Lin Nuan, van Zomeren Koen C, Plosch Torsten, Hofsink Naomi, van Veen Teelkien, Li Hui Ting, Lin Jiazhe, Zhou Xiaoling, Groen Henk, Tietge Uwe J F, Cantineau Astrid, Schirhagl Romana, Hoek Annemieke
Department of Obstetrics and Gynecology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
Department of Obstetrics and Gynecology, The First Affiliated Hospital of Shantou University Medical College, Shantou, China.
Hum Reprod Open. 2025 Feb 17;2025(2):hoaf007. doi: 10.1093/hropen/hoaf007. eCollection 2025.
Does FSH induce free radical generation with substantial oxidative damage in human cumulus granulosa cells (cGCs) and mural granulosa cells (mGCs)?
FSH of both physiological and supraphysiological concentrations induced free radical generation on subcellular levels, most notably in the mitochondria, while the elevated free radical load caused neglectable oxidative damage in both cGCs and mGCs.
FSH is fundamental for regulation of granulosa cell (GC) function and oocyte maturation, during which a physiological level of reactive oxygen species (ROS) is essential, while excessive amounts lead to oxidative damage. Potential adverse effects of high FSH doses on GCs may be mediated by ROS.
This prospective experimental study included patients who attended a reproductive medicine center in 2023. cGC and mGC were separately isolated and brought into culture on the day of oocyte retrieval, 36 h after ovulation induction with recombinant hCG (250 mg). Recombinant FSH, at different concentrations, mimicking physiological (6 mIU/ml) and supraphysiological (60 and 600 mIU/ml) conditions, was applied (n = 4 in each group).
PARTICIPANTS/MATERIALS SETTING METHODS: Women aged 20-35 years, undergoing ICSI with at least three follicles, were included. Quantum sensing of cGC and mGC free radicals, detected by either cytoplasm-located fluorescent nanodiamonds (FNDs) or mitochondria-targeted FNDs, was tracked for 2 h following FSH treatment in a magnetometry setup. Mitochondrial function analysis, as well as oxidative damage to DNA/RNA, lipids, and proteins, upon FSH exposure, was examined.
FSH-induced cytoplasmic and mitochondrial ROS increases in cGC and mGC ( < 0.01 in all concentrations after 2 h) while showing different patterns along time: cGC showed significantly larger cytoplasmic ROS change compared with mGC to physiological ( < 0.01) and supraphysiological ( < 0.05) concentrations of FSH. Significantly larger free radical changes were observed in the mitochondria compared to the cytoplasm in response to FSH (all concentrations in cGCs with < 0.05; supraphysiological concentrations in mGCs with < 0.05, < 0.001, respectively) after 2 h. Mitochondrial basal respiration and ATP production were significantly increased upon FSH exposure to supraphysiological concentrations in both cGCs ( < 0.01) and mGCs ( < 0.05). However, no oxidative damage to GC DNA/RNA, proteins, or lipids was found upon FSH exposure at any concentration except elevated lipid peroxidation in the FSH group of 600 mIU/ml ( < 0.05).
N/A.
The GCs came from females of different biological backgrounds and were stimulated before oocyte and GC retrieval, thereby increasing the risk of variation. In addition, the effects of long-term FSH exposure as well as the effects of the FSH-induced ROS on the oocyte remain to be investigated.
We demonstrate that FSH of both physiological and supraphysiological concentrations induces free radical generation at subcellular levels, most notably in the mitochondria, while the elevated free radical load causes neglectable oxidative damage in both cGCs and mGCs. Our results suggest that the 'FSH Ootoxicity' hypothesis would not seem to be mediated by ROS in human GCs.
