Lee A W T, Ng J K W, Liao J, Luk A C, Suen A H C, Chan T T H, Cheung M Y, Chu H T, Tang N L S, Zhao M P, Lian Q, Chan W Y, Chan D Y L, Leung T Y, Chow K L, Wang W, Wang L H, Chen N C H, Yang W J, Huang J Y, Li T C, Lee T L
Developmental and Regenerative Biology Program, School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, PR China.
Department of Chemical Pathology, and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, PR China.
Hum Reprod. 2021 Jun 18;36(7):1907-1921. doi: 10.1093/humrep/deab100.
What is the transcriptome signature associated with poor performance of rescue IVM (rIVM) oocytes and how can we rejuvenate them?
The GATA-1/CREB1/WNT signalling axis was repressed in rIVM oocytes, particularly those of poor quality; restoration of this axis may produce more usable rIVM oocytes.
rIVM aims to produce mature oocytes (MII) for IVF through IVM of immature oocytes collected from stimulated ovaries. It is not popular due to limited success rate in infertility treatment. Genetic aberrations, cellular stress and the absence of cumulus cell support in oocytes could account for the failure of rIVM.
STUDY DESIGN, SIZE, DURATION: We applied single-cell RNA sequencing (scRNA-seq) to capture the transcriptomes of human in vivo oocytes (IVO) (n = 10) from 7 donors and rIVM oocytes (n = 10) from 10 donors. The effects of maternal age and ovarian responses on rIVM oocyte transcriptomes were also studied. In parallel, we studied the effect of gallic acid on the maturation rate of mouse oocytes cultured in IVM medium with (n = 84) and without (n = 85) gallic acid.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Human oocytes were collected from donors aged 28-41 years with a body mass index of <30. RNA extraction, cDNA generation, library construction and sequencing were performed in one preparation. scRNA-seq data were then processed and analysed. Selected genes in the rIVM versus IVO comparison were validated by quantitative real-time PCR. For the gallic acid study, we collected immature oocytes from 5-month-old mice and studied the effect of 10-μM gallic acid on their maturation rate.
The transcriptome profiles of rIVM/IVO oocytes showed distinctive differences. A total of 1559 differentially expressed genes (DEGs, genes with at least 2-fold change and adjusted P < 0.05) were found to be enriched in metabolic processes, biosynthesis and oxidative phosphorylation. Among these DEGs, we identified a repression of WNT/β-catenin signalling in rIVM when compared with IVO oocytes. We found that oestradiol levels exhibited a significant age-independent correlation with the IVO mature oocyte ratio (MII ratio) for each donor. rIVM oocytes from women with a high MII ratio were found to have over-represented cellular processes such as anti-apoptosis. To further identify targets that contribute to the poor clinical outcomes of rIVM, we compared oocytes collected from young donors with a high MII ratio with oocytes from donors of advanced maternal age and lower MII ratio, and revealed that CREB1 is an important regulator. Thus, our study identified that GATA-1/CREB1/WNT signalling was repressed in both rIVM oocytes versus IVO oocytes and in rIVM oocytes of lower versus higher quality. Consequently we investigated gallic acid, as a potential antioxidant substrate in human rIVM medium, and found that it increased the mouse oocyte maturation rate by 31.1%.
Raw data from this study can be accessed through GSE158539.
LIMITATIONS, REASONS FOR CAUTION: In the rIVM oocytes of the high- and low-quality comparison, the number of samples was limited after data filtering with stringent selection criteria. For the oocyte stage identification, we were unable to predict the presence of oocyte spindle, so polar body extrusion was the only indicator.
This study showed that GATA-1/CREB1/WNT signalling was repressed in rIVM oocytes compared with IVO oocytes and was further downregulated in low-quality rIVM oocytes, providing us the foundation of subsequent follow-up research on human oocytes and raising safety concerns about the clinical use of rescued oocytes.
STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Collaborative Research Fund, Research Grants Council, C4054-16G, and Research Committee Funding (Research Sustainability of Major RGC Funding Schemes), The Chinese University of Hong Kong. The authors have no conflicts of interest to declare.
与挽救性体外成熟（rIVM）卵母细胞发育能力差相关的转录组特征是什么，以及如何使其恢复活力？
GATA-1/CREB1/WNT信号轴在rIVM卵母细胞中受到抑制，尤其是质量较差的卵母细胞；恢复该信号轴可能会产生更多可用的rIVM卵母细胞。
rIVM旨在通过对从受刺激卵巢中收集的未成熟卵母细胞进行体外成熟培养来产生用于体外受精（IVF）的成熟卵母细胞（MII期）。由于在不孕症治疗中的成功率有限，它并不常用。卵母细胞中的基因异常、细胞应激以及缺乏卵丘细胞支持可能是rIVM失败的原因。
研究设计、规模、持续时间：我们应用单细胞RNA测序（scRNA-seq）来获取7名供体的人类体内成熟卵母细胞（IVO）（n = 10）和10名供体的rIVM卵母细胞（n = 10）的转录组。还研究了母亲年龄和卵巢反应对rIVM卵母细胞转录组的影响。同时，我们研究了没食子酸对在添加（n = 84）和未添加（n = 85）没食子酸的IVM培养基中培养的小鼠卵母细胞成熟率的影响。
参与者/材料、设置、方法：从28至41岁、体重指数<30的供体收集人类卵母细胞。RNA提取、cDNA生成、文库构建和测序在一次制备中完成。然后对scRNA-seq数据进行处理和分析。通过定量实时PCR验证rIVM与IVO比较中选定的基因。对于没食子酸研究，我们从5月龄小鼠收集未成熟卵母细胞，并研究10 μM没食子酸对其成熟率的影响。
rIVM/IVO卵母细胞的转录组图谱显示出明显差异。共发现1559个差异表达基因（DEGs，变化至少2倍且校正P<0.05的基因）在代谢过程、生物合成和氧化磷酸化中富集。在这些DEGs中，我们发现与IVO卵母细胞相比，rIVM中WNT/β-连环蛋白信号受到抑制。我们发现雌二醇水平与每个供体的IVO成熟卵母细胞比例（MII比例）呈现出显著的与年龄无关的相关性。发现MII比例高的女性的rIVM卵母细胞具有诸如抗凋亡等过度表达的细胞过程。为了进一步确定导致rIVM临床结果不佳的靶点，我们将从MII比例高的年轻供体收集的卵母细胞与高龄且MII比例低的供体的卵母细胞进行比较，发现CREB1是一个重要的调节因子。因此，我们的研究发现GATA-1/CREB1/WNT信号在rIVM卵母细胞与IVO卵母细胞之间以及质量较低与较高的rIVM卵母细胞中均受到抑制。因此，我们研究了没食子酸作为人类rIVM培养基中潜在的抗氧化底物，发现它使小鼠卵母细胞成熟率提高了31.1%。
本研究的原始数据可通过GSE158539获取。
局限性、谨慎原因：在高质量与低质量rIVM卵母细胞的比较中，经过严格筛选标准的数据过滤后，样本数量有限。对于卵母细胞阶段的鉴定，我们无法预测卵母细胞纺锤体的存在，因此极体排出是唯一的指标。
本研究表明，与IVO卵母细胞相比，rIVM卵母细胞中GATA-1/CREB1/WNT信号受到抑制，并且在低质量rIVM卵母细胞中进一步下调，为我们后续对人类卵母细胞的研究提供了基础，并引发了对挽救卵母细胞临床应用的安全担忧。
研究资金/利益冲突：本研究得到了香港中文大学研究资助局协作研究基金C4054 - 16G以及研究委员会资助（研究资助局主要资助计划的研究可持续性）的支持。作者声明无利益冲突。
