| BackgroundRecurrent spontaneous abortion(RSA)refers to two or more consecutive pregnancy losses with the same spouse before 20 weeks of pregnancy.In addition,at least half of them are unexplained recurrent loss(URSA).The incidence of RSA accounts for about 1-2%of all pregnancies,which seriously endangers women's reproductive health and causes great physical and mental pain to patients and their families.The pathological factors of RSA are very complex.The research in this field mainly focuses on maternal factors,such as meiosis error,oocyte quality,endocrine abnormality,uterine structure,metabolic factors,prethrombotic state,infection and immunology.Given that men provide 50%of their DNA to developing embryos,it's not surprising that male factor causes pregnancy loss.At present,DNA damage is related to pregnancy loss,and the epigenetic state of sperm may be one of the causes of recurrent pregnancy loss.However,the research on male factors of RSA is not sufficient,which may be due to the short survival time of sperm in vitro,and the related research is difficult to carry out.In addition,sperm is highly differentiated cells,and it is difficult to carry out cell culture.In addition,the causes of recurrent pregnancy loss are complex,and stable male animal models are difficult to establish,which restrict the study of male factors of recurrent pregnancy loss.At present,the research on male of RSA is relatively lacking and there is a huge research space left.Therefore,the research on male factors is more important and urgent.Protein is the ultimate executor of various biological processes.Proteomics,as a new research method in the post genomic era,studies protein expression,function and interaction of tissue or cell from the overall level,and now has become one of the main means of life science research.Sperm protein detection has achieved high-throughput,sperm cells are easy to separate and purify in the laboratory,and easy to obtain,so it is very suitable for proteomics analysis.Several thousands of sperm proteins have been found by proteomics technology,most of which have clear functions and are mainly involved in biological processes such as cell energy metabolism,signal transduction and skeleton protein composition.Before this study,there was no proteomic study on sperm of RSA.In this study,we put forward a bold hypothesis that abnormal expression of sperm protein may lead to the occurrence of RSA.Over the past decade,proteomics technology has been evolving from 2-dimensional electrophoresis(2-DE)to two-dimensional 2D difference gel electrophoresis(2D-DIGE)to liquid chromatography tandem mass spectrometry(LC-MS/MS),the progress of technology promotes the development of proteomics.Isobaric tags for relative absolute quantification(ITRAQ)technology is a high-throughput screening technology commonly used in proteomics in recent years.It uses a variety of isotopic reagents to label protein polypeptides.Through high-precision mass spectrometer tandem analysis,it can simultaneously compare the protein expression between up to eight samples,and detect low abundance protein,strong basic protein,strong acid protein,protein with molecular weight less than 10kd or more than 200kD.At present,iTRAQ technology is widely used in spermatogenesis,asthenospermia and assisted reproductive technology,but it has not been reported in the sperm research of RSA patients.Research objectiveUsing iTRAQ differential proteomics strategy,this study explored the protein composition and differential expression protein of the sperm of male partners with recurrent loss,sought for potential useful biomarkers,and provided new ideas and theoretical basis for the diagnosis and treatment of RSAMethodsIn this study,iTRAQ coupled LC-MS/MS technology was used for the first time to study the sperm protein of RSA male spouses,identifing the components of sperm protein,establish the differential expression protein spectrum of sperm of RSA male spouses,and bioinformatics analysis of the differential expression protein.There were 10 males in RSA group,7 males in control group with normal reproductive history within 2 years.After abstinence for 2-7 days,the semen was obtained by masturbation,and the semen parameters were analyzed after liquefaction.Sperm were separated by 50%percoll gradient centrifugation and stored at-80?.Two groups of samples were mixed and labeled with iTRAQ reagent after the sperm proteins were extracted and quantified.The RSA group was labled with 121 and that of control group was labled with 116.After strong cation exchange and reversed-phase liquid chromatography separation,mass spectrometry identification was carried out.To search and identify the proteins from the mass spectrometry data,set the difference multiple as 1.5 times,and p-value?0.05 as the screening standard,and complete the screening of differential proteins.Gene Ontology analysis was carried out for differential proteins;protein protein interaction analysis was carried out by using string website to determine the interaction relationship between differential proteins;pathway significance enrichment analysis was carried out by using KEGG database to determine the main metabolic pathways involved in differential proteins.Westerm blot experiment to verify the mass spectrum resultsResultsIn this study,2350 proteins and 38 differentially expressed proteins were identified in sperm of male RSA spouses for the first time,25 of which were up-regulated and 13 down regulated,which could be used as potential biomarkers of RSA.Bioinformatics analysis shows that these differentially expressed proteins are mainly located in sperm membrane and nucleus.And they mainly perform biological regulation,metabolism,cell process,protein binding and different enzyme activity functions.Pathway enrichment analysis shows that the differentially expressed proteins are mainly related to fat and glucose metabolism pathways,and the up-regulated proteins are mainly involved in fatty acid synthesis and metabolism pathway Hexokinase(HK1),ATP-citrate synthase(ACLY)and fatty acid synthetase(FASN)are significantly enriched in the metabolic pathway,and form a metabolic interaction network in the protein interaction network,which can be used as biomarkers.Western blotting was used for further verifing the expression of three biomarkers HK1,ACLY and FASN,which were related to metabolism.The results were consistent with the mass spectrometry,which confirmed the proteomics research results of this project.ConclusionsITRAQ technology is a powerful tool for sperm protein research,which has a great advantage over the traditional 2DE technology.For the first time,this project systematically revealed the protein composition of sperm of male in RSA,identified 2350 proteins,found 38 differentially expressed proteins,and verified the interest proteins by immunoblotting.The differential proteins are mainly related to biological regulation,metabolism and cell processes,protein binding and different enzyme activities,as well as fat and glucose metabolic signaling pathways.The changes of sperm differential protein and the abnormal metabolism of fatty acids and glucose in male couples of uprl may be the cause of RSA.The results of this study will provide a basis for the study of the male pathogenic mechanism of unexplained recurrent abortion. |
PDF Full Text Request