Fetal ischemia monitoring with in vivo implanted electrochemical multiparametric microsensors

Under intrauterine growth restriction (IUGR), abnormal attainment of the nutrients and oxygen by the fetus restricts the normal evolution of the prenatal causing in many cases high morbidity being one of the top-ten causes of neonatal death. The current gold standards in hospitals to detect this relevant problem is the clinical observation by echography, cardiotocography and Doppler. These qualitative techniques are not conclusive and requires risky invasive fetal scalp blood testing and/or amniocentesis. We developed micro-implantable multiparametric electrochemical sensors for measuring ischemia in real time in fetal tissue and vascular. This implantable technology is designed to continuous monitoring for an early detection of ischemia to avoid potential fetal injury. Two miniaturized electrochemical sensors were developed based on oxygen and pH detection. The sensors were optimized in vitro under controlled concentration, to assess the selectivity and sensitivity required. The sensors were then validated in vivo in the ewe fetus model, by means of their insertion in the muscle leg and inside the iliac artery of the fetus. Ischemia was achieved by gradually obstructing the umbilical cord to regulate the amount of blood reaching the fetus. An important challenge in fetal monitoring is the detection of low levels of oxygen and pH changes under ischemic conditions, requiring high sensitivity sensors. Significant differences were observed in both; pH and pO2 sensors under changes from normoxia to hypoxia states in the fetus tissue and vascular with both sensors. Herein, we demonstrate the feasibility of the developed sensors for future fetal monitoring in medical applications.

DNA methylation dynamics of long noncoding RNA during human fetal development

Aim: To determine whether the promoters of long noncoding RNAs (lncRNAs) undergo dynamic changes in DNA methylation during fetal development. Methods: ANOVA and the tissue specificity index were used to identify and validate tissue-specific methylation sites. Age-associated DNA methylation signatures were identified by applying the elastic net method. Results: The lncRNA methylome landscape was characterized in four types of fetal tissue and at three gestational time points, and specific characteristics relative to the tissue of origin and developmental age were identified. Higher levels of lncRNA methylation might be involved in tissue differentiation. LncRNAs harboring age-associated methylation signatures may participate in the fetal developmental process. Conclusion: This study provides novel insights into the role of lncRNA methylomes in fetal tissue specification and development.

Placental Autophagy and Viral Replication Co-localize in Human and Non-human Primate Placentae Following Zika Virus Infection: Implications for Therapeutic Interventions

Background: Multiple studies have shown both induction and inhibition of autophagy during Zika virus (ZIKV) infection. While some have proposed mechanisms by which autophagic dysregulation might facilitate ZIKV vertical transmission, there is a lack of in situ data in human and non-human primate models. This is an especially pertinent question as autophagy-inhibitors, such as hydroxychloroquine, have been proposed as potential therapeutic agents aimed at preventing vertical transmission of ZIKV and other RNA viruses.Objectives: Given the paucity of pre-clinical data in support of either autophagic enhancement or inhibition of placental ZIKV viral infection, we sought to assess cellular, spatial, and temporal associations between placental ZIKV infection and measures of autophagy in human primary cell culture and congenital infection cases, as well as an experimental non-human primate (marmoset, Callithrix jacchus) model.Study Design: Primary trophoblast cells were isolated from human placentae (n = 10) and infected in vitro with ZIKV. Autophagy-associated gene expression (ULK-1, BECN1, ATG5, ATG7, ATG12, ATG16L1, MAP1LC3A, MAP1LC3B, p62/SQSTM1) was then determined by TaqMan qPCR to determine fold-change with ZIKV-infection. In in vivo validation experiments, autophagy genes LC3B and p62/SQSTM1 were probed using in situ hybridization (ISH) in the placentae of human Congenital Zika Syndrome (CZS) cases (n = 3) and ZIKV-infected marmoset placenta (n = 1) and fetal tissue (n = 1). Infected and uninfected villi were compared for mean density and co-localization of autophagic protein markers.Results: Studies of primary cultured human trophoblasts revealed decreased expression of autophagy genes ATG5 and p62/SQSTM1 in ZIKV-infected trophoblasts [ATG5 fold change (±SD) 0.734-fold (±0.722), p = 0.036; p62/SQSTM1 0.661-fold (±0.666), p = 0.029]. Histologic examination by ISH and immunohistochemistry confirmed spatial association of autophagy and ZIKV infection in human congenital infection cases, as well as marmoset placental and fetal tissue samples. When quantified by densitometric data, autophagic protein LC3B, and p62/SQSTM1 expression in marmoset placenta were significantly decreased in in situ ZIKV-infected villi compared to less-infected areas [LC3B mean 0.951 (95% CI, 0.930–0.971), p = 0.018; p62/SQSTM1 mean 0.863 (95% CI, 0.810–0.916), p = 0.024].Conclusion: In the current study, we observed that in the non-transformed human and non-human primate placenta, disruption (specifically down-regulation) of autophagy accompanies later ZIKV replication in vitro, in vivo, and in situ. The findings collectively suggest that dysregulated autophagy spatially and temporally accompanies placental ZIKV replication, providing the first in situ evidence in relevant primate pre-clinical and clinical models for the importance of timing of human therapeutic strategies aimed at agonizing/antagonizing autophagy. These studies have likely further implications for other congenitally transmitted viruses, particularly the RNA viruses, given the ubiquitous nature of autophagic disruption and dysregulation in host responses to viral infection during pregnancy.

