Cumulus cells surrounding oocytes with high developmental competence exhibit down-regulation of phosphoinositol 1,3 kinase/protein kinase B (PI3K/AKT) signalling genes involved in proliferation and survival.

STUDY QUESTION: Is the phosphoinositol 1,3-kinase/protein kinase B (PI3K/AKT) pathway expression profile in cumulus cells (CCs) a potential marker of oocyte competence and predictive of pregnancy outcome? SUMMARY ANSWER: Eleven genes (AKT1, ARHGEF7, BCL2L1, CCND1, E2F1, HRAS, KCNH2, PIK3C2A, SHC1, SOS1 and SPP1) in the PI3K/AKT pathway were significantly down-regulated in CCs from oocytes that went on to produce a pregnancy compared to CCs associated with a negative outcome. WHAT IS KNOWN ALREADY: The PI3K/AKT pathway plays a pivotal role in the interdependence and continuous feedback between the oocyte and CCs. STUDY DESIGN SIZE, DURATION: The expression analysis of 92 transcripts in the PI3K/AKT pathway in CCs from patients with negative or positive pregnancy outcome, after single embryo transfer, was performed. Mouse CCs target gene expression was conducted to associate the expression profile of PI3K/AKT pathway to oocyte developmental profile. PARTICIPANTS/MATERIALS, SETTING, METHODS: Fifty-five good prognosis IVF patients who had been referred to IVF or intracytoplasmic sperm injection treatment for male-factor infertility or tubal disease were enroled. CCs from single cumulus-oocyte complexes (COCs) from 16 patients who underwent a single embryo transfer were analyzed. Twenty-five CD-1 mice were used to assess gene expression in CCs associated with oocytes with different competence in relation to hCG priming. A total 220 human COCs were collected. The RNA extracted from CCs of 16 selected patients was used to analyze PI3K/AKT pathway gene expression employing a 96-well custom TaqMan Array. Expression data of CCs associated to positive IVF outcome were compared to data from negative outcome samples. Mice were sacrificed after 9, 12, 15, 21 and 24 h post-hCG administration to obtain CCs from MII oocytes with different developmental competence. Akt1, Bcl2l2 and Shc1 expression were tested in the collected mouse CCs. In addition, the expression of upstream regulator ESR1, the gene encoding for the oestrogen receptor ERß, and the downstream effectors of the pathway FOXO1, FOXO3 and FOXO4 was evaluated in human and mouse samples. MAIN RESULTS AND THE ROLE OF CHANCE: Transcripts involved in the PI3K Signaling Pathway were selectively modulated according to the IVF/ICSI outcome of the oocyte. Eleven transcripts in this pathway were significantly down-regulated in all samples of CCs from oocytes with positive when compared those with a negative outcome. These outcomes were confirmed in mouse CCs associated with oocytes at different maturation stages. Expression data revealed that the down-regulation of ESR1 could be related to oocyte competence and is likely to be the driver of expression changes highlighted in the PI3K/AKT pathway. LIMITATIONS REASONS FOR CAUTION: Small sample size and retrospective design. WIDER IMPLICATIONS OF THE FINDINGS: The CCs expression profile of PI3K/AKT signaling genes, disclosed a specific CCs gene signature related to oocyte competence. It could be speculated that CCs associated with competent oocytes have completed their role in sustaining oocyte development and are influencing their fate in response to metabolic and hormonal changes by de-activating anti-apoptotic signals. STUDY FUNDING/COMPETING INTEREST(S): Supported by Merck Serono an affiliate of Merck KGaA, Darmstadt, Germany (research grant for the laboratory session; Merck KGaA reviewed the manuscript for medical accuracy only before journal submission. The authors are fully responsible for the content of this manuscript, and the views and opinions described in the publication reflect solely those of the authors). The authors declare no conflict of interest.

Power frequency magnetic field promotes a more malignant phenotype in neuroblastoma cells via redox-related mechanisms.

In accordance with the classification of the International Agency for Research on Cancer, extremely low frequency magnetic fields (ELF-MF) are suspected to promote malignant progression by providing survival advantage to cancer cells through the activation of critical cytoprotective pathways. Among these, the major antioxidative and detoxification defence systems might be targeted by ELF-MF by conferring cells significant resistance against clinically-relevant cytotoxic agents. We investigated whether the hyperproliferation that is induced in SH-SY5Y human neuroblastoma cells by a 50 Hz, 1 mT ELF magnetic field was supported by improved defence towards reactive oxygen species (ROS) and xenobiotics, as well as by reduced vulnerability against both H2O2 and anti-tumor ROS-generating drug doxorubicin. ELF-MF induced a proliferative and survival advantage by activating key redox-responsive antioxidative and detoxification cytoprotective pathways that are associated with a more aggressive behavior of neuroblastoma cells. This was coupled with the upregulation of the major sirtuins, as well as with increased signaling activity of the erythroid 2-related nuclear transcription factor 2 (NRF2). Interestingly, we also showed that the exposure to 50 Hz MF as low as 100 µT may still be able to alter behavior and responses of cancer cells to clinically-relevant drugs.

