title: Role of Sirt3 in mitochondrial biogenesis and developmental competence of human in vitro matured oocytes.
abstract: OBJECTIVE
  Does Sirt3 dysfunction result in poor developmental outcomes for human oocytes after in vitro maturation (IVM)?
  
  
  CONCLUSIONS
  Inefficient Sirt3 expression induced decreased mitochondrial DNA copy number and biogenesis, and therefore impaired the developmental competence of human IVM oocytes.
  
  
  BACKGROUND
  Cytoplasmic immaturity in IVM oocytes may lead to reduced developmental competence. Mitochondrial dysfunction results in the accumulation of free radicals and leads to DNA mutations, protein damage, telomere shortening and apoptosis. SIRT3 (in the Sirtuin protein family) has emerged as a mitochondrial fidelity protein that directs energy generation and regulates reactive oxygen species scavenging proteins.
  
  
  METHODS
  In vivo matured metaphase II (IVO-MII) oocytes and IVM-MII oocytes were donated by 324 infertile patients undergoing assisted reproductive technology cycles (12 patients for 60 IVO oocytes, and 312 patients for 403 IVM oocytes). Five oocytes each in the germinal vesicle (GV), IVM and IVO groups were compared with respect to mRNA levels for Sirt1-7 mRNA, and five samples at each developmental stage were analysed for Sirt3 mRNA. IVM-MII oocytes were injected with in vitro transcribed mRNA (n = 59) or small interfering RNA (siRNA) (n = 78). In human and mouse, IVM, mRNA-injection IVM, and siRNA-injection IVM groups (n = 5 each) were analysed for mitochondrial DNA copy number and abundance of Sirt3 and Pgc1α (an inducer of mitochondrial biogenesis) mRNAs. Human blastocysts in the IVO (n = 12), IVM (n = 9), mRNA-injection IVM (n = 13) and siRNA-injection IVM (n = 6) groups were used to generate embryonic stem cells (ESCs). In addition, 587 IVO-MII and 1737 IVM-MII oocytes from 83 mice were collected to compare the preliminary human oocyte data with another species.
  
  
  METHODS
  mRNA abundance was analysed by single-cell real-time PCR. Karyotyping of human embryos was performed with an array comparative genomic hybridization method, and that of ESCs by cytogenetic analysis. The function of the Sirt3 gene was investigated using siRNA and in vitro transcribed mRNA injection. Markers of ESCs were identified using immunofluorescence.
  
  
  RESULTS
  A retrospective analysis revealed a higher spontaneous abortion rate (P < 0.01) and decrease in high quality embryo rate (P < 0.01) in patients with IVM versus controlled ovarian stimulation (COS) cycles. A decrease in abundance of Sirt3 mRNA (P < 0.01) and mitochondrial biogenesis (P < 0.05) were identified in human IVM compared with IVO oocytes. The developmental potential of human IVM-MII oocytes to the blastocyst stage was significantly reduced when Sirt3 mRNA was inhibited by siRNA (P < 0.05 versus IVM-MII group) but could be up-regulated by injection of Sirt3 mRNAs. Compared with IVO-MII group, comparable generation efficiency of human ESCs can be obtained using blastocysts from IVM-MII oocytes with Sirt3 mRNA injection. Sirt3 mRNA was significantly increased in mouse zygotes after IVF (P < 0.001 versus MII oocytes) but gradually declined until the blastocyst stage. In mice, lower Sirt3 mRNA levels were observed IVM-MII oocytes and preimplantation embryos compared with in vivo controls, and mitochondrial biogenesis and the developmental efficiency from oocytes to blastocyst were affected by the abundance of Sirt3 mRNA in accordance with human. Therefore a similar role for Sirt3 mRNA in IVM-MII oocytes was observed in mouse and human.
  
  
  CONCLUSIONS
  The couples in the study had a variety of different simple and complex factors causing infertility. Additional studies with a larger number of oocytes are required to confirm the present results owing to the limited number of human oocytes in the present study.
  
  
  CONCLUSIONS
  To our knowledge, this is the first study investigating a role of the Sirt3 gene in mitochondrial biogenesis and the developmental competence of human IVM-MII oocytes. The observation may help to improve clinical application of the IVM procedure.
  
  
  BACKGROUND
  This work was supported in part by the National Natural Science Foundation of Key Program (31230047), Ministry of Science and Technology of China Grants (973 program; 2014CB943203), the National Natural Science Foundation of General Program (31371521 and 81571400), Beijing Nova Program (xxjh2015011), and Specialized Research Fund for the Doctoral Program of Higher Education (20120001130008) and the National Natural Science Foundation of Young Scholar (31501201). The authors have declared that no conflict of interest exists.
authors.affiliation: Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100191, China.
authors.forename: Hong-Cui
authors.initials: HC
authors.lastname: Zhao
authors.affiliation: Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100191, China Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing 100191, China Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing 100191, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Ding
authors.affiliation: Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100191, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Ren
authors.affiliation: Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100191, China.
authors.forename: Tian-Jie
authors.initials: TJ
authors.lastname: Li
authors.affiliation: Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100191, China Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing 100191, China Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing 100191, China.
authors.forename: Rong
authors.initials: R
authors.lastname: Li
authors.affiliation: Key Laboratory for Major Obstetric Diseases of Guangdong Province, the Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Fan
authors.affiliation: Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100191, China Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing 100191, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Yan
authors.affiliation: Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100191, China Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing 100191, China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100191, China Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing 100191, China Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing 100191, China.
authors.forename: Mo
authors.initials: M
authors.lastname: Li
authors.affiliation: Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100191, China Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing 100191, China Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing 100191, China yuyang5012@hotmail.com jie.qiao@263.net.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100191, China Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing 100191, China Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing 100191, China yuyang5012@hotmail.com jie.qiao@263.net.
authors.forename: Jie
authors.initials: J
authors.lastname: Qiao
mesh_terms: D000328:Adult; D000818:Animals; D055028:Comparative Genomic Hybridization; D053595:Embryonic Stem Cells / Q000378:metabolism; D005260:Female; D018628:Gene Dosage; D006801:Humans; D007150:Immunohistochemistry; D059471:In Vitro Oocyte Maturation Techniques; D007621:Karyotyping; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D009865:Oocytes / Q000254:growth & development / Q000378:metabolism*; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha / Q000235:genetics / Q000378:metabolism; D011247:Pregnancy; D011256:Pregnancy Outcome; D034622:RNA Interference; D012189:Retrospective Studies; D059010:Single-Cell Analysis; D056566:Sirtuin 3 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
chemical_list: D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; C466093:SIRT3 protein, human; D056566:Sirtuin 3
keywords: apoptosis; embryo development; in vitro maturation; mitochondrial biogenesis; oocyte