes when the samples is left for two hours inroom temperature.In this study, 31 samples were used. Each sample was divided into two groups. The firstgroup was mixed with a SpermFreeze Solution™ and the second group was mixed withSperm CryoProtectTM II. All samples were frozen using cooling vapor and stored in tankswith liquid nitrogen. The concentration of motile sperm was measured after thawing andpreparation. SpermFreeze SolutionTM showed significant better results in the number ofsperm who survived after freezing and thawing process Also, the results showed that werewas no significant difference on concentration of motile sperm in samples after being left atroom temperature for two hours. In summary, an improvement in the process of preparationrecommends to be performed to reduce the mechanical stress to ensure a greater quantity ofmotile sperm after the whole process.

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

Aujourd’hui, au moins 5 millions d’enfants à travers le monde, sont nés suite au recours de leurs parents à l’AMP. Les traitements de l’infertilité ont significativement évolué, le plus souvent cela a eu lieu en dehors des protocoles expérimentaux classiques. L’exemple le plus marquant a été l’introduction de la FIV avec micro-injection intracytoplasmique d’un spermatozoïde (ICSI). Le manque d’évaluation de la santé des enfants nés de ces techniques reste la faiblesse de cette spécialité. Nous avons mis en place un suivi longitudinal d’une cohorte mono-centrique au sein de l’hôpital Saint-Joseph (Marseille). Le recueil a été fait par la collecte des photocopies des pages du carnet de santé des enfants et de questionnaires remplis par les parents. Notre étude est une des rares études françaises présentant un suivi à long terme, pouvant aller jusqu’à 5 ans, sur une cohorte à grande échelle. L’étude de l’IMC jusqu’à l’âge de 5 ans, n’a pas révélé d’effet de la FIV, comme cela a pu être pressenti dans la littérature. D’autres investigations méritent d’être conduites. Il est important de construire un système d’information cohérent autour de la santé des enfants nés après FIV à cause de l’apparition constante des nouvelles techniques dans cette spécialité, toutes étant potentiellement responsables de risques sur la santé future de l’enfant. La faisabilité de la collecte de données couvrant à la fois l’environnement maternel, conceptionnel et les indicateurs de santé de l’enfant doit être pensée à l’échelle nationale. A cette fin le développement des méthodes de liaison entre les différents registres existants en France serait une des solutions les plus opportunes. / Today, at least 5 million children worldwide were born following the enrollment of their parents in ART program. Infertility treatments have changed significantly; most often these changes took place outside traditional experimental protocols. The most striking event was when IVF with intracytoplasmic sperm injection (ICSI) was introduced in ART practices in 1995. The lack assessment of the health of children born after this technique remains the major weak in this discipline. We established a longitudinal monocentric follow-up study in Saint-Joseph Hospital (Marseille). The data were collected by asking parents to send copies of child health records and questionnaires filled out by them. This investigation is one of the few French studies involving a long-term follow- up to 5 years, in a large scale cohort. The study of BMI up to age 5 years didn’t show the suspected epigenetic influence of IVF reported in literature. Further investigations need to be conducted. It is important to build a coherent information system around the health of children born after IVF. The feasibility of collecting a series of data covering both maternal and conceptional environment, and child health indicators should be considered at the national level through the development of connection methods between different registers developed in France.

Les translocations chromosomiques et les autres types de réarrangements peuvent, bien qu'associées à un phénotype normal, mener à la transmission d'un contenu génétique déséquilibré à la descendance. Il n'est pas possible de distinguer les chromosomes équilibrés des déséquilibrés, ce qui empêche toute sélection dans le cadre d'une fécondation in vitro (FIV). Nous avons ainsi mené une série de projets de recherche dont le but a été de mettre en évidence des caractéristiques spécifiques de ces spermatozoïdes déséquilibrés, afin de pouvoir les distinguer au cours d'une FIV. Nous avons montré que les spermatozoïdes déséquilibrés présentaient des taux de fragmentation de l'ADN plus élevés, étaient moins denses, et avaient un volume nucléaire supérieur. Ces constatations ont mené au développement d'une procédure de sélection des spermatozoïdes équilibrés chez les porteurs de réarrangements chromosomiques. En utilisant le hypo-osmotic swelling test (HOST), nous avons montré qu'une morphologie flagellaire spécifique était associée à un contenu chromosomique équilibré. Nous proposons d'utiliser cette procédure de sélection dans le cadre d'une ICSI, afin d'améliorer le pronostic reproductif chez les couples concernés. Nous proposons également d'évaluer la proportion de spermatozoïdes déséquilibrés chez chaque patient porteur de réarrangement chromosomique. En effet, bien que ce taux varie d'un patient à l'autre, il est stable dans le temps pour un patient donné. Il est de plus un élément déterminant dans le choix d'une des options de prise en charge : reproduction naturelle, insémination artificielle avec test de migration survie (TMS), ICSI avec sélection par HOST, et diagnostic préimplantatoire (DPI). / Chromosomal translocations and other balanced rearrangements, although usually associated with a normal phenotype, can lead to the transmission of an abnormal unbalanced genetic content to the offspring. Balanced and unbalanced spermatozoa are indistinguishable, making it impossible to select them prior to in vitro fertilization. We conducted a series of research projects aimed at evidencing specific characterics for unbalanced spermatozoa, in a way to ultimately distinguish them from balanced ones during in vitro fertilization. We showed that unbalanced spermatozoa had higher DNA fragmentation rates, were less dense, and that their nuclear volume was higher. This led to developing a selection procedure for balanced spermatozoa in rearrangement carriers. Using the hypo-osmotic swelling test (HOST), we showed that a specific flagellar morphology was associated with balanced chromosomal status. We suggest using this procedure prior to ICSI in order to improve the reproductive prognosis in those patients. We also suggest evaluating the proportion of unbalanced spermatozoa in every patient with a chromosomal rearrangement. Although this proportion varies among patients, it is stable over time for a given patient. We believe this is a decisive element in choosing between the different available options : natural conception, artificial insemination with discontinuous gradient centrifugation, ICSI with HOST-based selection, and pre-implantation genetic diagnosis (PGD).