Early pregnancy biomarkers. Bos taurus

Low fertility remains a leading cause of poor productivity in dairy cattle. In this context, there is significant interest in developing novel tools for accurate early diagnosis of pregnancy. MicroRNAs (miRNAs) are short RNA molecules which are critically involved in regulating gene expression during both health and disease. MiRNAs have been shown to regulate ovarian function, uterine receptivity, embryonic development and placental function. Circulating miRNAs can provide useful biomarkers of tissue function and disease; importantly, differential miRNA profiles have been linked to pregnancy and preeclampsia in humans. This study sought to establish the potential of circulating miRNAs as biomarkers of early pregnancy in cattle. We applied Illumina small-RNA sequencing to profile miRNAs in plasma samples collected from eight non-pregnant heifers on Days 0, 8 and 16 of the oestrous cycle and 11 heifers on Days 16 and 24 of pregnancy. We sequenced a total of 46 samples and generated 9.2 million miRNA reads per sample. There were no differences in miRNA read abundance between any of the pregnant and non-pregnant time-points (FDR > 0.1). As a complementary approach, we analysed sample pools (3-4 samples/pool) corresponding to Days 0, 8 and 16 of the oestrous cycle and Day 24 of pregnancy (n = 3 pools/group) using Qiagen PCR arrays. A total of 16 miRNAs were differentially expressed (FDR < 0.1) in plasma between pregnant and non-pregnant animals. RT-qPCR validation using the same plasma samples confirmed that miR-26a was differentially upregulated on Day 16 pregnant relative to non-pregnant heifers (1.7-fold; P = 0.043), whereas miR-1249 tended to be upregulated in Day 16 pregnant heifers (1.6-fold; P = 0.081). Further validation in an independent group of heifers confirmed an increase in plasma miR-26a levels during early pregnancy, which was significant only on Day 24 (2.0-fold; P = 0.027). Through genome-wide analyses we have successfully profiled plasma miRNA populations associated with early pregnancy in cattle. We have identified miR-26a as a potential circulating biomarker of early pregnancy. Overall design: Sequencing of three sequencial samples from each of eight cycling animals (Days 0, 8 and 16, total 24 samples) and two samples from each of 11 pregnant animals (Days 16 and 24, total 22 samples). Main comparisons were between non-pregnant and pregnant groups

低繁殖率仍是导致奶牛生产性能低下的首要诱因。在此背景下，学界对开发可实现精准早期妊娠诊断的新型工具抱有浓厚兴趣。微小RNA（microRNAs，miRNAs）是一类短链RNA分子，在机体健康与疾病状态下均对基因表达调控发挥关键作用。已有研究证实，miRNAs可调控卵巢功能、子宫容受性、胚胎发育及胎盘功能。循环中的miRNAs可作为组织功能与疾病的有效生物标志物；尤为关键的是，人类妊娠与子痫前期均与差异表达的miRNA谱存在关联。本研究旨在探究循环miRNAs作为奶牛早期妊娠生物标志物的潜力。
本研究采用Illumina小RNA测序技术，对采集自8头未妊娠青年母牛发情周期第0、8、16天的血浆样本，以及11头妊娠青年母牛妊娠第16、24天的血浆样本进行miRNA谱分析。本研究共完成46份样本的测序，每份样本平均产生920万条miRNA读段。各妊娠与未妊娠时间点间的miRNA读段丰度均无显著差异（错误发现率False Discovery Rate, FDR>0.1）。
作为补充实验方案，本研究采用Qiagen PCR芯片技术，对对应发情周期第0、8、16天及妊娠第24天的混合样本（每组3-4份样本/混合池，每组设3个混合池）进行分析。妊娠与未妊娠动物的血浆中共有16种miRNAs存在差异表达（FDR<0.1）。
采用相同血浆样本开展的实时定量聚合酶链反应（Real-time Quantitative Polymerase Chain Reaction, RT-qPCR）验证实验证实，相较于未妊娠青年母牛，妊娠第16天的miR-26a呈现差异上调（1.7倍；P=0.043），而miR-1249在妊娠第16天的青年母牛中呈上调趋势（1.6倍；P=0.081）。在独立队列的青年母牛中开展的进一步验证实验证实，早期妊娠过程中血浆miR-26a水平升高，且该升高仅在妊娠第24天时达到显著水平（2.0倍；P=0.027）。
通过全基因组分析，本研究成功绘制了与奶牛早期妊娠相关的血浆miRNA表达谱，并鉴定出miR-26a可作为潜在的早期妊娠循环生物标志物。
实验设计：对8头发情周期正常的青年母牛分别采集3份样本（第0、8、16天，共计24份样本），对11头妊娠青年母牛分别采集2份样本（第16、24天，共计22份样本）进行测序。核心比较组为未妊娠组与妊娠组。