BACKGROUND The amniotic fluid (AF) cell-free transcriptome is modulated by physiologic and pathologic processes during pregnancy. AF gene expression changes with advancing gestation reflect fetal development and organ maturation; yet, defining normal expression and splicing patterns for biomarker discovery in obstetrics requires larger heterogeneous cohorts, evaluation of potential confounding factors, and novel analytical approaches. METHODS Women with a normal pregnancy who had an AF sample collected during midtrimester (n = 30) or at term gestation (n = 68) were included. Expression profiling at exon level resolution was performed using Human Transcriptome Arrays. Differential expression was based on moderated t-test adjusted p < 0.05 and fold change > 1.25; for differential splicing, a splicing index > 2 and adjusted p < 0.05 were required. Functional profiling was used to interpret differentially expressed or spliced genes. The expression of tissue-specific and cell-type specific signatures defined by single-cell genomics was quantified and correlated with covariates. In-silico validation studies were performed using publicly available datasets. RESULTS 1) 64,071 genes were detected in AF, with 11% of the coding and 6% of the non-coding genes being differentially expressed between midtrimester and term gestation. Expression changes were highly correlated with those previously reported (R > 0.79, p < 0.001) and featured increased expression of genes specific to the trachea, salivary glands, and lung and decreased expression of genes specific to the cardiac myocytes, uterus, and fetal liver, among others. 2) Single-cell RNA-seq signatures of the cytotrophoblast, Hofbauer cells, erythrocytes, monocytes, T and B cells, among others, showed complex patterns of modulation with gestation (adjusted p < 0.05). 3) In 17% of the genes detected, we found differential splicing with advancing gestation in genes related to brain development processes and immunity pathways, including some that were missed based on differential expression analysis alone. CONCLUSIONS This represents the largest AF transcriptomics study in normal pregnancy, reporting for the first time that single-cell genomic signatures can be tracked in the AF and display complex patterns of expression during gestation. We also demonstrate a role for alternative splicing in tissue-identity acquisition, organ development, and immune processes. The results herein may have implications for the development of fetal testing to assess placental function and fetal organ maturity.

中文翻译：

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羊水无细胞转录组：正常妊娠期间胎儿发育和胎盘细胞动力学的一瞥。

背景技术羊水（AF）无细胞转录组在怀孕期间受到生理和病理过程的调节。随着妊娠的进展，AF基因的表达变化反映了胎儿的发育和器官的成熟。但是，为产科中生物标志物的发现定义正常表达和剪接模式需要更大的异质队列，潜在混杂因素的评估和新颖的分析方法。方法包括在孕中期（n = 30）或足月妊娠（n = 68）采集房颤样本的正常妊娠妇女。使用人类转录组阵列以外显子水平的分辨率进行表达谱分析。差异表达是基于经调节的t检验调整的p <0.05和倍数变化> 1.25；对于差分拼接，拼接索引> 2且调整后的p <0。必须为05。功能分析用于解释差异表达或剪接的基因。量化由单细胞基因组学定义的组织特异性和细胞类型特异性标记的表达，并将其与协变量相关联。使用公开可用的数据集进行计算机内验证研究。结果1）在房颤中检测到64,071个基因，其中11％的编码基因和6％的非编码基因在孕中期和足月妊娠之间差异表达。表达变化与先前报道的高度相关（R> 0.79，p <0.001），其特征在于气管，唾液腺和肺特异基因的表达增加，而心肌，子宫和胎儿肝脏特异的基因表达下降等等。2）滋养细胞，霍夫鲍尔细胞，红细胞，单核细胞，T和B细胞等的单细胞RNA-seq信号显示了妊娠调节的复杂模式（调整后的p <0.05）。3）在17％的检测到的基因中，我们发现与大脑发育过程和免疫途径相关的基因中，随着妊娠的进展而出现的差异剪接，包括仅基于差异表达分析而遗漏的一些。结论这是正常妊娠中最大的房颤转录组学研究，首次报道可在房颤中追踪单细胞基因组特征并在妊娠期间显示复杂的表达模式。我们还展示了替代剪接在组织身份获取，器官发育和免疫过程中的作用。