Yifan Liu1, Ming Han2,3, Xiaoshuang Li4,5, Hui Wang1, Minyue Ma1, Shihui Zhang1, Yifan Guo1, Shuling Wang1, Yuanfen Wang1, Na Duan6, Bing Xu1, Jingwen Yin6, Yuanqing Yao1. 1. Department of Obstetrics and Gynecology, Chinese PLA General Hospital and Chinese PLA Medical School, 28 Fuxing Road, Beijing 100853, China. 2. Peking University Ditan Teaching Hospital, 8 Jingshun East Street, Beijing 100015, China. 3. Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, 8 Jingshun East Street, Beijing 100015, China. 4. Naval Clinical College, Anhui Medical University, Meishan Road, Hefei 230032, China. 5. Department of Obstetrics and Gynecology, PLA Navy General Hospital, 6 Fucheng Road, Beijing 100048, China. 6. Clinical Medical College, Nankai University, 94 Weijin Road, Tianjin 300071, China.
Abstract
STUDY QUESTION: What changes in the mitochondria of human mural granulosa cells (mGCs) with maternal aging? SUMMARY ANSWER: The mitochondrial membrane potential (MMP) and the ability of oxidative phosphorylation (OXPHOS) of mGCs declines with reproductive aging, accompanied with more abnormal mitochondria. WHAT IS KNOWN ALREADY: Mitochondria play an important role in the dialogue between the mGCs and oocytes. However, the underlying mechanism of mitochondrial dysfunction in mGCs in aging is still poorly understood. STUDY DESIGN SIZE, DURATION: In total, 149 infertile women underwent IVF in the ART Centre of the Chinese PLA General Hospital, China from September 2016 to May 2017. Two age groups were investigated: the young group (<38 years old) and the old group (≥38 years old). PARTICIPANTS/MATERIALS, SETTING, METHODS: The mitochondrial ultrastructure of mGCs was observed by transmission electron microscopy, and real-time quantitative polymerase chain reaction was applied to quantify the mitochondrial DNA (mtDNA) copy number, 4977-bp deleted DNA and mRNA expression of mitochondrial ATP synthases ATP5A1 and ATP5I. MMP was detected by flow cytometry and fluorescence microscopy, respectively. Reactive oxygen species (ROS) was tested by flow cytometry. A luminometer was used to measure the ATP levels and western blot to analyse the OXPHOS complex. MAIN RESULTS AND THE ROLE OF CHANCE: In the young group, mitochondria were mostly round or oval, with a few intact parallel tubular-vesicular cristae and homogenous matrix density, while elongated mitochondria were mainly observed in the old group, which had numerous cristae and more high-density matrix particles. Abnormal mitochondria were more common in aging women (P = 0.012). mtDNA relative copy number was positively correlated with maternal age (r = 0.294, P = 0.009) and we found no one with 4977-bp deleted mitochondria. JC-1 (dye used as an indicator of MMP) ratio in the old group was significantly lower than the young group (3.01 ± 0.21 vs 3.85 ± 0.27, P = 0.033). Intracellular ROS levels between the groups did not differ significantly (P = 0.191). The intracellular ATP level in the young group was 1.75-fold higher than that of the advanced-age group (7.17 ± 1.16 vs 4.15 ± 0.60, P = 0.025). The protein expression of ATP5A1, as one of five proteins of OXPHOS, decreased with aging (P < 0.001). ATP5A1 mRNA expression was negatively correlated with aging (r = -0.341, P = 0.012). LIMITATIONS REASONS FOR CAUTION: The quantity of mGCs from some individual patient, especially an advanced-age individual, was small, which cannot meet the demands of all the detections. WIDER IMPLICATIONS OF THE FINDINGS: mGCs dysfunction with aging is mainly linked to impaired mitochondrial function, especially OXPHOS function. Improving the OXPHOS ability in mGCs should be the focus in resolving infertility among advanced age women and making mGCs the proper mitochondria donor cells in the autologous mitochondria transplantation to oocytes. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the grants of the National High Technology Research and Development Program of China, 863 Program No. SS2015AA020402, and the Key Projects of Military Medical Research, No. BWS11J058. There were no competing interests.
STUDY QUESTION: What changes in the mitochondria of human mural granulosa cells (mGCs) with maternal aging? SUMMARY ANSWER: The mitochondrial membrane potential (MMP) and the ability of oxidative phosphorylation (OXPHOS) of mGCs declines with reproductive aging, accompanied with more abnormal mitochondria. WHAT IS KNOWN ALREADY: Mitochondria play an important role in the dialogue between the mGCs and oocytes. However, the underlying mechanism of mitochondrial dysfunction in mGCs in aging is still poorly understood. STUDY DESIGN SIZE, DURATION: In total, 149 infertile women underwent IVF in the ART Centre of the Chinese PLA General Hospital, China from September 2016 to May 2017. Two age groups were investigated: the young group (<38 years old) and the old group (≥38 years old). PARTICIPANTS/MATERIALS, SETTING, METHODS: The mitochondrial ultrastructure of mGCs was observed by transmission electron microscopy, and real-time quantitative polymerase chain reaction was applied to quantify the mitochondrial DNA (mtDNA) copy number, 4977-bp deleted DNA and mRNA expression of mitochondrial ATP synthases ATP5A1 and ATP5I. MMP was detected by flow cytometry and fluorescence microscopy, respectively. Reactive oxygen species (ROS) was tested by flow cytometry. A luminometer was used to measure the ATP levels and western blot to analyse the OXPHOS complex. MAIN RESULTS AND THE ROLE OF CHANCE: In the young group, mitochondria were mostly round or oval, with a few intact parallel tubular-vesicular cristae and homogenous matrix density, while elongated mitochondria were mainly observed in the old group, which had numerous cristae and more high-density matrix particles. Abnormal mitochondria were more common in aging women (P = 0.012). mtDNA relative copy number was positively correlated with maternal age (r = 0.294, P = 0.009) and we found no one with 4977-bp deleted mitochondria. JC-1 (dye used as an indicator of MMP) ratio in the old group was significantly lower than the young group (3.01 ± 0.21 vs 3.85 ± 0.27, P = 0.033). Intracellular ROS levels between the groups did not differ significantly (P = 0.191). The intracellular ATP level in the young group was 1.75-fold higher than that of the advanced-age group (7.17 ± 1.16 vs 4.15 ± 0.60, P = 0.025). The protein expression of ATP5A1, as one of five proteins of OXPHOS, decreased with aging (P < 0.001). ATP5A1 mRNA expression was negatively correlated with aging (r = -0.341, P = 0.012). LIMITATIONS REASONS FOR CAUTION: The quantity of mGCs from some individual patient, especially an advanced-age individual, was small, which cannot meet the demands of all the detections. WIDER IMPLICATIONS OF THE FINDINGS: mGCs dysfunction with aging is mainly linked to impaired mitochondrial function, especially OXPHOS function. Improving the OXPHOS ability in mGCs should be the focus in resolving infertility among advanced age women and making mGCs the proper mitochondria donor cells in the autologous mitochondria transplantation to oocytes. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the grants of the National High Technology Research and Development Program of China, 863 Program No. SS2015AA020402, and the Key Projects of Military Medical Research, No. BWS11J058. There were no competing interests.