A prospective randomized study to compare four different mineral oils used to culture human embryos in IVF/ICSI treatments

Objective(s)

We aimed to evaluate and compare the embryo quality at early cleavage stages using different oils overlaying media to culture human embryos during IVF/ICSI treatments.

Study design

A total of 500 IVF/ICSI treatments from 500 women were analyzed in a prospective randomized study. Oocytes/embryos were treated into microdroplets of appropriate media overlaid with (i) Mineral Oil (CryoBioSystem, L’Aigle, France) (group 1, n = 129), (ii) Liquid Paraffin (Medicult, Lyon, France) (group 2, n = 126), (iii) Nidoil (Nidacon International, Guthenburg, Sweden) (group 3, n = 126) and (iv) Ovoil (Vitrolife, Kungsbacka, Sweden) (group 4, n = 119). Comparisons between groups were done using two by two post hoc tests, with 5% significance. The primary endpoint was the embryo quality, defined as good or top quality when embryos were with (i) less than 20% of fragmentation and (ii) 3–5/4 cells at day 2 or 6–10/8 cells at day 3, respectively.

Results

At day 2, the embryo quality was similar in all groups. However, the mean number of top quality embryos at day 3 was statistically higher into the group 4 (1.4 ± 1.8) compared to the group 1 (0.9 ± 1.0; p = 0.03) and 2 (0.8 ± 1.3; p = 0.05). Furthermore, a significant increase of the mean number of good quality embryos was observed at day 3 into the group 4 (2.6 ± 2.6) compared to the group 1 (1.6 ± 1.6; p = 0.02).

Conclusion(s)

The embryo quality could be modified according to commercial oils used to overlay culture media.     

Cochlear implants and ex vivo BDNF gene therapy protect spiral ganglion neurons

Spiral ganglion neurons often degenerate in the deaf ear, compromising the function of cochlear implants. Cochlear implant function can be improved by good preservation of the spiral ganglion neurons, which are the target of electrical stimulation by the implant. Brain derived neurotrophic factor (BDNF) has previously been shown to enhance spiral ganglion survival in experimentally deafened ears. Providing enhanced levels of BDNF in human ears may be accomplished by one of several different methods. The goal of these experiments was to test a modified design of the cochlear implant electrode that includes a coating of fibroblast cells transduced by a viral vector with a BDNF gene insert. To accomplish this type of ex vivo gene transfer, we transduced guinea pig fibroblasts with an adenovirus with a BDNF gene cassette insert, and determined that these cells secreted BDNF. We then attached BDNF-secreting cells to the cochlear implant electrode via an agarose gel, and implanted the electrode in the scala tympani. We determined that the BDNF expressing electrodes were able to preserve significantly more spiral ganglion neurons in the basal turns of the cochlea after 48 days of implantation when compared to control electrodes. This protective effect decreased in the higher cochlear turns. The data demonstrate the feasibility of combining cochlear implant therapy with ex vivo gene transfer for enhancing spiral ganglion neuron survival.     

Identification of novel susceptibility genes in childhood‐onset systemic lupus erythematosus using a uniquely designed candidate gene pathway platform

Objective

Childhood‐onset systemic lupus erythematosus (SLE) presents a unique subgroup of patients for genetic study. The present study was undertaken to identify susceptibility genes contributing to SLE, using a novel candidate gene pathway microarray platform to investigate gene expression in patients with childhood‐onset SLE and both of their parents.

Methods

Utilizing bioinformatic tools, a platform of 9,412 single‐nucleotide polymorphisms (SNPs) from 1,204 genes was designed and validated. Molecular inversion probes and high‐throughput SNP technologies were used for assay development. Seven hundred fifty three subjects, corresponding to 251 full trios of childhood‐onset SLE families, were genotyped and analyzed using transmission disequilibrium testing (TDT) and multitest corrections.

