Androgen Signaling in the Placenta

The placenta is a multifunctional, transitory organ that mediates transport of nutrients and waste, gas exchange, and endocrine signaling. In fact, placental secretion of hormones is critical for maintenance of pregnancy, as well as growth and development of healthy offspring. In this chapter, the role of androgens in placental development and function is highlighted. First, a brief summary will be provided on the different mammalian placental types followed by an overview of placental steroidogenesis. Next, the chapter will focus on genomic and non-genomic androgen signaling pathways. Finally, an overview will be provided on the current status of androgen signaling in the placenta during normal and abnormal pregnancies

Non-Coding RNA Sequencing of Equine Endometrium During Maternal Recognition of Pregnancy.

Maternal recognition of pregnancy (MRP) in the mare is not well defined. In a non-pregnant mare, prostaglandin F2α (PGF) is released on day 14 post-ovulation (PO) to cause luteal regression, resulting in loss of progesterone production. Equine MRP occurs prior to day 14 to halt PGF production. Studies have failed to identify a gene candidate for MRP, so attention has turned to small, non-coding RNAs. The objective of this study was to evaluate small RNA (<200 nucleotides) content in endometrium during MRP. Mares were used in a cross-over design with each having a pregnant and non-mated cycle. Each mare was randomly assigned to collection day 11 or 13 PO (n = 3/day) and endometrial biopsies were obtained. Total RNA was isolated and sequencing libraries were prepared using a small RNA library preparation kit and sequenced on a HiSeq 2000. EquCab3 was used as the reference genome and DESeq2 was used for statistical analysis. On day 11, 419 ncRNAs, representing miRNA, snRNA, snoRNA, scaRNA, and vaultRNA, were different between pregnancy statuses, but none on day 13. Equine endometrial ncRNAs with unknown structure and function were also identified. This study is the first to describe ncRNA transcriptome in equine endometrium. Identifying targets of these ncRNAs could lead to determining MRP

Expression of miRNAs in ovine fetal gonads: potential role in gonadal differentiation

<p>Abstract</p> <p>Background</p> <p>Gonadal differentiation in the mammalian fetus involves a complex dose-dependent genetic network. Initiation and progression of fetal ovarian and testicular pathways are accompanied by dynamic expression patterns of thousands of genes. We postulate these expression patterns are regulated by small non-coding RNAs called microRNAs (miRNAs). The aim of this study was to identify the expression of miRNAs in mammalian fetal gonads using sheep as a model.</p> <p>Methods</p> <p>We determined the expression of 128 miRNAs by real time PCR in early-gestational (gestational day (GD) 42) and mid-gestational (GD75) sheep ovaries and testes. Expression data were further examined and validated by bioinformatic analysis.</p> <p>Results</p> <p>Expression analysis revealed significant differences between ovaries and testes among 24 miRNAs at GD42, and 43 miRNAs at GD75. Bioinformatic analysis revealed that a number of differentially expressed miRNAs are predicted to target genes known to be important in mammalian gonadal development, including <it>ESR1, CYP19A1</it>, and <it>SOX9</it>. In situ hybridization revealed <it>miR-22 </it>localization within fetal testicular cords. As estrogen signaling is important in human and sheep ovarian development, these data indicate that miR-22 is involved in repressing estrogen signaling within fetal testes.</p> <p>Conclusions</p> <p>Based on our results we postulate that gene expression networks underlying fetal gonadal development are regulated by miRNAs.</p

Sex Reversal in C57BL/6J XY Mice Caused by Increased Expression of Ovarian Genes and Insufficient Activation of the Testis Determining Pathway