Abstract P231: Aspirin Reverses Placental Gene Expression Changes Associated With Preeclampsia

Aspirin is the only medication recommended for preventing preeclampsia, a hypertensive disorder of pregnancy. However, the mechanism(s) by which aspirin prevents preeclampsia are unknown. To better understand the mechanism by which aspirin prevents preeclampsia, we compared expression of genes involved in signal transduction, cardiac remodeling, ion transport, stress and immune response, apoptosis, sarcomere structure, transcriptional regulation, and cell cycle regulation between placental tissues from 4 different cohorts: preeclamptic women, preeclamptic women who took aspirin during pregnancy, non-preeclamptic women, and non-preeclamptic women who took aspirin (N=3 per cohort). Tissues were obtained from the University of Iowa Maternal Fetal Tissue Bank (IRB# 200910784). When comparing whole thickness samples, 27 genes had a greater than 5-fold change in expression in the tissues of women with preeclampsia compared to those without. For example, CDKN1B whose protein inhibits the cell cycle, is increased in preeclampsia (-3.7ΔΔCt, 13.4-fold increase). However, in women who took aspirin, both those that developed preeclampsia and those who did not, CDKN1B expression was similar to women without preeclampsia who did not take aspirin indicating a decrease in gene expression in response to aspirin. However, aspirin also increases expression of several genes associated with preeclampsia. For example, we identified that RGS2 and PLAC1 in the decidual layer of the placenta were increased in response to aspirin. Decreases in expression of both genes is associated with preeclampsia. However, 19 genes whose expression is increased in preeclampsia compared to controls had no response to aspirin. Identifying specific genes and pathways that serve as targets of aspirin during pregnancy provides valuable insight into the mechanisms by which aspirin reduces the occurrence of preeclampsia and may help to identify who would benefit most from its use. Additionally, it may identify targets for the development of other preeclampsia preventative or therapeutic medications.

​Mineral Analysis in the Amniotic Fluid of Gaddi Sheep during Different Stages of Gestation

Background: Amniotic fluid surrounds the fetus and is considered as an important source of certain trace elements essential for fetal tissue growth and metabolism. The concentration of these trace elements in amniotic fluid changes with the progression of gestation. The objective of the present study was to evaluate the concentration of some micro and macro minerals in the amniotic fluid of Gaddi sheep and their relation to fetal age and weight. Methods: Amniotic fluid was collected from fifty normal gravid uteri for mineral analysis. The foetii were weighed and their crown rump length was measured and then they were divided into four groups i.e. group I (31-60 days), group II (61-90 days), group III (91-120 days) and group IV (121-till term) on the basis of their gestational age. Result: The mean concentration of sodium and potassium showed a declining trend with the progression of gestation. The level of chloride decreased and that of phosphorus increased significantly from group III to IV. Statistical analysis revealed no significant variation in the concentration of copper among different age groups. Significant correlation was observed between the concentration of minerals and fetal age and fetal weight. Based on the changes in the concentration of minerals in the amniotic fluid, it may be possible to detect the early deviations in the metabolism and thereby appropriate farm management practices could be adopted.

Comparison of Genome-Wide DNA Methylation Profiles of Human Fetal Tissues Conceived by in vitro Fertilization and Natural Conception

BackgroundAssisted reproductive technology (ART) might induce adverse pregnancy outcomes and increase the risk of metabolic diseases in offspring’ later life with unknown reasons. Here we evaluated the global methylation level and methylation profile of fetal tissue from elective terminations of pregnancy (ETP) after natural conception and multifetal pregnancy reduction (MFPR) after in vitro fertilization and embryo transfer (IVF-ET).ResultsGlobal methylation levels were comparable between the fetal tissue of ETP after natural conception group and MFPR after IVF-ET group. The methylation levels were lower in the hypermethylated regions of the MFPR group than in the ETP group, while the methylation levels were higher in the hypomethylated regions of the MFPR group. Heatmap visualization and hierarchical clustering of the candidate differentially methylated regions (DMRs) showed differences between the DMRs in the ETP and MFPR samples. We identified 196 differentially methylated regions that matched 164 genes between the ETP and MFPR groups. In the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, skeletal system morphogenesis and diabetes mellitus ranked first. Ingenuity Pathway Analysis (IPA) revealed 8 diseases and functional annotations associated with IVT-ET. In the MFPR group, the final validation showed lower methylation levels in gene bodies of bone morphogenetic protein 4 (BMP4), higher methylation levels in the 1st exon and 5′UTR of thyroid peroxidase (TPO), and higher methylation levels in TSS1500 and TSS200 of interleukin 1 beta (IL1B).ConclusionsART does not alter global DNA methylation level, but influences DNA methylation variation in specific regions of human fetus in the early stage of life. Further studies are warranted to clarify the potential role of DNA methylation alterations in the gene expression profile.

An automatic multi-tissue human fetal brain segmentation benchmark using the Fetal Tissue Annotation Dataset

It is critical to quantitatively analyse the developing human fetal brain in order to fully understand neurodevelopment in both normal fetuses and those with congenital disorders. To facilitate this analysis, automatic multi-tissue fetal brain segmentation algorithms are needed, which in turn requires open datasets of segmented fetal brains. Here we introduce a publicly available dataset of 50 manually segmented pathological and non-pathological fetal magnetic resonance brain volume reconstructions across a range of gestational ages (20 to 33 weeks) into 7 different tissue categories (external cerebrospinal fluid, grey matter, white matter, ventricles, cerebellum, deep grey matter, brainstem/spinal cord). In addition, we quantitatively evaluate the accuracy of several automatic multi-tissue segmentation algorithms of the developing human fetal brain. Four research groups participated, submitting a total of 10 algorithms, demonstrating the benefits the dataset for the development of automatic algorithms.