Prenatal fetal head and placental gene expression in a mouse model of advanced maternal age

Advanced maternal age is a risk factor for neurodevelopmental disorders in offspring. The present study aimed to investigate differences in placental and fetal head gene expression between old and young C57BL6/J female mice. Conceptuses were collected at E12.5 and processed for transcriptomic mRNA gene expression microarray analyses. The most significant differences between old and young females were found in gene expression of the fetal heads (398 differentially expressed genes, DEG) as compared with the placenta (51 DEG). Fetuses with their placentas were collected from pregnant BTBR and B6 advanced maternal aged females (42-65 weeks old, AMA) and young (10-23 weeks old, YMA) females. RNA for transcriptomic analyses was extracted from placentas and fetal heads following the manufacturers’ instructions. Total RNA extraction was conducted using the Universal RNA Purification Kit (Eurx, Poland), and included a DNase treatment (Eurx, Poland). The quantity and purity of isolated nucleic acids were validated using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, USA). RNA integrity of selected samples was additionally validated using a Bioanalyzer 2100 microcapillary electrophoresis device (Agilent, USA). Genome-wide transcriptomic analysis was performed using SurePrint G3 Mouse GE v2 8x60K microarrays (GPL21163, Agilent Technologies, USA) and Agilent Technologies Reagent Set according to the manufacturer’s instructions. 100 ng of RNA of each sample (n=4 fetuses and placentas per maternal age group per tissue, only male offspring were selected for microarrays purposes) was labeled using Low Input QuickAmp Labeling Kit One-Color (Agilent Technologies, USA) and hybridized on the microarrays according to the manufacturer’s protocol. Statistical analysis of raw data was carried out using the default protocol of the Partek Genomics Suite. Microarrays analyses were conducted in n=4 conceptuses per maternal age group, obtained from 4 different litter of old and young mothers.

产妇高龄是子代神经发育障碍的一项危险因素。本研究旨在探究年轻与高龄C57BL6/J品系雌性小鼠的胎盘及胎头基因表达差异。于胚胎期12.5天（embryonic day 12.5, E12.5）收集胚胎，并开展转录组mRNA基因表达微阵列分析。相较于胎盘组的51个差异表达基因（differentially expressed genes, DEG），胎头组的基因表达差异更为显著，共涉及398个DEG。研究对象为妊娠的BTBR及B6品系雌鼠：其中高龄产妇组（advanced maternal aged, AMA）雌鼠周龄为42~65周，年轻对照组（young maternal aged, YMA）雌鼠周龄为10~23周，收集其妊娠子代的胎鼠及胎盘。按照试剂盒说明书，从胎盘及胎头中提取用于转录组分析的RNA。总RNA提取采用通用RNA纯化试剂盒（Eurx，波兰），并配套进行DNase处理（Eurx，波兰）。使用Nanodrop 1000分光光度计（赛默飞世尔科技，美国Thermo Fisher Scientific）对提取的核酸的浓度与纯度进行验证。选取部分样本，额外采用Bioanalyzer 2100微毛细管电泳装置（安捷伦科技，美国Agilent Technologies）验证RNA完整性。按照试剂盒说明书，采用SurePrint G3小鼠基因表达芯片v2 8x60K（GPL21163，安捷伦科技，美国Agilent Technologies）及安捷伦试剂套装开展全基因组转录组分析。每个样本取100 ng RNA（按母体年龄组及组织类型分组，每组每个组织对应4个胎鼠及胎盘样本，且仅选取雄性子代用于微阵列实验），采用低起始量QuickAmp单色标记试剂盒（安捷伦科技，美国Agilent Technologies）进行标记，并按照试剂盒流程完成芯片杂交。采用Partek Genomics Suite软件的默认流程对原始数据进行统计分析。本研究的微阵列分析共纳入每个母体年龄组的4个胚胎样本，分别来自高龄与年轻雌鼠的4窝子代。