Sex reversal can occur in XY humans with only a single functional WT1 or SF1 allele or a duplication of the chromosome region containing WNT4. In contrast, XY mice with only a single functional Wt1, Sf1, or Wnt4 allele, or mice that over-express Wnt4 from a transgene, reportedly are not sex-reversed. Because genetic background plays a critical role in testis differentiation, particularly in C57BL/6J (B6) mice, we tested the hypothesis that Wt1, Sf1, and Wnt4 are dosage sensitive in B6 XY mice. We found that reduced Wt1 or Sf1 dosage in B6 XYB6 mice impaired testis differentiation, but no ovarian tissue developed. If, however, a YAKR chromosome replaced the YB6 chromosome, these otherwise genetically identical B6 XY mice developed ovarian tissue. In contrast, reduced Wnt4 dosage increased the amount of testicular tissue present in Sf1+/− B6 XYAKR, Wt1+/− B6 XYAKR, B6 XYPOS, and B6 XYAKR fetuses. We propose that Wt1B6 and Sf1B6 are hypomorphic alleles of testis-determining pathway genes and that Wnt4B6 is a hypermorphic allele of an ovary-determining pathway gene. The latter hypothesis is supported by the finding that expression of Wnt4 and four other genes in the ovary-determining pathway are elevated in normal B6 XX E12.5 ovaries. We propose that B6 mice are sensitive to XY sex reversal, at least in part, because they carry Wt1B6 and/or Sf1B6 alleles that compromise testis differentiation and a Wnt4B6 allele that promotes ovary differentiation and thereby antagonizes testis differentiation. Addition of a “weak” Sry allele, such as the one on the YPOS chromosome, to the sensitized B6 background results in inappropriate development of ovarian tissue. We conclude that Wt1, Sf1, and Wnt4 are dosage-sensitive in mice, this dosage-sensitivity is genetic background-dependant, and the mouse strains described here are good models for the investigation of human dosage-sensitive XY sex reversal

Clinical outcome in decompression alone versus decompression and instrumented fusion in patients with isthmic spondylolisthesis:A prospective cohort study

OBJECTIVE In the surgical treatment of isthmic spondylolisthesis, it is debatable whether instrumented fusion is mandatory in addition to decompression. The objective of this prospective cohort study was to assess the long-term effect of decompression alone compared with decompression and instrumented fusion in patients who underwent the intervention of their own preference. The results were compared with those in patients who underwent randomly assigned treatment. METHODS The authors performed a prospective observational multicenter cohort study, including 91 patients with isthmic spondylolisthesis assigned to undergo either decompression alone (n = 44) or decompression and fusion (n = 47). The main outcomes were the Roland-Morris Disability Questionnaire (RDQ) scores and the patient's perceived recovery at the 2-year follow-up. Secondary outcomes were visual analog scale (VAS) leg pain and back pain scores and the reoperation rate. A meta-analysis was performed for data from this cohort study (n = 91) and from a randomized controlled trial (RCT) previously reported by the authors (n = 84). Subgroup analyses were performed on these combined data for age, sex, weight, smoking, and Meyerding grade. RESULTS At the 12-week follow-up, improvements of RDQ scores were comparable for the two procedures (decompression alone [D group] 4.4, 95% CI 2.3-6.5; decompression and fusion [DF group] 5.8, 95% CI −4.3 to 1.4; p = 0.31). Likewise, VAS leg pain scores (D group 35.0, 95% CI 24.5-45.6; DF group 47.5, 95% CI 37.4-57.5; p = 0.09) and VAS back pain scores (D group 23.5, 95% CI 13.3-33.7; DF group 34.0, 95% CI 24.1-43.8; p = 0.15) were comparable. At the 2-year follow-up, there were no significant differences between the two groups in terms of scores for RDQ (difference −3.1, 95% CI −6.4 to 0.3, p = 0.07), VAS leg pain (difference −7.4, 95% CI −22.1 to 7.2, p = 0.31), and VAS back pain (difference −11.4, 95% CI −25.7 to 2.9, p = 0.12). In contrast, patient-perceived recovery from leg pain was significantly higher in the DF group (79% vs 51%, p = 0.02). Subgroup analyses did not demonstrate a superior outcome for decompression alone compared with decompression and fusion. Nine patients (20.5%) underwent reoperation in total, all in the D group. The meta-analysis including both the cohort and RCT populations yielded an estimated pooled mean difference in RDQ of −3.7 (95% CI −5.94 to −1.55, p = 0.0008) in favor of decompression and fusion at the 2-year follow-up. CONCLUSIONS In patients with isthmic spondylolisthesis, at the 2-year follow-up, patients who underwent decompression and fusion showed superior functional outcome and perceived recovery compared with those who underwent decompression alone. No subgroups benefited from decompression alone. Therefore, decompression and fusion is recommended over decompression alone as a primary surgical treatment option in isthmic spondylolisthesis.</p

