microRNA signature is altered in both human epididymis and seminal microvesicles following vasectomy.

BACKGROUND: Vasectomized men who wish for restoration of fertility commonly face problems to conceive despite high surgical success rates of vasovasostomy, a surgery routinely used to retrieve duct patency. In human, vasectomy has consequences on the epididymal transcriptome and is responsible for non-reversible changes that affect the biochemical parameters and functions of ejaculated sperm in vasovasotomized men. Since microRNAs (miRNAs) are key regulators of post-transcriptional gene expression, we explored the consequences of vasectomy on miRNA pattern in the human epididymis and determined the repercussion and reversibility of these changes in seminal microvesicles (SMVs) from semen of vasovasostomized men. METHOD: miRNA signatures from a unique set of human epididymal segments (caput, corpus, cauda) provided by controls and vasectomized donors as well as miRNA content of SMVs isolated from semen of normal, vasectomized and vasovasostomized donors were determined by using high-throughput microarray technology. Epididymal miRNA signature was correlated with gene expression profiles by employing an integrated genomic approach. RESULTS: While the segmented pattern of miRNA expression was maintained along the human epididymis following vasectomy, the array of miRNAs from each anatomical region significantly differed between normal and vasectomized donors. Greatest changes were observed in cauda epididymidis and were negatively correlated with transcription factors. Vasectomy also impacted expression of miRNAs found in SMVs from normal donors, including miRNAs of epididymal origin contained in epididymosomes. Among seminal miRNAs sequelae, 52 were reversible according to miRNA expression level following duct patency retrieval. In contrast, 66 of miRNAs found in SMVs were not retrieved in SMVs after vasovasostomy. CONCLUSION: We showed that vasectomy has a substantial impact on the miRNA signature of the epididymis and SMVs, and that vasovasostomy does not restore all molecular players found in normal fertile individuals. According to the critical role played by miRNAs in the control of male fertility, we believe that miRNA sequelae occurring upstream and downstream of the vasectomy site may be in part responsible for the reduced fertility outcome reported after surgically successful vasectomy reversal. Human epididymides from 3 control (26, 47 and 50 years old. GEO accession number GSE35522) and 3 vasectomized donors (45, 47 and 48 years old), were collected and dissected into 3 anatomical regions (caput, corpus and cauda). Seminal microvesicles (SMVs) were isolated from the semen of normal (pool of 3 donnors), vasectomized (pool of 3 donnors) and vasovasostomized donors (pool of 5 donnors). A cross-comparison between the miRNA signature found in different segments and seminal vesicles was performed using GeneChip miRNA Array 2.0 (Affimetrix, Santa Clara, CA).

### 背景
希望恢复生育能力的输精管结扎术后男性，即便输精管吻合术（vasovasostomy，一种常规用于恢复输精管道通畅的手术）的手术成功率较高，仍常面临难以受孕的问题。在人体中，输精管结扎会对附睾转录组产生影响，并引发不可逆的改变，进而影响输精管吻合术后男性射出精子的生化参数与功能。鉴于微小核糖核酸（microRNAs, miRNAs）是转录后基因表达的关键调控因子，本研究探究了输精管结扎对人体附睾miRNA表达谱的影响，并明确了此类改变在输精管吻合术后男性精液微囊泡（seminal microvesicles, SMVs）中的效应与可逆性。
### 方法
本研究采用高通量微阵列技术，对来自正常对照与输精管结扎供者的一组独特人体附睾节段（附睾头、附睾体、附睾尾）的miRNA表达谱，以及从正常、输精管结扎及输精管吻合术供者精液中分离得到的SMVs的miRNA含量进行了检测。本研究通过整合基因组学方法，将附睾miRNA表达谱与基因表达谱进行了关联分析。
### 结果
尽管输精管结扎术后，人体附睾仍维持了原有的节段性miRNA表达模式，但正常供者与输精管结扎供者各解剖区域的miRNA表达谱存在显著差异。附睾尾的miRNA表达改变最为显著，且与转录因子呈负相关。输精管结扎同样会影响正常供者SMVs中的miRNA表达，包括附睾小体中源自附睾的miRNA。在精液miRNA的后遗症中，有52种miRNA的表达水平在输精管道通畅恢复后可恢复正常；与之相反，SMVs中检出的66种miRNA在输精管吻合术后未能恢复至正常水平。
### 结论
本研究证实，输精管结扎会对附睾与SMVs的miRNA表达谱产生显著影响，且输精管吻合术无法使正常可育个体体内的所有分子调控因子恢复至初始状态。鉴于miRNA在男性生育调控中的关键作用，我们认为，发生于输精管结扎位点上下游的miRNA后遗症，可能是手术成功的输精管吻合术后生育结局仍欠佳的部分原因。
本研究收集了3名正常对照供者（年龄分别为26岁、47岁与50岁，基因表达综合数据库（GEO）登录号：GSE35522）与3名输精管结扎供者（年龄分别为45岁、47岁与48岁）的人体附睾组织，并将其解剖为3个解剖区域（附睾头、附睾体与附睾尾）。从正常（3名供者混合样本）、输精管结扎（3名供者混合样本）及输精管吻合术供者（5名供者混合样本）的精液中分离得到SMVs。本研究采用GeneChip miRNA Array 2.0芯片（Affymetrix，美国加利福尼亚州圣克拉拉），对不同附睾节段与SMVs的miRNA表达谱进行了交叉比较分析。