Polycystic ovary syndrome (PCOS), characterized by increased ovarian androgen biosynthesis, anovulation, and infertility, affects 5-7% of reproductive-age women. Genome-wide association studies ...identified PCOS candidate loci that were replicated in subsequent reports, including DENND1A, which encodes a protein associated with clathrin-coated pits where cell-surface receptors reside. However, these studies provided no information about functional roles for DENND1A in the pathogenesis of PCOS. DENND1A protein was located in the cytoplasm as well as nuclei of theca cells, suggesting a possible role in gene regulation. DENND1A immunostaining was more intense in the theca of PCOS ovaries. Using theca cells isolated and propagated from normal cycling and PCOS women, we found that DENND1A variant 2 (DENND1A.V2) protein and mRNA levels are increased in PCOS theca cells. Exosomal DENND1A.V2 RNA was significantly elevated in urine from PCOS women compared with normal cycling women. Forced overexpression of DENND1A.V2 in normal theca cells resulted in a PCOS phenotype of augmented CYP17A1 and CYP11A1 gene transcription, mRNA abundance, and androgen biosynthesis. Knock-down of DENND1A.V2 in PCOS theca cells reduced androgen biosynthesis and CYP17A1 and CYP11A1 gene transcription. An IgG specific to DENND1A.V2 also reduced androgen biosynthesis and CYP17 and CYP11A1 mRNA when added to the medium of cultured PCOS theca cells. We conclude that the PCOS candidate gene, DENND1A, plays a key role in the hyperandrogenemia associated with PCOS. These observations have both diagnostic and therapeutic implications for this common disorder.
The road to low-dose aspirin therapy for the prevention of preeclampsia began in the 1980s with the discovery that there was increased thromboxane and decreased prostacyclin production in placentas ...of preeclamptic women. At the time, low-dose aspirin therapy was being used to prevent recurrent myocardial infarction and other thrombotic events based on its ability to selectively inhibit thromboxane synthesis without affecting prostacyclin synthesis. With the discovery that thromboxane was increased in preeclamptic women, it was reasonable to evaluate whether low-dose aspirin would be effective for preeclampsia prevention. The first clinical trials were very promising, but then two large multi-center trials dampened enthusiasm until meta-analysis studies showed aspirin was effective, but with caveats. Low-dose aspirin was most effective when started <16 weeks of gestation and at doses >100 mg/day. It was effective in reducing preterm preeclampsia, but not term preeclampsia, and patient compliance and patient weight were important variables. Despite the effectiveness of low-dose aspirin therapy in correcting the placental imbalance between thromboxane and prostacyclin and reducing oxidative stress, some aspirin-treated women still develop preeclampsia. Alterations in placental sphingolipids and hydroxyeicosatetraenoic acids not affected by aspirin, but with biologic actions that could cause preeclampsia, may explain treatment failures. Consideration should be given to aspirin's effect on neutrophils and pregnancy-specific expression of protease-activated receptor 1, as well as additional mechanisms of action to prevent preeclampsia.
Polycystic ovary syndrome (PCOS) is a common endocrine disorder characterized by hyperandrogenemia of ovarian thecal cell origin, resulting in anovulation/oligo-ovulation and infertility. Our ...previous studies established that ovarian theca cells isolated and propagated from ovaries of normal ovulatory women and women with PCOS have distinctive molecular and cellular signatures that underlie the increased androgen biosynthesis in PCOS. To evaluate differences between gene expression in single-cells from passaged cultures of theca cells from ovaries of normal ovulatory women and women with PCOS, we performed single-cell RNA sequencing (scRNA-seq). Results from these studies revealed differentially expressed pathways and genes involved in the acquisition of cholesterol, the precursor of steroid hormones, and steroidogenesis. Bulk RNA-seq and microarray studies confirmed the theca cell differential gene expression profiles. The expression profiles appear to be directed largely by increased levels or activity of the transcription factors SREBF1, which regulates genes involved in cholesterol acquisition (
,
,
,
,
, and
), and GATA6, which regulates expression of genes encoding steroidogenic enzymes (
) in concert with other differentially expressed transcription factors (
,
). This study provides insights into the molecular mechanisms underlying the hyperandrogenemia associated with PCOS and highlights potential targets for molecular diagnosis and therapeutic intervention.
