Reproductive Medical Center, Peking University People's Hospital, Peking University, Beijing, China.
Hum Reprod. 2021 Oct 18;36(11):2904-2915. doi: 10.1093/humrep/deab203.
What is the relationship between mitochondria of granulosa cells (GCs) and age and ovarian function in the patients under the POSEIDON classification?
Our results revealed obvious abnormal mitochondrial-related changes in low prognosis IVF population, where age and the function of ovarian reserve exerted a divergent effect on mitochondrial content and function.
Mitochondria have an important role in the cross-talk between GCs and oocytes. However, factors affecting mitochondria of GCs and related mechanisms are still poorly understood.
STUDY DESIGN, SIZE, DURATION: GCs samples were obtained from 119 infertile women undergoing IVF from September 2020 to February 2021. Six groups were investigated by the POSEIDON stratification: young with normal prognosis (C1), aging with normal prognosis (C2), young and low prognosis group with normal ovarian reserve (NOR) (G1), aging and low prognosis group with NOR (G2), young and low prognosis group with diminished ovarian reserve (DOR) (G3), and aging and low prognosis group with DOR (G4).
PARTICIPANTS/MATERIALS, SETTING, METHODS: The morphology of GC mitochondria was observed by transmission electron microscopy. MtDNA copy number and mitochondrial replication-related genes were detected by real-time quantitative PCR (qPCR). Mitochondrial membrane potential (MMP) and cytosolic reactive oxygen species (ROS) were detected by confocal microscopy. Cellular glycolysis and aerobic respiratory capacity were analyzed by Seahorse XFe96 Analyzer, and related gene expression and protein levels were assessed by qPCR and Western blot.
Compared to the normal prognosis groups, mitochondrial morphology was impaired in the low prognosis groups, where the young groups (G1, G3) with low prognosis showed phenotypes undergoing oxidative stress (round, vacuolated, swollen with decreased matrix density) and the aging groups (G2, G4) revealed typical aging characteristics (an irregular shape with heterogeneous matrix density and cord-like cristae). Additionally, the degree of corresponding change and damage was more obvious in patients with DOR (G3, G4) regardless of age. For mitochondrial content, the mtDNA copy number in GCs was significantly negatively correlated with age in the low prognosis groups (β = -0.373, P = 0.005). Interestingly, the relationship between mtDNA copy number and anti-Mullerian hormone score differed between the two age groups with low prognosis, with a negative correlation in the young groups (β = -0.639, P = 0.049) and a positive correlation in the aging groups (β = 0.505, P = 0.039). In addition, significantly reduced mitochondrial activity (MMP, ROS) and cell metabolism (both glycolysis and OXPHOS) were observed in the low prognosis groups, with the most obvious decrease being observed in the DOR population. However, the metabolism of the GCs in normal prognosis aging women (C2) shifted from OXPHOS to anaerobic glycolysis.
LIMITATIONS, REASONS FOR CAUTION: Owing to the difficulties involved in primary GC collection and culture, the sample size was limited.
Mitochondrial abnormality is closely linked to the low prognostic outcome in IVF patients. Supplementing the functional mitochondrial content or improving mitochondrial function by autologous mitochondrial transfer or mitochondrial-related regulating drugs may help improve the clinical outcomes in patients with a low prognosis, especially for those with DOR.
STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (No. 21737001), the Peking University Clinical Medicine + X Youth Project (PKU2020LCXQ011), the Research and Development Program of Peking University People's Hospital (No. RDH2017-03; No. RDX2019-06) and the Application of Clinical Features of Capital Special Subject (Z171100001017130). There were no competing interests.
This study was registered with the Chinese Clinical Trial Register (Clinical Trial Number: ChiCTR2100045531).
在 POSEIDON 分类下，颗粒细胞（GC）的线粒体与年龄和卵巢功能之间存在什么关系？
我们的结果显示，低预后 IVF 人群中存在明显的异常线粒体相关变化，其中年龄和卵巢储备功能对线粒体含量和功能产生了不同的影响。
线粒体在 GC 和卵母细胞之间的相互作用中起着重要作用。然而，影响 GC 线粒体的因素和相关机制仍知之甚少。
研究设计、规模、持续时间：2020 年 9 月至 2021 年 2 月，从 119 名接受 IVF 的不孕妇女中获得 GC 样本。通过 POSEIDON 分层对 6 组进行研究：年轻且预后正常组（C1）、衰老且预后正常组（C2）、年轻且卵巢储备正常低预后组（NOR）（G1）、衰老且卵巢储备正常低预后组（NOR）（G2）、年轻且卵巢储备减少低预后组（DOR）（G3）和衰老且卵巢储备减少低预后组（DOR）（G4）。
参与者/材料、设置、方法：通过透射电子显微镜观察 GC 线粒体的形态。通过实时定量 PCR（qPCR）检测 mtDNA 拷贝数和线粒体复制相关基因。通过共聚焦显微镜检测线粒体膜电位（MMP）和细胞质活性氧（ROS）。通过 Seahorse XFe96 分析仪分析细胞糖酵解和有氧呼吸能力，并通过 qPCR 和 Western blot 评估相关基因表达和蛋白水平。
与预后正常组相比，低预后组的线粒体形态受损，其中低预后的年轻组（G1、G3）表现出氧化应激的表型（圆形、空泡化、基质密度降低的肿胀），而衰老组（G2、G4）则表现出典型的衰老特征（形状不规则，基质密度不均匀，嵴呈索状）。此外，DOR 患者（G3、G4）的相应变化和损伤程度更为明显，无论年龄大小。对于线粒体含量，GC 中的 mtDNA 拷贝数与低预后组的年龄呈显著负相关（β=-0.373，P=0.005）。有趣的是，mtDNA 拷贝数与抗苗勒管激素评分之间的关系在两个低预后年龄组之间存在差异，年轻组呈负相关（β=-0.639，P=0.049），而衰老组呈正相关（β=0.505，P=0.039）。此外，在低预后组中观察到线粒体活性（MMP、ROS）和细胞代谢（糖酵解和 OXPHOS 均）显著降低，DOR 组的降低最为明显。然而，正常预后衰老女性（C2）的 GC 代谢从 OXPHOS 转变为无氧糖酵解。
局限性、谨慎的原因：由于原发性 GC 采集和培养的困难，样本量有限。
线粒体异常与 IVF 患者的低预后结果密切相关。通过自体线粒体转移或与线粒体相关的调节药物补充功能性线粒体含量或改善线粒体功能，可能有助于改善低预后患者的临床结局，特别是对于 DOR 患者。
研究基金/利益冲突：本工作得到了国家自然科学基金（No.21737001）、北京大学临床医学+X 青年项目（PKU2020LCXQ011）、北京大学人民医院研发计划（No.RDH2017-03；No.RDX2019-06）和首都临床特色应用研究（Z171100001017130）的支持。没有竞争利益。
本研究在中国临床试验注册中心注册（临床试验编号：ChiCTR2100045531）。