Developmental profile and sexually dimorphic expression of kiss1 and kiss1r in the fetal mouse brain

The hypothalamic-pituitary-gonadal axis (HPG) is a complex neuroendocrine circuit involving multiple levels of regulation. Kisspeptin neurons play essential roles in controlling the HPG axis from the perspectives of puberty onset, oscillations of gonadotropin releasing hormone (GnRH) neuron activity and the pre-ovulatory LH surge. The current studies focus on the expression of kisspeptin during murine fetal development using in situ hybridization (ISH), quantitative reverse transcription real-time PCR (QPCR) and immunocytochemistry. Expression of mRNA coding for kisspeptin (KISS1) and its receptor KISS1R was observed at embryonic (E) day 13 by ISH. At E13 and other later ages examined, Kiss1 signal in individual cells within the arcuate nucleus (ARC) appeared stronger in females than males. ISH examination of agonadal steroidogenic factor-1 (Sf1) knockout mice revealed that E17 XY knockouts resembled wild-type XX females. These findings raise the possibility that gonadal hormones modulate the expression of Kiss1 in the ARC prior to birth. The sex and genotype differences were tested quantitatively by QPCR experiments in dissected hypothalami from mice at E17 and adulthood. Females had significantly more Kiss1 than males at both ages, even though the number of cells detected by ISH was similar. In addition, QPCR revealed a significant difference in the amount of Kiss1 mRNA in Sf1 mice with wild-type (WT) XY mice expressing less than XY knockouts (KO) and XX mice of both genotypes. The detection of immunoreactive KISS1 in perikarya of the ARC at E17 indicates that early mRNA is translated to peptide. The functional significance of this early expression of Kiss1 awaits elucidation

Transcript abundance of stromal and thecal cell related genes during bovine ovarian development

