Análise proteômica do plasma seminal de homens com obesidade com e sem alteração nos parâmetros espermáticos
Data
2024-09-10
Autores
Carvalho, Renata Cristina de 
Orientadores
Fraietta, Renato
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Objetivo: Comparar a expressão de proteínas seminais entre amostras de homens com obesidade com e sem alterações nos parâmetros espermáticos. Ainda, verificar se as proteínas justificam a alteração espermática e se tal prejuízo também é refletido nos aspectos funcionais do espermatozoide. Métodos: Neste estudo observacional, homens com idade entre 20 e 50 anos foram distribuídos em 3 grupos de acordo com sua porcentagem de gordura corporal e características espermáticas, sendo: grupo controle eutrófico, obesidade sêmen normal e obesidade sêmen alterado. A coleta da amostra seminal aconteceu após 2 a 7 dias de abstinência ejaculatória; com ela realizou-se a análise seminal convencional, que auxiliou na distribuição dos grupos, análise funcional dos espermatozoides (integridade do DNA, atividade mitocondrial e integridade do acrossoma) e, com a alíquota restante, centrifugações foram aplicadas para separação do plasma seminal. Assim, o plasma seminal obtido foi utilizado para avaliação dos níveis proteicos; para tal, quantificamos o total de proteínas, preparamos cada amostra e realizamos a análise proteômica sem alvo definido usando espectrometria de massas (LC-MS/MS). A análise estatística dos dados gerais dos participantes e das variáveis seminais foi realizada pelo software SPSS incluindo avaliação de normalidade, homogeneidade, além de teste General Linear Model (GLM) com post-hoc de Sidak, sempre considerando p menor que 0,05. Por fim, os dados gerados pela proteômica foram utilizados para identificação das proteínas diferencialmente representadas entre os grupos (teste ANOVA com post-hoc LSD realizado pelo software MetaboAnalyst). Resultados: 73 homens participaram do estudo (19 no grupo controle eutrófico, 33 em obesidade sêmen normal e 21 em obesidade sêmen alterado) apresentando uma média de idade de 31,05, 33,88 e 36,67 anos, respectivamente. Conforme esperado, os grupos de obesidade apresentaram maior peso, porcentagem de gordura corporal e IMC em relação ao grupo eutrófico, mas sem diferença entre si. A análise seminal convencional indicou resultados significativos para as variáveis motilidade progressiva, motilidade não progressiva, imóveis, concentração e contagem espermática, morfologia, vitalidade e total de espermatozoides móveis morfologicamente normais (MMN) sendo que os piores valores foram observados sempre no grupo obesidade sêmen alterado com exceção da variável motilidade não progressiva na qual os resultados significativos foram entre os grupos de eutróficos e obesidade sêmen normal (este último apresentando menor porcentagem de espermatozoides com motilidade não progressiva). Em relação aos aspectos funcionais dos espermatozoides, o grupo obesidade sêmen alterado apresentou maior porcentagem de espermatozoides com DNA totalmente fragmentado e maior prejuízo na atividade mitocondrial espermática; já a integridade do acrossoma foi pior para os grupos de obesidade em relação aos homens eutróficos. Por fim, na análise proteômica foram encontradas 746 proteínas sendo que 30 delas se mostraram diferencialmente representadas entre os grupos; no grupo obesidade sêmen alterado verificamos que 3 proteínas estavam praticamente ausentes (Kunitz-type protease inhibitor 3, Ovochymase-2, Dipeptidase 3). Além disso, as outras proteínas apresentaram resultados significativos variados entre os grupos sempre com menor concentração nos grupos de obesidade e ainda menor no grupo de obesidade sêmen alterado. Conclusões: Homens com obesidade e alteração nas características espermáticas podem apresentar também maior prejuízo nos aspectos funcionais dos seus gametas e nas vias moleculares seminais.
