Super-Resolution-Chip: an in-vitro platform that enables super-resolution microscopy of co-cultures and 3D systems.

The development of organs-on-a-chip platforms has revolutionized in-vitro cellular culture by allowing cells to be grown in an environment that better mimics human physiology. However, there is still a challenge in integrating those platforms with advanced imaging technology. This is extremely important when we want to study molecular changes and subcellular processes on the level of a single molecule using super-resolution microscopy (SRM), which has a resolution beyond the diffraction limit of light. Currently, existing platforms that include SRM have certain limitations, either as they only support 2D monocultures, without flow or as they demand a lot of production and handling. In this study, we developed a Super-Res-Chip platform, consisting of a 3D-printed chip and a porous membrane, that could be used to co-culture cells in close proximity either in 2D or in 3D while allowing SRM on both sides of the membrane. To demonstrate the functionality of the device, we co-cultured in endothelial and epithelial cells and used direct stochastic optical reconstruction microscopy (dSTORM) to investigate how glioblastoma cells affect the expression of the gap-junction protein Connexin43 in endothelial cells grown in 2D and in 3D. Cluster analysis of Connexin43 distribution revealed no difference in the number of clusters, their size, or radii, but did identify differences in their density. Furthermore, the spatial resolution was high also when the cells were imaged through the membrane (20-30 nm for x-y) and 10-20 nm when imaged directly both for 2D and 3D conditions. Overall, this chip allows to characterize of complex cellular processes on a molecular scale in an easy manner and improved the capacity for imaging in a single molecule resolution complex cellular organization.

Term Idiopathic Polyhydramnios, and Labor Complications.

Background and Aim: Polyhydramnios is associated with an increased risk of various adverse pregnancy outcomes, yet complications during labor have not been sufficiently studied. We assessed the labor and perinatal outcomes of idiopathic polyhydramnios during term labor. Methods: Retrospective cohort study at a tertiary medical center between 2010 and 2014. Women with idiopathic polyhydramnios defined as an amniotic fluid index (AFI) greater than 24 cm or a deep vertical pocket (DVP) > 8 cm (cases) were compared with women with a normal AFI (5-24 cm) (controls). Statistics: Descriptive, means ± SDs, medians + IQR. Comparisons: chi-square, Fisher's exact test, Mann-Whitney Test, multivariate logistic models. Results: During the study period 11,065 women had ultrasound evaluation completed by a sonographer within two weeks of delivery. After excluding pregnancies complicated by diabetes (pre-gestational or gestational), fetal anomalies, IUFD, multifetal pregnancies, elective cesarean deliveries (CD) or missing data, we included 750 cases and 7000 controls. The degree of polyhydramnios was mild in 559 (75.0%) cases (AFI 24-30 cm or DVP 8-12 cm), moderate in 137 (18.0%) cases (30-35 cm or DVP 12-15 cm) and severe in 54 (7.0%) cases (AFI >35 cm or DVP > 15 cm). Idiopathic polyhydramnios was associated with a higher rate of CD 9.3% vs. 6.2%, p = 0.004; a higher rate of macrosomia 22.8% vs. 7.0%, p < 0.0001; and a higher rate of neonatal respiratory complications 2.0% vs. 0.8%, p = 0.0001. A multivariate regression analysis demonstrated an independent relation between polyhydramnios and higher rates of CD, aOR 1.62 (CI 1.20-2.19 p = 0.002) and composite adverse neonatal outcome aOR 1.28 (CI 1.01-1.63 p = 0.043). Severity of polyhydramnios was significantly associated with higher rates of macrosomia and CD (p for trend <0.01 in both). Conclusions: The term idiopathic polyhydramnios is independently associated with macrosomia, CD and neonatal complications. The severity of polyhydramnios is also associated with macrosomia and CD.

Loss of heterochromatin and retrotransposon silencing as determinants in oocyte aging.

Mammalian oocyte quality reduces with age. We show that prior to the occurrence of significant aneuploidy (9M in mouse), heterochromatin histone marks are lost, and oocyte maturation is impaired. This loss occurs in both constitutive and facultative heterochromatin marks but not in euchromatic active marks. We show that heterochromatin loss with age also occurs in human prophase I-arrested oocytes. Moreover, heterochromatin loss is accompanied in mouse oocytes by an increase in RNA processing and associated with an elevation in L1 and IAP retrotransposon expression and in DNA damage and DNA repair proteins nuclear localization. Artificial inhibition of the heterochromatin machinery in young oocytes causes an elevation in retrotransposon expression and oocyte maturation defects. Inhibiting retrotransposon reverse-transcriptase through azidothymidine (AZT) treatment in older oocytes partially rescues their maturation defects and activity of the DNA repair machinery. Moreover, activating the heterochromatin machinery via treatment with the SIRT1 activating molecule SRT-1720, or overexpression of Sirt1 or Ezh2 via plasmid electroporation into older oocytes causes an upregulation in constitutive heterochromatin, downregulation of retrotransposon expression, and elevated maturation rates. Collectively, our work demonstrates a significant process in oocyte aging, characterized by the loss of heterochromatin-associated chromatin marks and activation of specific retrotransposons, which cause DNA damage and impair oocyte maturation.