Role of Sirt3 in mitochondrial biogenesis and developmental competence of human in vitro matured oocytes.

STUDY QUESTION: Does Sirt3 dysfunction result in poor developmental outcomes for human oocytes after in vitro maturation (IVM)? SUMMARY ANSWER: Inefficient Sirt3 expression induced decreased mitochondrial DNA copy number and biogenesis, and therefore impaired the developmental competence of human IVM oocytes. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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. LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTERESTS: 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.

Assessment of ocular biomechanics using dynamic ultra high-speed Scheimpflug imaging in keratoconic and normal eyes.

PURPOSE: To introduce several new ocular biomechanical parameters for comparison between keratoconic and normal eyes using an analysis method based on corneal dynamic deformation video recorded by corneal visualization Scheimpflug technology (Corvis ST; Oculus Optikgeräte GmbH, Wetzlar, Germany). METHODS: This comparative study comprised 52 keratoconic eyes of 43 patients with keratoconus and 52 normal eyes of 52 controls. An analysis method (PolyU [Labview 2009; National Instrument, Austin, TX]) was developed to introduce several new ocular biomechanical parameters and to compare the difference between keratoconic and normal eyes. The repeatability of the new parameters measurement was evaluated and compared with the Corvis ST measurement. Receiver operating characteristic curves were used to establish a cutoff value for the new biomechanical parameters. RESULTS: Intraclass correlation coefficients of the deformation amplitude, peak distance, corneal concave radius of curvature, maximum deformation area, maximum corneal inward velocity and outward velocity (Vin, max and Vout, max) were high in both the keratoconic and normal eyes (all intraclass correlation coefficients > 0.75). The measurement agreement of the PolyU analysis method and Corvis ST was good. Most of the biomechanical parameters of patients with keratoconus were significantly different from those of the controls. In the receiver operating characteristic analysis, the Vin, max was the best predictive parameter with an area under the curve of 0.79. CONCLUSIONS: The corneal deformation video recorded by the Corvis ST provides useful information for the study of ocular biomechanics. Most of the new ocular biomechanical parameters were significantly different between keratoconic and normal eyes. Further research is needed to develop more comprehensive clinical applications with these new ocular biomechanical parameters.

Combining prostate-specific antigen density with prostate imaging reporting and data system score version 2.1 to improve detection of clinically significant prostate cancer: A retrospective study.

Globally, Prostate cancer (PCa) is the second most common cancer in the male population worldwide, but clinically significant prostate cancer (CSPCa) is more aggressive and causes to more deaths. The authors aimed to construct the risk category based on Prostate Imaging Reporting and Data System score version 2.1 (PI-RADS v2.1) in combination with Prostate-Specific Antigen Density (PSAD) to improve CSPCa detection and avoid unnecessary biopsy. Univariate and multivariate logistic regression and receiver-operating characteristic (ROC) curves were performed to compare the efficacy of the different predictors. The results revealed that PI-RADS v2.1 score and PSAD were independent predictors for CSPCa. Moreover, the combined factor shows a significantly higher predictive value than each single variable for the diagnosis of CSPCa. According to the risk stratification model constructed based on PI-RADS v2.1 score and PSAD, patients with PI-RADS v2.1 score of ≤2, or PI-RADS V2.1 score of 3 and PSA density of <0.15 ng/mL2, can avoid unnecessary of prostate biopsy and does not miss clinically significant prostate cancer.

Age and Sex Effects on the Active Stiffness of Vastus Intermedius under Isometric Contraction.

Previously, a novel technique was proposed to quantify the relationship between the muscle stiffness and its nonfatigue contraction intensity. The method extended the measured range of isometric contraction to 100% maximum voluntary contraction (MVC) using an ultrasonic shear wave measurement setup. Yet, it has not been revealed how this relationship could be affected by factors like age or sex. To clarify these questions, vastus intermedius (VI) stiffness of 40 healthy subjects was assessed under 11 step levels of isometric contraction. The subjects were divided into four groups: young males, young females, elderly males, and elderly females (n = 10 for each). In a relaxed state, no significant difference was observed between the male and female subjects (p = 0.156) nor between the young and elderly subjects (p = 0.221). However, when performing isometric contraction, the VI stiffness of males was found to be significantly higher than that of females at the same level (p < 0.001), and that of the young was higher than the elderly (p < 0.001). Meanwhile, for two knee joint angles used, the stiffness measured at a 90° knee joint angle was always significantly larger than that measured at 60° (p < 0.001). Recognizing the active muscle stiffness of VI contributes to body stability, and these results may provide insight into the age and sex bias in musculoskeletal studies, such as those on fall risks.

Shear modulus estimation on vastus intermedius of elderly and young females over the entire range of isometric contraction.

Elderly people often suffer from sarcopenia in their lower extremities, which gives rise to the increased susceptibility of fall. Comparing the mechanical properties of the knee extensor/flexors on elderly and young subjects is helpful in understanding the underlying mechanisms of the muscle aging process. However, although the stiffness of skeletal muscle has been proved to be positively correlated to its non-fatiguing contraction intensity by some existing methods, this conclusion has not been verified above 50% maximum voluntary contraction (MVC) due to the limitation of their measurement range. In this study, a vibro-ultrasound system was set up to achieve a considerably larger measurement range on muscle stiffness estimation. Its feasibility was verified on self-made silicone phantoms by comparing with the mechanical indentation method. The system was then used to assess the stiffness of vastus intermedius (VI), one of the knee extensors, on 10 healthy elderly female subjects (56.7 ± 4.9 yr) and 10 healthy young female subjects (27.6 ± 5.0 yr). The VI stiffness in its action direction was confirmed to be positively correlated to the % MVC level (R2 = 0.999) over the entire range of isometric contraction, i.e. from 0% MVC (relaxed state) to 100% MVC. Furthermore, it was shown that there was no significant difference between the mean VI shear modulus of the elderly and young subjects in a relaxed state (p > 0.1). However, when performing step isometric contraction, the VI stiffness of young female subjects was found to be larger than that of elderly participants (p < 0.001), especially at the relatively higher contraction levels. The results expanded our knowledge on the mechanical property of the elderly's skeletal muscle and its relationship with intensity of active contraction. Furthermore, the vibro-ultrasound system has a potential to become a powerful tool for investigating the elderly's muscle diseases.