| Backgrounds:Human sperm cryopreservation is the professional work of the human sperm bank,including donors group and autologous sperm cryopreservation group.The former is the core business of human sperm bank in China,and the latter has been gradually received attention.Objectives:We also evaluate the current situation of fertility preservation for male in China.In addition,we study the related mechanism of human sperm freezing injury and explore ways to improve sperm quality after freezing-thawing procedure.Methods:First,we performed a retrospective study by collecting available information from the Beijing human sperm bank,and compared the semen parameters of patients with different tumors before tumor treatment.Second,we focused on the changes of sperm mitochondrial structure and function before and after freezing-thawing procedure,by electron microscopy,laser confocal microscopy,flow cytometry and other molecular biology experimental techniques.Second,we identify changes of potential protein and metabolite in sperm before and after freezing-thawing procedure,by Data-Independent Acquisition-based quantitative proteomics and targeted metabolomics.Subsequently,based on the result of the two parts of this study,we selected L-camitine as a component of cryoprotectant,to explore its effect on the cryopreserved sperm.Results:From July 2006 to December 2017,a total of 295 patients successfully completed autologous semen cryopreservation,among whom the cancer or other disease accounted for 63.1%(186/295).Twenty male patients withdrew their frozen semen for subsequent ART,which resulted in 43 treatment cycles by ART and 16 babies were successfully delivered.Our data showed that patients with sarcoma or leukemia could significantly decrease the total sperm count and sperm motility.In patients of testicular tumor,the total sperm count decreased,but there was no significant difference in sperm motility.Patients with testis cancer or leukemia had signifieantly worse recovery rate of sperm after freezing-thawing procedure.Our results indicated that looser mitochondrial structure,wider intermembrane space,and showed vacuolated degeneration of sperm after freezing-thawing procedure were founded.Sperm mitochondrial membrane potential and ATP were also decreased.Mitochondrial DNA copy numbers of sperm after freezing-thawing procedure were increased,but its integrity was not changeable.Expression of sperm Bax was down-regulated and sperm Bcl-2 was up-regulated after freezing-thawing proeedure.In addition,the differential expression of proteins and metabolites of sperm before and after freezing-thawing procedure was investigated by integrated proteomics and targeted metabolomics.The results showed 174 dysregulated proteins(76 up-regulated proteins,98 down-regulated proteins)and 16 differentially expressed metabolites(7 metabolites down-regulated and 9 metabolites up-regulated).Through bioinformatics analysis,we found that the freezing-thawing procedure resulted in the dysfunction of sperm glycolysis:the four differentially expressed proteins:Glucose-6-phosphate isomerase(GPI),Lactate Dehydrogenase B(LDHB),Alcohol Dehydrogenase 5(ADH5)and Phosphoglycerate mutase 1(PGAM1);and four differentially expressed related metabolites,L-lactic acid,phosphoenolpyruvate,D-glucose and dihydroxyacetone phosphate.Our results demonstrated that L-carnitine(LC)could improve cryopreserved sperm motility and movement parameters,and could reduce the damage of sperm plasma membrane and nuclear DNA caused by freezing injury.However,it had no obvious protective effect on sperm acrosome integrity.Our results also revealed that LC may protect the function of sperm mitochondria through anti-oxidation:increasing mitochondrial membrane potential and reducing ROS.Additionally,LC may improve cryopresevated sperm quality by regulating lactic acid and pyruvate.Conclusions:1.Sperm cryopreservation is an effective way for male fertility preservation.Semen quality declines in men with testicular tumors,leukemia or sarcomas.2.The freezing-thawing procedure leads to structural disorder and dysfunction of sperm mitochondria.3.Freezing damage can cause differential expression of sperm proteins and metabolites.Bioinformatics results suggest that the freezing-thawing procedure can lead to sperm glycolytic function disorder4.L.-carnitine as a component of cryoprotectant can improve sperm function. |
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