Age-related changes in the mitochondria of human mural granulosa cells.

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.

VEGF111b, a C-terminal splice variant of VEGF-A and induced by mitomycin C, inhibits ovarian cancer growth.

BACKGROUND: Alternative splicing of VEGF-A gives rise to two families - the pro-angiogenic VEGFxxx family and the anti-angiogenic VEGFxxxb family that differ by only six amino acids at their C-terminal end. The first verified and widely reported VEGFxxxb family member is VEGF165b, and here VEGF165b is a positive control. METHORDS: VEGF111b mRNA was detected in ovarian cancer cell lines SKOV3 and OVCAR3 by RT-PCR. Western blot was used to detect VEGF111b and VEGF165b protein in the CMs and lysates of OVCAR3 cells. MTT and colony formation assay were used to detect the short-term and long-term proliferation inhibition ability of ovarian cancer cells with VEGF111b overexpression. Cell-cycle analysis was performed to further characterize VEGF111b inhibition effects. VEGF111b signaling on ovarian cancer cells were determined by western blot. The expression levels of Ki67, PCNA, CD31 and VEGF in VEGF111b overexpression xenograft model were detected by immunohistochemistry. RESULTS: Under the effect of mitomycin C, we identify a new member of VEGFxxxb family-VEGF111b in ovarian cancer cell lines. SKOV3 and OVCAR cells were transfected with empty lentivirus, VEGF111b or VEGF165b lentivirus. VEGF111b and VEGF165b overexpression inhibits proliferation of the ovarian cancer cells, but inhibition effect of VEGF111b is slightly less efficient than VEGF165b. Cell cycle analysis was further used to elucidate the mechanism involved in the inhibition effect. Further, we detected the expression of VEGF-R2 in SKOV3 and OVCAR3 cells, and shown that VEGF111b might bind to conventional VEGF-R2 with the results of reducing VEGF-R2 tyrosine phosphorylation and downstream signaling to have anti-tumor effects. In vivo VEGF111b overexpression inhibits ovarian cancer growth in xenograft mice. CONCLUSION: Our results show that VEGF111b, as a new member of VEGFxxxb family, with similar properties to VEGF165b, plays potent anti-tumor effect in vitro and in vivo that can target the VEGF-R2 and its signaling pathway to inhibit ovarian tumor growth. This also opens a new avenue for treating ovarian cancer.

[A meta-analysis of preventing bone mineral loss in patients with endometriosis treated by gonadotrophin-releasing hormone analogues with add-back therapy].

OBJECTIVE: To evaluate the role and efficacy of preventing bone mineral loss in patients with endometriosis treated by gonadotrophin-releasing hormone analogues (GnRH-a) combined with add-back therapy. METHODS: Prospective, randomized controlled studies of the use of GnRHa with add-back therapy in treatment of endometriosis were enrolled in this study from Medline, Embase, Cochrane library, China National Knowledge Internet (CNKI), Chinese Biological Medicine Disk (CBM) and Data Base of Wanfang.After quality assessment and data extraction, meta-analysis were conducted in the change of BMD, reproductive hormone (E2) and visual pain score(VAS) by Stata 11.0 software. RESULTS: A total of 785 patients from 13 randomized controlled trail (RCT) studies enrolled in this study after exclude no following up, poor quality and repeat published studies.377 patients were in group of GnRH-a with add-back treatment and 408 patients were in group of GnRna alone.The findinds were showed in meta-analysis: (1) there was a significant difference in percentage change of bone mineral density (BMD) between two groups, the add-back therapy was more effective in prevention of bone loss which was (SMD = 0.223, 95%CI:0.003 to 0.443, P = 0.047). (2) There was no significant difference in the level of reproductive hormone between two groups (SMD = -0.053, 95% CI:-0.479 to 0.373, P = 0.807). (3) There was also no significant difference in the visual pain score between the two groups (SMD = -0.157, 95% CI: -0.474 to 0.160, P = 0.332). CONCLUSIONS: GnRH-a with add-back therapy have been shown to be more effective in preventing loss of BMD than GnRH-a treatment alone.However, the long term effect of preventing BMD should be studied.

Preliminary Safety and Potential Effect of 6B11-OCIK Adoptive Cell Therapy Against Platinum-Resistant Recurrent or Refractory Ovarian Cancer.

Ovarian cancer is a leading cause of death among gynecological malignancies, and novel therapies are urgently needed. Here we report preliminary findings on the potential safety and efficacy of 6B11-OCIK, an adoptive cell therapy of autologous T cells induced by the humanized anti-idiotypic antibody 6B11 minibody plus dendritic cells and cytokines, against platinum-resistant recurrent or refractory ovarian cancer in three patients. We found that 6B11-OCIK treatment was safe and well tolerated after five cycles of intravenous infusion with an initial dose of 1-2×109 cells and a dose-climbing strategy. Hemoglobin, platelets, white cell count, creatinine or liver enzyme values, coagulation function, kidney and heart function were not significantly affected over the duration of therapy. Two of the three enrolled patients showed potentially drug-related grade 1 and 2 weakness, and no other adverse events were observed. Of the three enrolled patients, one had stable disease and two showed disease progression. The patient with favorable clinical efficacy had better immune response as measured by 6B11-OCIK proliferation capacity, activation ability of CD3+CD8+ tumor-specific cytotoxic T lymphocytes and CD3+CD56+ cytokine-induced killer cells, and tumor cell killing efficiency. Changes in circulating tumor cells after treatment were consistent with serum level CA125 in the patient with stable disease (both decreased), while differences were observed in the two patients with disease progression (increased CA125 in both and decreased CTC in the patient with better immune response), suggesting that variation of circulating tumor cells was more consistent with immune response and reflected efficacy directly. This preliminary study suggested that autologous 6B11-OCIK treatment was safe and had potential clinical efficacy against ovarian cancer. Patients with better immune response had more favorable efficacy. In addition to imaging, CA125 and immunophenotypes, CTC monitoring may represent a potential indicator of immunotherapy response.

[Diversity of nirS-type denitrifying bacteria under different nitrogen fertilizer management in wheat soil].

OBJECTIVE: To study the effect of inorganic nitrogen fertilizer on nirS-type denitrifiers' community in the wheat soil. METHODS: We constructed nirS gene libraries of two different treatments of soil: soil fertilized with inorganic nitrogen fertilizer (B-UN) and without nitrogen fertilizer (B-NN). And we used restriction fragment length polymorphism (RFLP) method to analyze the nirS-type denitrifiers' diversity. RESULTS: There were 27 operational taxa units (OTUs) in each treatment after Msp I and Afa I digestion and only nine OTUs existed in both treatments. The ecological indexes such as Shannon-Wiene index, Simpon index, richness index and evenness index of two different soils were nearly equivalent. However, significant difference was found between the OTU clusters from clone libraries of different treatments. Eleven representative clones were sequenced. The similarities of ten sequences were from 73% to 95% to the sequences of the database. There was one sequence which had no similar nucleotide identities in database. CONCLUSION: The application of inorganic nitrogen fertilizer altered significantly the nirS-type denitrifiers' community in the wheat soil.