Results

Family‐based TDT showed a significant association of SLE with a N673S polymorphism in the P‐selectin gene (SELP) (P = 5.74 × 10⁻⁶) and a C203S polymorphism in the interleukin‐1 receptor–associated kinase 1 gene (IRAK1) (P = 9.58 × 10⁻⁶). These 2 SNPs had a false discovery rate for multitest correction of <0.05, and therefore a >95% probability of being considered as proven. Furthermore, 7 additional SNPs showed q values of <0.5, suggesting association with SLE and providing a direction for followup studies. These additional genes notably included TNFRSF6 (Fas) and IRF5, supporting previous findings of their association with SLE pathogenesis.

Conclusion

SELP and IRAK1 were identified as novel SLE‐associated genes with a high degree of significance, suggesting new directions in understanding the pathogenesis of SLE. The overall design and results of this study demonstrate that the candidate gene pathway microarray platform used provides a novel and powerful approach that is generally applicable in identifying genetic foundations of complex diseases.

     

Increased expression of type I collagen induced by microfibril‐associated glycoprotein 2: Novel mechanistic insights into the molecular basis of dermal fibrosis in scleroderma

Objective

Mutations in fibrillin 1, a key component of extracellular microfibrils, are associated with connective tissue disorders such as Marfan's syndrome or skin fibrosis in the tight skin mouse model of scleroderma. Previous studies have suggested that fibrillin 1 mediates skin fibrosis via its interface with associated microfibrillar proteins and type I collagen; in particular, microfibril‐associated glycoprotein 2 (MAGP‐2), an extracellular matrix protein that binds to fibrillins and the αvβ3 integrin, is increased in TSK mouse and human scleroderma skin. Because the function of MAGP‐2 in the biologic processes of the matrix remains unknown, this study investigated whether MAGP‐2 regulates type I collagen.

Methods

Fibroblast cultures conditionally overexpressing MAGP‐2 were developed. Cells were analyzed by Western blotting, Northern blotting, pulse‐chase analysis, and immunofluorescence to assess the effect of MAGP‐2 on type I collagen.

Results

Cells overexpressing MAGP‐2 formed increased MAGP‐2 matrix and showed a 3‐fold increase in intracellular type I procollagen. This increase was associated with increased levels of type I collagen in the medium and matrix. Increased type I collagen colocalized with the MAGP‐2 matrix. MAGP‐2 overexpression had no effect on type I procollagen messenger RNA, but markedly increased the half‐life of type I procollagen. MAGP‐2 induced type I collagen even under conditions in which no MAGP‐2 matrix was detectable, and did not require the presence of the RGD motif of MAGP‐2 in its integrin‐binding site.

Conclusion

This study shows that MAGP‐2 stabilizes type I procollagen, identifying an important function of MAGP‐2 in extracellular matrix homeostasis. It also suggests that MAGP‐2 might mediate skin fibrosis in TSK mice and in patients with scleroderma.

     

A novel dynamic matrix detachment model reveals a shift from apoptosis to necrosis in melanoma cells

Anchorage-independence is a hallmark of invasive cancer. The setback of the classical poly-HEMA static matrix detachment (SMD) anoikis model is the absence of dynamic fluid circulation, resulting in cell aggregates. We addressed this problem by developing a novel 3D cell culture dynamic matrix detachment (DMD) model with a turbulent-free laminar flow, yielding a very low shear stress. In this study, we focused on melanoma cells where apoptosis was evaluated both via annexin V flow cytometry and caspase cleavage. The DMD model was superior to SMD in the induction of melanoma cell death and in revealing a shift from apoptosis to necrotic cell death, as evident by failure to activate caspase 9 and a decrease in annexin V stain. Combination of DMD with cisplatin could further accentuate necrotic cell death in cisplatin-resistant melanoma cells. Thus, the DMD model may be a useful matrix deprivation model to identify necrotic vs. apoptotic cell death pathways.     