Objective: To compare the expression of seminal proteins between samples from men with obesity with and without changes in sperm parameters. Furthermore, to verify if the proteins justify the sperm alteration and if such damage is also reflected in the functional aspects of the sperm. Methods: In this observational study, men aged between 20 and 50 years were distributed into 3 groups according to their percentage of body fat and sperm features. These groups were called control eutrophic, obesity normal semen and obesity altered semen. The seminal sample collection occurred after 2 to 7 days of ejaculatory abstinence; with this sample, conventional semen analysis was performed, which assisted in group distribution, and sperm functional analysis (DNA integrity, mitochondrial activity and acrosome integrity). With the remained sample, centrifugation was performed to obtain seminal plasma. Then, the seminal plasma obtained was used to evaluate protein levels. To this end, we quantified the total proteins, prepared each sample and performed shotgun proteomic analysis using mass spectrometry (LC-MS/MS). Statistical analysis of participants' general data and seminal variables was performed using SPSS software and included evaluation of normality, homogeneity, besides to the General Linear Model (GLM) test with Sidak post-hoc, always considering p less than 0.05. Finally, the data resulted from proteomics were used to identify the proteins differentially represented between the groups (ANOVA test with post-hoc LSD was performed by the MetaboAnalyst software). Results: 73 men participated in the study (19 in the eutrophic control, 33 in obesity normal semen and 21 in obesity altered semen group) with a mean age of 31.05, 33.88 and 36.67 years, respectively. As expected, weight values, BMI, and fat percentage were higher in the obesity groups; however, there was no statistical difference between those with and without alteration of sperm features. Conventional semen analysis indicated significant results for the variables progressive motility, non-progressive motility, immobile, sperm concentration and count, morphology, viability and total motile morphologically normal sperm (MMN) with the worst values always being observed in the obesity altered semen group with the exception of the non-progressive motility variable in which the significant result was between the eutrophic and obesity normal semen group (the latter presenting a lower percentage of sperm with non-progressive motility). In relation to sperm functional aspects, the obesity altered semen group presented a higher percentage of sperm with completely fragmented DNA and greater impairment in sperm mitochondrial activity. The acrosome integrity was worse for the obesity groups compared to eutrophic men. Finally, in the proteomic analysis, 746 proteins were found in which 30 were differentially represented between the groups. In the obesity altered semen group we found that 3 proteins were practically absent (Kunitz-type protease inhibitor 3, Ovochymase-2, Dipeptidase 3). Furthermore, the other proteins showed significant results that varied between the groups, always with lower concentrations in the obesity groups and even lower concentrations in the obesity altered semen group. Conclusions: Men with obesity and altered sperm features may also present greater damage in their gametes’ functional aspects and in their seminal molecular pathways.
Objective: To compare the expression of seminal proteins between samples from men with obesity with and without changes in sperm parameters. Furthermore, to verify if the proteins justify the sperm alteration and if such damage is also reflected in the functional aspects of the sperm. Methods: In this observational study, men aged between 20 and 50 years were distributed into 3 groups according to their percentage of body fat and sperm features. These groups were called control eutrophic, obesity normal semen and obesity altered semen. The seminal sample collection occurred after 2 to 7 days of ejaculatory abstinence; with this sample, conventional semen analysis was performed, which assisted in group distribution, and sperm functional analysis (DNA integrity, mitochondrial activity and acrosome integrity). With the remained sample, centrifugation was performed to obtain seminal plasma. Then, the seminal plasma obtained was used to evaluate protein levels. To this end, we quantified the total proteins, prepared each sample and performed shotgun proteomic analysis using mass spectrometry (LC-MS/MS). Statistical analysis of participants' general data and seminal variables was performed using SPSS software and included evaluation of normality, homogeneity, besides to the General Linear Model (GLM) test with Sidak post-hoc, always considering p less than 0.05. Finally, the data resulted from proteomics were used to identify the proteins differentially represented between the groups (ANOVA test with post-hoc LSD was performed by the MetaboAnalyst software). Results: 73 men participated in the study (19 in the eutrophic control, 33 in obesity normal semen and 21 in obesity altered semen group) with a mean age of 31.05, 33.88 and 36.67 years, respectively. As expected, weight values, BMI, and fat percentage were higher in the obesity groups; however, there was no statistical difference between those with and without alteration of sperm features. Conventional semen analysis indicated significant results for the variables progressive motility, non-progressive motility, immobile, sperm concentration and count, morphology, viability and total motile morphologically normal sperm (MMN) with the worst values always being observed in the obesity altered semen group with the exception of the non-progressive motility variable in which the significant result was between the eutrophic and obesity normal semen group (the latter presenting a lower percentage of sperm with non-progressive motility). In relation to sperm functional aspects, the obesity altered semen group presented a higher percentage of sperm with completely fragmented DNA and greater impairment in sperm mitochondrial activity. The acrosome integrity was worse for the obesity groups compared to eutrophic men. Finally, in the proteomic analysis, 746 proteins were found in which 30 were differentially represented between the groups. In the obesity altered semen group we found that 3 proteins were practically absent (Kunitz-type protease inhibitor 3, Ovochymase-2, Dipeptidase 3). Furthermore, the other proteins showed significant results that varied between the groups, always with lower concentrations in the obesity groups and even lower concentrations in the obesity altered semen group. Conclusions: Men with obesity and altered sperm features may also present greater damage in their gametes’ functional aspects and in their seminal molecular pathways.
Descrição
Citação
CARVALHO, Renata Cristina de. Análise proteômica do plasma seminal de homens com obesidade com e sem alteração nos parâmetros espermáticos. 2024. 122 f. Tese (Doutorado em Urologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP). São Paulo, 2024.