The impact of maternal age on gene expression during the GV to MII transition in euploid human oocytes.
The impact of maternal age on gene expression during the GV to MII transition in euploid human oocytes.
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母亲年龄对整倍体人卵母细胞GV向MII转化过程中基因表达的影响。
DOI：
10.1093/humrep/deab226
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发表时间：
2021-12-27
期刊：
影响因子：
--
通讯作者：
Picton HM
中科院分区：
文献类型：
--
作者：
Ntostis P;Iles D;Kokkali G;Vaxevanoglou T;Kanavakis E;Pantou A;Huntriss J;Pantos K;Picton HM
在整倍体人卵母细胞从生发泡（GV）到中期II（MII）的转变过程中，是否存在与年龄相关的基因表达差异？观察到从GV到MII卵母细胞的胚胎相关转录物的减少，随着年龄的增长，MII卵母细胞的减少幅度更大。早期胚胎发育依赖于卵子发生过程中卵母细胞内积累和储存的母体转录物。卵母细胞的转录活性决定了其最终的发育潜力，可能受年龄的影响，并解释了与年轻母亲年龄（YMA）相比，高龄母亲年龄（AMA）卵母细胞的能力降低。已经在人类和动物卵母细胞中研究了基因表达;然而，RNA测序可以进一步了解YMA和AMA患者的GV和体内成熟的MII整倍体卵母细胞的转录组谱。15名在一个IVF单位接受不孕症治疗的妇女同意参加这项研究。捐献来自6名21-26岁女性（YMA队列）的5个GV和5个MII卵母细胞以及来自6名41-44岁接受IVF治疗的女性（AMA队列）的5个GV和6个MII卵母细胞。样本是在4个月的时间范围内收集的。分离RNA并在单细胞水平上进行深度测序。所有供体提供GV或MII卵母细胞。hCG注射后38 h从供体卵母细胞中进行卵丘解剖，将裸露的卵母细胞插入补充有RNA酶抑制剂的裂解缓冲液中。将样品储存在-80 °C下，直至进一步使用。使用寡脱氧胸苷（dT）引发方法（SMART-Seq v4超低输入RNA测定法; Takara Bio，日本）和Nextera XT DNA文库制备测定法（Illumina，美国）进行从GV和MII卵母细胞分离的RNA的文库制备，然后进行深度测序。利用FastQC、HISAT 2、StringTie和edgeR等软件进行数据处理、质量评价和生物信息学分析，沿着功能注释分析，利用scploid R软件包进行倍性鉴定。在YMA和AMA队列中对单个GV和MII卵母细胞进行深度测序后，发现数百个转录本以显著不同的水平表达。当比较YMA和AMA MII卵母细胞转录组时，其中最重要的与线粒体结构和功能有关，包括生物学过程，线粒体呼吸链复合物I组装和线粒体翻译终止（错误发现率（FDR）6.0E−10至1.2E−7）。这些结果表明，随着年龄的增长，YMA MII队列的能量潜力更高。在YMA MII队列中显著更高的其他生物过程包括参与翻译过程的转录本（FDR 1.9E−2）。缺乏这些转录本可能导致AMA MII卵母细胞受精前或受精后蛋白质合成不当。RNA测序数据以登录号GSE 164371保藏在Gene Expression Omnibus（https：//www.ncbi.nlm.nih.gov/geo）中。样本量相对较小可能是谨慎的原因。然而，RNA测序结果显示具有低组内变异的同质聚类，并且来自每组至少三名不同女性的五到六个生物重复使样本量的潜在影响最小化。了解衰老对卵母细胞转录组的影响可以突出GV到MII转变的机制，并确定证明MII卵母细胞质量良好的生物标志物。这些知识有可能指导AMA患者的IVF方案。这项工作得到了医学研究理事会的支持（MRC资助号MR/K 020501/1）。
Are there age-related differences in gene expression during the germinal vesicle (GV) to metaphase II (MII) stage transition in euploid human oocytes? A decrease in mitochondrial-related transcripts from GV to MII oocytes was observed, with a much greater reduction in MII oocytes with advanced age. Early embryonic development is dependent on maternal transcripts accumulated and stored within the oocyte during oogenesis. Transcriptional activity of the oocyte, which dictates its ultimate developmental potential, may be influenced by age and explain the reduced competence of advanced maternal age (AMA) oocytes compared with the young maternal age (YMA). Gene expression has been studied in human and animal oocytes; however, RNA sequencing could provide further insights into the transcriptome profiling of GV and in vivo matured MII euploid oocytes of YMA and AMA patients. Fifteen women treated for infertility in a single IVF unit agreed to participate in this study. Five GV and 5 MII oocytes from 6, 21–26 years old women (YMA cohort) and 5 GV and 6 MII oocytes from 6, 41–44 years old women (AMA cohort) undergoing IVF treatment were donated. The samples were collected within a time frame of 4 months. RNA was isolated and deep sequenced at the single-cell level. All donors provided either GV or MII oocytes. Cumulus dissection from donated oocytes was performed 38 h after hCG injection, denuded oocytes were inserted into lysis buffer supplemented with RNase inhibitor. The samples were stored at −80°C until further use. Isolated RNA from GV and MII oocytes underwent library preparation using an oligo deoxy-thymidine (dT) priming approach (SMART-Seq v4 Ultra Low Input RNA assay; Takara Bio, Japan) and Nextera XT DNA library preparation assay (Illumina, USA) followed by deep sequencing. Data processing, quality assessment and bioinformatics analysis were performed using source-software, mainly including FastQC, HISAT2, StringTie and edgeR, along with functional annotation analysis, while scploid R package was employed to determine the ploidy status. Following deep sequencing of single GV and MII oocytes in both YMA and AMA cohorts, several hundred transcripts were found to be expressed at significantly different levels. When YMA and AMA MII oocyte transcriptomes were compared, the most significant of these were related to mitochondrial structure and function, including biological processes, mitochondrial respiratory chain complex I assembly and mitochondrial translational termination (false discovery rate (FDR) 6.0E−10 to 1.2E−7). These results indicate a higher energy potential of the YMA MII cohort that is reduced with ageing. Other biological processes that were significantly higher in the YMA MII cohort included transcripts involved in the translation process (FDR 1.9E−2). Lack of these transcripts could lead to inappropriate protein synthesis prior to or upon fertilisation of the AMA MII oocytes. The RNA sequencing data were deposited in the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo), under the accession number: GSE164371. The relatively small sample size could be a reason for caution. However, the RNA sequencing results showed homogeneous clustering with low intra-group variation and five to six biological replicates derived from at least three different women per group minimised the potential impact of the sample size. Understanding the effects of ageing on the oocyte transcriptome could highlight the mechanisms involved in GV to MII transition and identify biomarkers that characterise good MII oocyte quality. This knowledge has the potential to guide IVF regimes for AMA patients. This work was supported by the Medical Research Council (MRC Grant number MR/K020501/1).
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影响因子：
4
作者：
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通讯作者：
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影响因子：
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作者：
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作者：
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