Antibodies against the cancer-testis antigen CTSP-1 are frequently found in prostate cancer patients and are an independent prognostic factor for biochemical-recurrence

Cancer-testis (CT) antigens are immunogenic proteins expressed in normal gametogenic tissues and in different types of tumors. CTSP-1 is a CT antigen frequently expressed in prostate tumors, and capable of eliciting humoral response in prostate cancer patients. Here, we analyzed the presence of anti-CTSP-1 antibodies in 147 patients with localized prostate cancer and determined its prognostic value for predicting biochemical-recurrence after radical prostatectomy. Anti-CTSP-1 antibodies were detected in 25% of the patients and a significant correlation (p = 0.017) between CTSP-1 protein expression and the presence of specific humoral response was observed. No association was found between the presence of antibodies and the pathological variables analyzed. On univariate analysis, patients without antibodies against CTSP-1 had a lower biochemical-recurrence free survival than did those with anti-CTSP-1 antibodies, although the difference between the groups was not statistically significant (57 vs. 75%, p = 0.075). However, the presence of antibodies against CTSP-1 was significantly associated with a better prognosis in patients with higher Gleason score (36 vs. 80%, p = 0.028). On multivariate analysis, antibodies against CTSP-1 were associated with a better prognosis (Hazard ratio = 0.41, 95% IC 0.18-0.90 p = 0.039), being the third most powerful prognostic factor among Gleason score and preoperative PSA levels. CTSP-1 should be considered a promising candidate for prostate cancer immunotherapy, since it is frequently expressed in prostate tumors and capable of eliciting humoral immune response in prostate cancer patients. Our results also suggest that humoral response against CTSP-1 could be used as a prognostic marker, especially among patients with a high Gleason score.     

Paxillin is a target for somatic mutations in lung cancer: implications for cell growth and invasion

Lung cancer is characterized by abnormal cell growth and invasion, and the actin cytoskeleton plays a major role in these processes. The focal adhesion protein paxillin is a target of a number of oncogenes involved in key signal transduction and important in cell motility and migration. In lung cancer tissues, we have found that paxillin was highly expressed (compared with normal lung), amplified (12.1%, 8 of 66) and correlated with increased MET and epidermal growth factor receptor (EGFR) gene copy numbers, or mutated (somatic mutation rate of 9.4%, 18 of 191). Paxillin mutations (19 of 21) were clustered between LD motifs 1 and 2 and the LIM domains. The most frequent point mutation (A127T) enhanced lung cancer cell growth, colony formation, focal adhesion formation, and colocalized with Bcl-2 in vitro. Gene silencing from RNA interference of mutant paxillin led to reduction of cell viability. A murine in vivo xenograft model of A127T paxillin showed an increase in tumor growth, cell proliferation, and invasion. These results establish an important role for paxillin in lung cancer.     

Effects of isometric handgrip training on cardiac autonomic profile: A systematic review and meta‐analysis study

Meta‐analyses have shown that isometric handgrip training reduces blood pressure in normotensive and hypertensive subjects. However, the effects on cardiac autonomic modulation are still controversial. Thus, the aim of this systematic review and meta‐analysis was to analyse the effects of isometric handgrip training on cardiac autonomic modulation in normotensive and hypertensive subjects. For this, Medline, Cinhal, Embase, Spordiscus and PEdro were searched for relevant studies published until December 2018. Randomized controlled trials investigating the effect of isometric handgrip training on heart rate variability parameters were considered eligible. Parameters were obtained in time (standard deviation of all the RR intervals‐SDNN, root mean square of successive differences between the normal adjacent RR intervals‐RMSSD and the percentage of adjacent intervals with more than 50 ms‐PNN50) and frequency domain (low frequency‐LF, high frequency‐HF and sympathovagal balance‐LF/HF). Mean difference (MD) and 95% confidence interval (95% CI) were calculated using an inverse variance method with a random effects model. Seven trials were included in the systematic review and meta‐analysis, totalling 86 participants. No significant effect was observed in heart rate variability parameters after isometric handgrip training (4 trials to SDNN: MD = ?1.44 ms and 95% CI = ?8.02, 5.14 ms; RMSSD: MD = ?1.48 ms and 95% CI = ?9.41, 6.45 ms; PNN50: MD = 0.85% and 95% CI = ?1.10, 2.81%; 7 trials to LF: ?0.17 n.u. and 95% CI = ?6.32, 5.98 n.u.; HF: MD = 0.17 n.u. and 95% CI = ?5.97, 6.30 n.u.; and LF/HF: MD = 0.13 and 95% CI = ?0.34, 0.59). In conclusion, current literature indicates that isometric handgrip training does not improve heart rate variability.