STUDY FUNDING/COMPETING INTERESTS: This study is supported by the Abel Tasman Talent Program (ATTP) of the Graduate School of Medical Sciences of the University Medical Center Groningen/University of Groningen, The Netherlands, as well as an XS grant from NWO. Unrelated to the current work, A.H. is a member of an advisory board on the development and application of a lifestyle App for patients with infertility from Ferring Pharmaceutical Company, The Netherlands. R.S. is the founder of QT Sense B.V., who commercialize quantum sensing equipment. This article has no direct relation to the work of QT Sense B.V. The remaining authors have no conflicts of interest.
促卵泡激素（FSH）是否会在人卵丘颗粒细胞（cGCs）和壁层颗粒细胞（mGCs）中诱导自由基生成并造成实质性氧化损伤？
生理浓度和超生理浓度的FSH均在亚细胞水平诱导自由基生成，最显著的是在线粒体中，而自由基负荷升高在cGCs和mGCs中均造成可忽略不计的氧化损伤。
FSH对颗粒细胞（GC）功能和卵母细胞成熟的调节至关重要，在此过程中，生理水平的活性氧（ROS）必不可少，而过量的ROS会导致氧化损伤。高剂量FSH对GCs的潜在不良影响可能由ROS介导。
研究设计、规模、持续时间：这项前瞻性实验研究纳入了2023年到一家生殖医学中心就诊的患者。在注射重组人绒毛膜促性腺激素（250mg）诱导排卵36小时后的取卵日，分别分离cGC和mGC并进行培养。应用不同浓度的重组FSH，模拟生理（6mIU/ml）和超生理（60和600mIU/ml）条件（每组n = 4）。
参与者/材料、设置、方法：纳入年龄在20 - 35岁、接受至少有三个卵泡的卵胞浆内单精子注射（ICSI）的女性。在磁力测量装置中，用位于细胞质的荧光纳米金刚石（FNDs）或靶向线粒体的FNDs检测cGC和mGC自由基的量子传感，在FSH处理后跟踪2小时。检测FSH暴露后线粒体功能分析以及对DNA/RNA、脂质和蛋白质的氧化损伤。
FSH诱导cGC和mGC中细胞质和线粒体ROS增加（2小时后所有浓度下P均<0.01），同时随时间呈现不同模式：与mGC相比，cGC对生理浓度（P<0.01）和超生理浓度（P<0.05）的FSH显示出显著更大的细胞质ROS变化。2小时后，与细胞质相比，FSH刺激后线粒体中观察到显著更大的自由基变化（cGCs中所有浓度下P<0.05；mGCs中超生理浓度下P分别<0.05、<0.001）。FSH暴露于超生理浓度时，cGCs（P<0.01）和mGCs（P<0.05）中线粒体基础呼吸和ATP产生均显著增加。然而，除600mIU/ml的FSH组脂质过氧化升高（P<0.05）外，任何浓度的FSH暴露均未发现对GC DNA/RNA、蛋白质或脂质的氧化损伤。
无。
局限性、谨慎理由：GCs来自不同生物学背景的女性，并且在取卵和GCs获取之前受到刺激，从而增加了变异风险。此外，FSH长期暴露的影响以及FSH诱导的ROS对卵母细胞的影响仍有待研究。
我们证明生理浓度和超生理浓度的FSH均在亚细胞水平诱导自由基生成，最显著的是在线粒体中，而自由基负荷升高在cGCs和mGCs中均造成可忽略不计的氧化损伤。我们的结果表明，“FSH卵毒性”假说似乎在人GCs中不由ROS介导。
研究资金/利益冲突：本研究得到荷兰格罗宁根大学医学中心/格罗宁根大学医学科学研究生院的阿贝尔·塔斯曼人才计划（ATTP）以及荷兰科学研究组织（NWO）的XS资助。与当前工作无关，A.H.是荷兰辉凌制药公司为不育症患者开发和应用生活方式应用程序的咨询委员会成员。R.S.是QT Sense B.V.的创始人，该公司将量子传感设备商业化。本文与QT Sense B.V.的工作无直接关系。其余作者无利益冲突。