<div><p>Movement and expansion of mesonephric-derived stroma appears to be very important in the development of the ovary. Here, we examined the expression of 24 genes associated with stroma in fetal ovaries during gestation (n = 17; days 58–274) from <i>Bos taurus</i> cattle. RNA was isolated from ovaries for quantitative RT-PCR. Expression of the majority of genes in TGFβ signalling, stromal transcription factors (<i>NR2F2</i>, <i>AR)</i>, and some stromal matrix genes (<i>COL1A1</i>, <i>COL3A1</i> and <i>FBN1</i>, but not <i>FBN3</i>) showed a positive linear increase with gestational age. Expression of genes associated with follicles (<i>INSL3</i>, <i>CYP17A1</i>, <i>CYP11A1</i> and <i>HSD3B1</i>), was low until mid-gestation and then increased with gestational age. <i>LHCGR</i> showed an unusual bimodal pattern; high levels in the first and last trimesters. <i>RARRES1</i> and <i>IGFBP3</i> also increased with gestational age. To relate changes in gene expression in stromal cells with that in non stromal cells during development of the ovary we combined the data on the stromal genes with another 20 genes from non stromal cells published previously and then performed hierarchical clustering analysis. Three major clusters were identified. Cluster 1 genes (<i>GATA4</i>, <i>FBN3</i>, <i>LHCGR</i>, <i>CYP19A1</i>, <i>ESR2</i>, <i>OCT4</i>, <i>DSG2</i>, <i>TGFB1</i>, <i>CCND2</i>, <i>LGR5</i>, <i>NR5A1</i>) were characterised by high expression only in the first trimester. Cluster 2 genes (<i>FSHR</i>, <i>INSL3</i>, <i>HSD3B1</i>, <i>CYP11A1</i>, <i>CYP17A1</i>, <i>AMH</i>, <i>IGFBP3</i>, <i>INHBA</i>) were highly expressed in the third trimester and largely associated with follicle function. Cluster 3 (<i>COL1A1</i>, <i>COL3A1</i>, <i>FBN1</i>, <i>TGFB2 TGFB3</i>, <i>TGFBR2</i>, <i>TGFBR3</i>, <i>LTBP2</i>, <i>LTBP3</i>, <i>LTBP4</i>, <i>TGFB1I1</i>, <i>ALDH1A1</i>, <i>AR</i>, <i>ESR1</i>, <i>NR2F2</i>) had much low expression in the first trimester rising in the second trimester and remaining at that level during the third trimester. Cluster 3 contained members of two pathways, androgen and TGFβ signalling, including a common member of both pathways namely the androgen receptor cofactor TGFβ1 induced transcript 1 protein (<i>TGFB1I1</i>; hic5). <i>GATA4</i>, <i>FBN3</i> and <i>LHCGR</i>, were highly correlated with each other and were expressed highly in the first trimester during stromal expansion before follicle formation, suggesting that this could be a critical phase in the development of the ovarian stroma.</p></div

Foreshore Assessment using Space Technology

Poster presented in SENTINEL-2 for Science Workshop (ESA-ESRIN, Frascati, Italy) on the 20th, 21st and 22nd May 2014. See more information about this workshop in http://seom.esa.int/S2forScience2014FORESHORE ASSESSMENT USING SPACE TECHNOLOGY. Vegetated foreshores and floodplains provide important ecosystem services. These include benefits for coastal defence: the foreshores attenuate waves, enhance sedimentation and reduce erosion, mitigating flood risk. At present, this function of wetland ecosystems is hardly incorporated in coastal defence schemes. Yet considering foreshores and floodplains in water safety assessments and in levee design can result in considerable cost reductions for flood risk management. In the EU-funded project FAST (Foreshore Assessment using Space Technology, 2014-2018), we aim to develop a GMES/Copernicus downstream service, combining Sentinel satellite data and in situ data to integrate the functioning of wetlands in flood risk management strategies. Sentinel data will be used to retrieve relevant biophysical parameters to characterise the foreshore, such as the morphology/topography of the foreshores, the density/biomass and type of vegetation, and the sediment type. Because of their location at the dynamic interface of land and water, foreshores and floodplains pose particular challenges for satellite observation and analysis, both regarding spatial and spectral resolution (due to the large environmental gradients and heterogeneity) and regarding temporal resolution (due to the large temporal variation as a result of, for example, tidal inundation). Information on some key variables is not readily available, and applicability of some relevant biophysical parameters and indicators developed for the terrestrial realm (such as Leaf Area Index, NDVI) remain to be tested for the characterisation of foreshores. The advent of the superspectral Sentinel-2 MSI opens up the potential to detect such characteristics from a spaceborne platform in a consistent way, based on an appropriate spectral, spatial and temporal resolution, with substantial (regional) coverage. However, a thorough assessment is required to optimize and validate retrieval schemes for the spatial characterisation of foreshores. At eight case-study sites across Europe (Spain, Romania, United Kingdom and the Netherlands), ground referencing will be executed to aid the retrieval of information from Sentinel imagery. Validation includes measurements of sediment type and vegetation density/biomass and hyperspectral reflectance of vegetation types. The in situ and remote sensing data will be combined with measurements on wave attenuation and erosion/deposition regimes. The results will provide insight in forshore stability and in the buffering function of foreshores and floodplains. From the collected data general relationships between foreshore and floodplain characteristics and flood risk mitigation properties will be derived. These will be implemented in a GIS based tool for integrating wetlands into cost efficient and safe flood risk management strategies.This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration.POSTE