The human placenta has been traditionally viewed as sterile, and microbial invasion of this organ has been associated with adverse pregnancy outcomes. Yet, recent studies that utilized sequencing ...techniques reported that the human placenta at term contains a unique microbiota. These conclusions are largely based on the results derived from the sequencing of placental samples. However, such an approach carries the risk of capturing background-contaminating DNA (from DNA extraction kits, polymerase chain reaction reagents, and laboratory environments) when low microbial biomass samples are studied.
To determine whether the human placenta delivered at term in patients without labor who undergo cesarean delivery harbors a resident microbiota (“the assemblage of microorganisms present in a defined niche or environment”).
This cross-sectional study included placentas from 29 women who had a cesarean delivery without labor at term. The study also included technical controls to account for potential background-contaminating DNA, inclusive in DNA extraction kits, polymerase chain reaction reagents, and laboratory environments. Bacterial profiles of placental tissues and background technical controls were characterized and compared with the use of bacterial culture, quantitative real-time polymerase chain reaction, 16S ribosomal RNA gene sequencing, and metagenomic surveys.
(1) Twenty-eight of 29 placental tissues had a negative culture for microorganisms. The microorganisms retrieved by culture from the remaining sample were likely contaminants because corresponding 16S ribosomal RNA genes were not detected in the same sample. (2) Quantitative real-time polymerase chain reaction did not indicate greater abundances of bacterial 16S ribosomal RNA genes in placental tissues than in technical controls. Therefore, there was no evidence of the presence of microorganisms above background contamination from reagents in the placentas. (3) 16S ribosomal RNA gene sequencing did not reveal consistent differences in the composition or structure of bacterial profiles between placental samples and background technical controls. (4) Most of the bacterial sequences obtained from metagenomic surveys of placental tissues were from cyanobacteria, aquatic bacteria, or plant pathogens, which are microbes unlikely to populate the human placenta. Coprobacillus, which constituted 30.5% of the bacterial sequences obtained through metagenomic sequencing of placental samples, was not identified in any of the 16S ribosomal RNA gene surveys of these samples. These observations cast doubt as to whether this organism is really present in the placenta of patients at term not in labor.
With the use of multiple modes of microbiologic inquiry, a resident microbiota could not be identified in human placentas delivered at term from women without labor. A consistently significant difference in the abundance and/or presence of a microbiota between placental tissue and background technical controls could not be found. All cultures of placental tissue, except 1, did not yield bacteria. Incorporating technical controls for potential sources of background-contaminating DNA for studies of low microbial biomass samples, such as the placenta, is necessary to derive reliable conclusions.
Highlights • GWAS have identified FHSR, LHCGR, INSR , and DENND1A as PCOS candidate genes. • Overexpression of DENND1A variant 2 increases androgen biosynthesis in theca cells from normal cycling ...women, converting them to a PCOS phenotype. • DENND1A, LHCGR, INSR , and RAB5B form a hierarchal signaling network that can influence androgen synthesis. • DENND1A variant 2 is a new diagnostic and therapeutic target for PCOS.
Sperm differentiation encompasses a complex sequence of morphological changes that takes place in the seminiferous epithelium. In this process, haploid round spermatids undergo substantial structural ...and functional alterations, resulting in highly polarized sperm. Hallmark changes during the differentiation process include the formation of new organelles, chromatin condensation and nuclear shaping, elimination of residual cytoplasm, and assembly of the sperm flagella. To achieve these transformations, spermatids have unique mechanisms for protein trafficking that operate in a coordinated fashion. Microtubules and filaments of actin are the main tracks used to facilitate the transport mechanisms, assisted by motor and non-motor proteins, for delivery of vesicular and non-vesicular cargos to specific sites. This review integrates recent findings regarding the role of protein trafficking in sperm differentiation. Although a complete characterization of the interactome of proteins involved in these temporal and spatial processes is not yet known, we propose a model based on the current literature as a framework for future investigations.
