Avaliação do impacto da criopreservação na qualidade espermática
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2019-02-12
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A criopreservação tem sido amplamente utilizada na área da procriação medicamente assistida, tendo sofrido um grande desenvolvimento nos últimos 60 anos. As tecnologias de criopreservação representam a possibilidade de ultrapassar tradicionais obstáculos da reprodução como a idade e disponibilidade das células reprodutivas femininas e masculinas. Embora não exista um protocolo estandardizado para criopreservação de sémen, a literatura reporta resultados positivos, mas muitas das investigações focaram-se essencialmente na análise da sobrevivência e viabilidade das células após o processo de criopreservação, e na taxa de nados-vivos resultantes da utilização destas mesmas células. Neste trabalho, avaliaram-se os parâmetros espermáticos, danos no DNA, por Ensaio do Cometa e TUNEL, e a percentagem de espermatozoides com mitocôndrias ativas nas amostras, antes e após criopreservação. Avaliou-se também a influência do período de armazenamento das amostras em azoto líquido nos parâmetros espermáticos, bem como a importância do crioprotetor, da taxa de arrefecimento das amostras e da qualidade espermática a fresco, no processo de criopreservação. Verificou-se que o congelamento e o inerente descongelamento das células, por si só, levou a um extenso dano celular, com diminuições significativas na motilidade, vitalidade, morfologia típica e mitocôndrias ativas dos espermatozoides, e a aumentos significativos dos danos no DNA detetados por Ensaio do Cometa, mas, no geral, não pela técnica de TUNEL. O tempo de armazenamento não mostrou afetar significativamente a qualidade espermática, e o impacto da criopreservação nos parâmetros espermáticos e na percentagem de espermatozoides com mitocôndrias ativas dependeu do método de criopreservação aplicado, ao contrário do impacto da criopreservação nos danos no DNA. Notou-se ainda uma tendência para o impacto da criopreservação na qualidade espermática ser tanto menor, quanto melhor a qualidade espermática das amostras a fresco. Neste trabalho, e uma vez que ainda existe alguma controvérsia em torno da influência do estilo de vida na fertilidade dos indivíduos, avaliou-se a repercussão dos hábitos e exposição ocupacional de risco na qualidade espermática, com ênfase no índice de massa corporal, dado a obesidade ser um problema de saúde crescente a nível mundial, do qual se concluiu que a obesidade e riscos associados ao estilo de vida têm um impacto negativo na qualidade espermática. É urgente o melhoramento das técnicas de criopreservação de sémen, principalmente para casos de pacientes com qualidade espermática muito reduzida.
Cryopreservation has been widely used in assisted reproductive treatments, having undergone great development in the last 60 years. Cryopreservation technologies represent the possibility of overcoming traditional obstacles to reproduction such as age and the availability of female and male reproductive cells. Although there is no standardized protocol for semen cryopreservation and the literature reports positive results, many of the investigations focus primarily on the analysis of survival and viability of the cells after the cryopreservation process, and on the rate of live births resulting from the use of these same cells. In this work, the spermatic parameters, the DNA damage, assessed by the Comet Assay and TUNEL technique, and the percentage of spermatozoa with active mitochondria in the samples were evaluated, before and after cryopreservation. The influence of the storage period of the samples in liquid nitrogen on the spermatic parameters, as well as the importance of the cryoprotectant, the cooling rate of the samples and the fresh sperm quality in the cryopreservation process were also evaluated. Freezing and inherent thawing of cells alone has been found to lead to extensive cell damage, with significant decreases in motility, vitality, morphologically normal spermatozoa and spermatozoa with active mitochondria, and significant increases in DNA damage detected by Comet Assay, non the less, in general, not by TUNEL technique. The storage time did not show significant effect on sperm quality, and the impact of cryopreservation on spermatic parameters and the percentage of spermatozoa with active mitochondria depended on the cryopreservation method applied, contrary to the impact of cryopreservation on DNA damage. There was also a tendency for the greater the quality of fresh samples, the less the spermatic quality was affected by cryopreservation. In this study, and since there is still some controversy about the influence of the lifestyle on male fertility, the repercussion of habits and occupational exposure of risk in sperm quality was evaluated, with emphasis on body mass index, given the obesity is a growing health problem worldwide, from which it has been concluded that obesity and lifestyle risks have a negative impact on sperm quality. There is an urgent need to enhance semen cryopreservation techniques, especially for patients with a very low spermatic quality.
Cryopreservation has been widely used in assisted reproductive treatments, having undergone great development in the last 60 years. Cryopreservation technologies represent the possibility of overcoming traditional obstacles to reproduction such as age and the availability of female and male reproductive cells. Although there is no standardized protocol for semen cryopreservation and the literature reports positive results, many of the investigations focus primarily on the analysis of survival and viability of the cells after the cryopreservation process, and on the rate of live births resulting from the use of these same cells. In this work, the spermatic parameters, the DNA damage, assessed by the Comet Assay and TUNEL technique, and the percentage of spermatozoa with active mitochondria in the samples were evaluated, before and after cryopreservation. The influence of the storage period of the samples in liquid nitrogen on the spermatic parameters, as well as the importance of the cryoprotectant, the cooling rate of the samples and the fresh sperm quality in the cryopreservation process were also evaluated. Freezing and inherent thawing of cells alone has been found to lead to extensive cell damage, with significant decreases in motility, vitality, morphologically normal spermatozoa and spermatozoa with active mitochondria, and significant increases in DNA damage detected by Comet Assay, non the less, in general, not by TUNEL technique. The storage time did not show significant effect on sperm quality, and the impact of cryopreservation on spermatic parameters and the percentage of spermatozoa with active mitochondria depended on the cryopreservation method applied, contrary to the impact of cryopreservation on DNA damage. There was also a tendency for the greater the quality of fresh samples, the less the spermatic quality was affected by cryopreservation. In this study, and since there is still some controversy about the influence of the lifestyle on male fertility, the repercussion of habits and occupational exposure of risk in sperm quality was evaluated, with emphasis on body mass index, given the obesity is a growing health problem worldwide, from which it has been concluded that obesity and lifestyle risks have a negative impact on sperm quality. There is an urgent need to enhance semen cryopreservation techniques, especially for patients with a very low spermatic quality.
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Dissertação de Mestrado em Biotecnologia para as Ciências da Saúde apresentada à Universidade de Trás-os-Montes e Alto Douro
Palavras-chave
criopreservação , infertilidade masculina , análise espermática , danos no DNA , mitocôndrias ativas , estilo de vida