Blood volume measurement with indocyanine green pulse spectrophotometry: dose and site of dye administration

(1) To determine the optimal administration site and dose of indocyanine green (ICG) for blood volume measurement using pulse spectrophotometry, (2) to assess the variation in repeated blood volume measurements for patients after subarachnoid hemorrhage and (3) to evaluate the safety and efficacy of this technique in patients who were treated for an intracranial aneurysm. Four repeated measurements of blood volume (BV) were performed in random order of bolus dose (10 mg or 25 mg ICG) and venous administration site (peripheral or central) in eight patients admitted for treatment of an intracranial aneurysm. Another five patients with subarachnoid hemorrhage underwent three repeated BV measurements with 25 mg ICG at the same administration site to assess the coefficient of variation. The mean +/- SD in BV was 4.38 +/- 0.88 l (n = 25) and 4.69 +/- 1.11 l (n = 26) for 10 mg and 25 mg ICG, respectively. The mean +/- SD in BV was 4.59 +/- 1.15 l (n = 26) and 4.48 +/- 0.86 l (n = 25) for central and peripheral administration, respectively. No significant difference was found. The coefficient of variance of BV measurement with 25 mg of ICG was 7.5% (95% CI: 3-12%). There is no significant difference between intravenous administration of either 10 or 25 mg ICG, and this can be injected through either a peripheral or central venous catheter. The 7.5% coefficient of variation in BV measurements determines the detectable differences using ICG pulse spectrophotometr

Oxygen tension regulates the miRNA profile and bioactivity of exosomes released from extravillous trophoblast cells - liquid biopsies for monitoring complications of pregnancy

Our understanding of how cells communicate has undergone a paradigm shift since the recent recognition of the role of exosomes in intercellular signaling. In this study, we investigated whether oxygen tension alters the exosome release and miRNA profile from extravillous trophoblast (EVT) cells, modifying their bioactivity on endothelial cells (EC). Furthermore, we have established the exosomal miRNA profile at early gestation in women who develop pre-eclampsia (PE) and spontaneous preterm birth (SPTB). HTR-8/SVneo cells were used as an EVT model. The effect of oxygen tension (i.e. 8% and 1% oxygen) on exosome release was quantified using nanocrystals (Qdot®) coupled to CD63 by fluorescence NTA. A real-time, live-cell imaging system (Incucyte™) was used to establish the effect of exosomes on EC. Plasma samples were obtained at early gestation (<18 weeks) and classified according to pregnancy outcomes. An Illumina TrueSeq Small RNA kit was used to construct a small RNA library from exosomal RNA obtained from EVT and plasma samples. The number of exosomes was significantly higher in EVT cultured under 1% compared to 8% oxygen. In total, 741 miRNA were identified in exosomes from EVT. Bioinformatic analysis revealed that these miRNA were associated with cell migration and cytokine production. Interestingly, exosomes isolated from EVT cultured at 8% oxygen increased EC migration, whilst exosomes cultured at 1% oxygen decreased EC migration. These changes were inversely proportional to TNF-α released from EC. Finally, we have identified a set of unique miRNAs in exosomes from EVT cultured at 1% oxygen and exosomes isolated from the circulation of mothers at early gestation, who later developed PE and SPTB. We suggest that aberrant exosomal signalling by placental cells is a common aetiological factor in pregnancy complications characterised by incomplete SpA remodeling and is therefore a clinically relevant biomarker of pregnancy complications.Grace Truong, Dominic Guanzon, Vyjayanthi Kinhal, Omar Elfeky, Andrew Lai, Sherri Longo, Zarin Nuzhat, Carlos Palma, Katherin Scholz-Romero, Ramkumar Menon, Ben W. Mol, Gregory E. Rice, Carlos Salomo