Impact of vitrification on the mitochondrial activity and redox homeostasis of human oocyte.

STUDY QUESTION: Do the extreme conditions of vitrification affect mitochondrial health and reactive oxygen species (ROS) levels of human oocytes? SUMMARY ANSWER: Vitrification of discarded human oocytes shifts the intracellular redox potential towards oxidation but does not alter the mitochondrial potential or intracellular ROS levels. WHAT IS KNOWN ALREADY: Recent studies have reflected increased ROS levels in warmed young oocytes and have highlighted the temporal dynamic loss of mitochondrial potential that could, therefore, lead to a decrease in ATP production, impairing embryo development. Mitochondrial function can also be evaluated in vivo by the FAD/NAD(P)H autofluorescence ratio, which reflects the respiratory chain activity and is considered as a marker of the intracellular redox state. STUDY DESIGN, SIZE, DURATION: A total of 629 discarded Metaphase II (MII) oocytes collected from June 2013 to April 2014 were included in this control (fresh oocytes, n= 270) versus treatment (vitrified oocytes, n= 359) study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Discarded MII oocytes were donated to research by young (<27 years old) and reproductively aged (>36 years old) women who underwent ovarian stimulation for IVF at a university-affiliated private fertility clinic. Redox state was assessed by measuring the FAD/NAD(P)H autofluorescence ratio, while ROS and mitochondrial activity were reported by in vivo labelling with carboxy-H2DCFDA and JC-1, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: Young and aged oocytes showed high and similar survival rates (81.8 versus 83.1%, not significant). Confocal microscopy revealed that the FAD/NAD(P)H ratio was significantly higher in vitrified oocytes than in fresh oocytes, suggesting a significant shift towards the oxidized state in oocytes after vitrification, regardless of the maternal age. Mitochondrial distribution was not affected by vitrification. Furthermore, it was not possible to resolve any difference in mitochondrial potential using JC-1 potentiometric dye or in reactive oxygen species (ROS) production (assessed with H2-DCFDA staining) between fresh and vitrified oocytes. Therefore, measurement of intracellular redox potential by autofluorescence imaging may be a more sensitive method to assess oxidative stress or mitochondrial demise in human oocytes because it showed a higher resolving power than JC-1 staining and displayed less variability than H2-DCFDA staining. LIMITATIONS, REASONS FOR CAUTION: Owing to sample availability, MII discarded oocytes (in vitro matured oocytes and unfertilized oocytes 20 h after ICSI) were included in the study. These discarded oocytes do not necessarily reflect the physiological condition of the MII human oocyte. WIDER IMPLICATIONS OF THE FINDINGS: Although vitrified oocytes yield comparable clinical outcomes compared with fresh oocytes, lower cleavage and blastocyst rates can be observed during in vitro culture. Data here obtained suggest that the redox state of human oocytes could be affected by vitrification. Therefore, the importance of adding protective antioxidant molecules to the vitrification solution and to the post-warming culture medium to improve embryo cleavage deserves some research. STUDY FUNDING/COMPETING INTERESTS: This research project was supported by the Valencian Government (Val+i+D program, M.N.-C.), INCLIVA Foundation for health research (G.S.-A.) and by the University of L'Aquila and Regione Abruzzo ('Reti per l'Alta Formazione' - P.O.F.S.E. Abruzzo 2007-2013 G.D.E.). No conflicts of interest were declared.

Regular and Moderate Exercise Counteracts the Decline of Antioxidant Protection but Not Methylglyoxal-Dependent Glycative Burden in the Ovary of Reproductively Aging Mice.

Population aging results in urgent needs of interventions aimed at ensuring healthy senescence. Exercise often results in healthy aging, yet many molecular mechanisms underlying such effects still need to be identified. We here investigated whether the age-dependent accumulation of oxidative and methylglyoxal- (MG-) related molecular damage could be delayed by moderate exercise in the mouse ovary, an organ that first exhibits impaired function with advancing age in mammals. CD1 female mice underwent two- or four-month treadmill-based running through the transition from adult to middle age, when ovaries show signs of senescence, and markers of protection against reactive oxygen species (ROS) and MG were measured. The long-term exercise reduced the protein oxidative damage in the ovaries (P < 0.01), and this was linked to the preservation of the glutathione peroxidase protection against ROS (P < 0.001), as well as to the increased glutathione availability (P < 0.001). Conversely, even though the age-related deactivation of the MG-targeting systems was partially prevented by the long-term running programme (P < 0.001), exercised mice were not protected from the age-dependent glycative burden. In summary, lately initiated regular and moderate exercise limited some changes occurring in the ovaries of middle-aged mice, and this might help to develop nonpharmacological cointerventions to reduce the vulnerability of mammalian ovaries towards redox dysfunctions.