Center for Reproductive Medicine, Zhongshan Hospital, NHC Key Lab of Reproduction Regulation, Shanghai Institute for Biomedical and Pharmaceutical Technologies, Pharmacy School, Fudan University, Shanghai, China.
State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China.
Hum Reprod. 2023 Sep 5;38(9):1723-1732. doi: 10.1093/humrep/dead155.
What are the differences in gene expression of cumulus cells (CCs) between young women with diminished ovarian reserve (DOR) and those of similar age with normal ovarian reserve (NOR)?
Gene expression and metabolome profiling analysis demonstrate that the de novo serine synthesis pathway (SSP) is increased in the CCs of young women with DOR.
The incidence of DOR has risen, tending to present at younger ages. Its mechanisms and aetiologies are still poorly understood. Abnormal metabolism is present in luteinized CCs of patients with DOR. Previous studies have revealed that mitochondrial dysfunction and impaired oxidative phosphorylation in CCs are related to DOR in women of advanced age. The pathogenic mechanisms likely differ between young women with DOR and cases associated with advanced maternal age. Several studies have examined amino acid metabolism in the follicle, with a focus on embryo development, but less information is available about CCs. The physiological significance of de novo serine synthesis in follicles and oocytes remains largely unknown.
STUDY DESIGN, SIZE, DURATION: CC samples were obtained from 107 young infertile women (age <38 years) undergoing ICSI, from July 2017 to June 2019, including 54 patients with DOR and 53 patients with NOR.
PARTICIPANTS/MATERIALS, SETTING, METHODS: Oocyte development data were analysed retrospectively. Comprehensive genome-wide transcriptomics of CCs was performed. Differentially expressed genes (DEGs) were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to categorize the functions of the DEGs and identify significantly enriched pathways. The transcript and protein levels of key enzymes involved in serine synthesis were verified in additional samples using quantitative real-time PCR (qRT-PCR) (n = 10) and capillary western blotting (n = 36). Targeted metabolomics of amino acids in CC extracts was performed by ultrahigh-performance liquid MS (UHPLC-MS/MS).
The number of oocytes (2.4 ± 2.2 versus 12.1 ± 5.3) and metaphase II oocytes (2.1 ± 2.0 versus 9.9 ± 4.9) retrieved was significantly decreased in the DOR versus the NOR group, respectively (P < 0.0001). The rates of fertilization (80.7% versus 78.8%), viable embryos (73.7% versus 72.5%), and high-quality embryos (42.8% versus 49.0%) did not differ between the DOR and NOR groups, respectively (P > 0.05). A total of 95 DEGs were found by transcriptome sequencing. GO and KEGG analyses demonstrated that the DEGs were linked to amino acid metabolism and suggested significantly higher activity of the de novo SSP in the CCs of young women with DOR. Further qRT-PCR and capillary western blotting revealed that key enzymes (PHGDH, PSAT1, PSPH, and SHMT2) involved in de novo serine synthesis were upregulated, and UHPLC-MS/MS analysis showed increases in serine and glycine (a downstream product of serine) levels in the CCs of young patients with DOR. Our data clearly demonstrate that the de novo SSP, which diverts 3-phosphoglycerate from glycolysis to serine synthesis, was upregulated in young DOR CCs.
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LIMITATIONS, REASONS FOR CAUTION: Regarding the reproductive capacity of young patients DOR, the pregnancy outcomes were not analysed. The sample size was limited, and only women undergoing ICSI were examined since this was a prerequisite for the acquisition of CCs, which may cause selection bias. The exact mechanisms by which the SSP in CCs regulates ovarian reserve still require further study.
Our research presents new evidence that alterations of the SSP in CCs of young infertile women are associated with DOR. We believe this is a significant contribution to the field, which should be key for understanding the cause and mechanisms of ovarian hypofunction in young women.
STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from the Ministry of Science and Technology of China (2018YFC1005001) and National Natural Science Foundation of China (31601197). There were no competing interests.
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年轻卵巢储备功能降低（DOR）女性和相似年龄卵巢储备正常（NOR）女性的卵丘细胞（CC）的基因表达有何不同？
基因表达和代谢组学分析表明，年轻 DOR 女性的 CC 中从头丝氨酸合成途径（SSP）增加。
DOR 的发病率上升，且趋于年轻化。其机制和病因仍知之甚少。DOR 患者的黄体化 CC 中存在异常代谢。先前的研究表明，CC 中线粒体功能障碍和氧化磷酸化受损与高龄女性的 DOR 有关。年轻 DOR 女性与与高龄相关的病例的发病机制可能不同。一些研究检查了卵泡中氨基酸代谢，重点是胚胎发育，但关于 CC 的信息较少。卵泡和卵母细胞中从头丝氨酸合成的生理意义在很大程度上仍然未知。
研究设计、规模、持续时间：2017 年 7 月至 2019 年 6 月，从 107 名接受 ICSI 的年轻不孕女性（年龄<38 岁）中获得 CC 样本，包括 54 例 DOR 患者和 53 例 NOR 患者。
参与者/材料、设置、方法：回顾性分析卵母细胞发育数据。对 CC 进行全基因组转录组分析。鉴定差异表达基因（DEGs）。进行基因本体论（GO）和京都基因与基因组百科全书（KEGG）分析，对 DEGs 的功能进行分类，并确定显著富集的途径。使用实时定量 PCR（qRT-PCR）（n=10）和毛细管 Western 印迹（n=36）在额外样本中验证参与丝氨酸合成的关键酶的转录和蛋白水平。通过超高效液相 MS（UHPLC-MS/MS）对 CC 提取物中的氨基酸进行靶向代谢组学分析。
DOR 组与 NOR 组相比，获得的卵母细胞（2.4±2.2 与 12.1±5.3）和中期 II 卵母细胞（2.1±2.0 与 9.9±4.9）数量显著减少（P<0.0001）。受精率（80.7%与 78.8%）、活胚胎率（73.7%与 72.5%）和优质胚胎率（42.8%与 49.0%）在 DOR 组与 NOR 组之间无差异（P>0.05）。通过转录组测序共发现 95 个 DEGs。GO 和 KEGG 分析表明，DEGs 与氨基酸代谢有关，并表明年轻 DOR 女性的 CC 中从头 SSP 的活性显著升高。进一步的 qRT-PCR 和毛细管 Western 印迹显示，参与从头丝氨酸合成的关键酶（PHGDH、PSAT1、PSPH 和 SHMT2）上调，UHPLC-MS/MS 分析显示 DOR 年轻患者的 CC 中丝氨酸和甘氨酸（丝氨酸的下游产物）水平升高。我们的数据清楚地表明，年轻 DOR CC 中的从头 SSP，即 3-磷酸甘油酸从糖酵解转移到丝氨酸合成，上调。
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局限性、谨慎的原因：关于年轻 DOR 患者的生殖能力，未分析妊娠结局。样本量有限，仅检查了接受 ICSI 的女性，因为这是获得 CC 的前提条件，这可能会导致选择偏差。CC 中 SSP 调节卵巢储备的确切机制仍需要进一步研究。
我们的研究提供了新的证据，表明年轻不孕妇女 CC 中的 SSP 改变与 DOR 有关。我们认为这是该领域的一项重大贡献，对于理解年轻女性卵巢功能低下的原因和机制至关重要。
研究资助/利益冲突：这项工作得到了中国科技部（2018YFC1005001）和国家自然科学基金（31601197）的支持。没有竞争利益。
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