人类精液的冷冻保存是辅助生殖技术（ART）实验室和精子库中常见的做法。然而，有关长期冷冻保存对精液的影响的信息仅限于临床数据摘要和描述。为了进行这项研究，我们准备了4个新鲜精液标本、4个冷冻保存至少1年的标本、3个冷冻保存至少5年的标本、4个冷冻保存至少10年的标本和3个冷冻保存至少15年的标本。我们从每个样本中提取总RNA，进行扩增、标记，并与miRBase数据库中已知的初级微小RNA（miRNA）进行比对，以预测新的miRNA。我们发现，冷冻保存会导致miRNA表达的改变，并且随着存储时间的增加，这些改变变得更加显著。同时，我们还发现，let-7d-3p、let-7c-5p和let-7i-3p miRNA在经过冷冻解冻的人类精子中的表达会随着冷冻保存时间的变化而动态改变。此外，我们分析了冷冻保存表达的miRNA及其靶向mRNA的时间依赖动态，并发现其中一半的靶基因在卵母细胞中表达。这些交叉基因主要富集在癌症和细胞骨架信号通路中。我们的发现表明，长期储存改变了冷冻保存的人类精液的miRNA表达谱。此外，随着存储时间的增加，对人类精子的影响在miRNA方面变得更加显著，这可能对后续的受精和胚胎发育产生影响。

The global practice of cryopreservation of human semen is commonplace in Assisted Reproductive Technology (ART) labs and sperm banks. However, information on the effects of long-term cryopreservation on semen is limited to clinical data summaries and descriptions. For this study, we prepared 4 semen specimens of fresh semen, 4 specimens cryostored for at least 1 year, 3 specimens cryostored for at least 5 years, 4 specimens cryostored for at least 10 years, and 3 specimens cryostored for at least 15 years. Total RNA was extracted from each sample, amplified, labeled, and mapped to the known primary microRNA (miRNA) in the miRBase database, enabling the prediction of novel miRNAs. We found that cryopreservation can lead to changes in miRNA expression, and with the increase in storage time, these changes became more pronounced. Meanwhile, the expression of let-7d-3p, let-7c-5p and let-7i-3p miRNAs changed dynamically over cryostorage time in frozen-thawed human sperm. Furthermore, we analyzed the time-dependent dynamics of cryostorage-expressed miRNAs and their target mRNAs and found that half of the target genes were expressed in oocytes. These intersection genes were mainly enriched in cancer and cytoskeletal signaling pathways. Our findings showed that the miRNA expression profile of cryopreserved human semen is modified by long-term storage. Furthermore, as the storage time increases, the impact on human sperm becomes more pronounced in terms of miRNAs, which may have an effect on subsequent fertilization and embryonic development.