The extracellular matrix (ECM) plays an important role in determining cell and organ function: (1) it is an organizing substrate that provides tissue tensile strength; (2) it anchors cells and ...influences cell morphology and function via interaction with cell surface receptors; and (3) it is a reservoir for growth factors. Alterations in the content and the composition of the ECM determine its physical and biological properties, including strength and susceptibility to degradation. The ECM components themselves also harbor cryptic matrikines, which when exposed by conformational change or proteolysis have potent effects on cell function, including stimulating the production of cytokines and matrix metalloproteinases (MMPs). Collectively, these properties of the ECM reflect a dynamic tissue component that influences both tissue form and function. This review illustrates how defects in ECM synthesis and metabolism and the physiological process of ECM turnover contribute to changes in the fetal membranes that precede normal parturition and contribute to the pathological events leading to preterm premature rupture of membranes (PPROM).
Evidence from family and twin-based studies provide strong support for a significant contribution of maternal and fetal genetics to the timing of parturition and spontaneous preterm birth. However, ...there has been only modest success in the discovery of genes predisposing to preterm birth, despite increasing sophistication of genetic and genomic technology. In contrast, DNA variants associated with other traits/diseases have been identified. For example, there is overwhelming evidence that suggests that the nature and intensity of an inflammatory response in adults and children are under genetic control. Because inflammation is often invoked as an etiologic factor in spontaneous preterm birth, the question of whether spontaneous preterm birth has a genetic predisposition in the case of pathologic inflammation has been of long-standing interest to investigators. Here, we review various genetic approaches used for the discovery of preterm birth genetic variants in the context of inflammation-associated spontaneous preterm birth. Candidate gene studies have sought genetic variants that regulate inflammation in the mother and fetus; however, the promising findings have often not been replicated. Genome-wide association studies, an approach to the identification of chromosomal loci responsible for complex traits, have also not yielded compelling evidence for DNA variants predisposing to preterm birth. A recent genome-wide association study that included a large number of White women (>40,000) revealed that maternal loci contribute to preterm birth. Although none of these loci harbored genes directly related to innate immunity, the results were replicated. Another approach to identify DNA variants predisposing to preterm birth is whole exome sequencing, which examines the DNA sequence of protein-coding regions of the genome. A recent whole exome sequencing study identified rare mutations in genes encoding for proteins involved in the negative regulation (dampening) of the innate immune response (eg, CARD6, CARD8, NLRP10, NLRP12, NOD2, TLR10) and antimicrobial peptide/proteins (eg, DEFB1, MBL2). These findings support the concept that preterm labor, at least in part, has an inflammatory etiology, which can be induced by pathogens (ie, intraamniotic infection) or “danger signals” (alarmins) released during cellular stress or necrosis (ie, sterile intraamniotic inflammation). These findings support the notion that preterm birth has a polygenic basis that involves rare mutations or damaging variants in multiple genes involved in innate immunity and host defense mechanisms against microbes and their noxious products. An overlap among the whole exome sequencing-identified genes and other inflammatory conditions associated with preterm birth, such as periodontal disease and inflammatory bowel disease, was observed, which suggests a shared genetic substrate for these conditions. We propose that whole exome sequencing, as well as whole genome sequencing, is the most promising approach for the identification of functionally significant genetic variants responsible for spontaneous preterm birth, at least in the context of pathologic inflammation. The identification of genes that contribute to preterm birth by whole exome sequencing, or whole genome sequencing, promises to yield valuable population-specific biomarkers to identify the risk for spontaneous preterm birth and potential strategies to mitigate such a risk.
Characterizing microbial communities via next-generation sequencing is subject to a number of pitfalls involving sample processing. The observed community composition can be a severe distortion of ...the quantities of bacteria actually present in the microbiome, hampering analysis and threatening the validity of conclusions from metagenomic studies. We introduce an experimental protocol using mock communities for quantifying and characterizing bias introduced in the sample processing pipeline. We used 80 bacterial mock communities comprised of prescribed proportions of cells from seven vaginally-relevant bacterial strains to assess the bias introduced in the sample processing pipeline. We created two additional sets of 80 mock communities by mixing prescribed quantities of DNA and PCR product to quantify the relative contribution to bias of (1) DNA extraction, (2) PCR amplification, and (3) sequencing and taxonomic classification for particular choices of protocols for each step. We developed models to predict the "true" composition of environmental samples based on the observed proportions, and applied them to a set of clinical vaginal samples from a single subject during four visits.
We observed that using different DNA extraction kits can produce dramatically different results but bias is introduced regardless of the choice of kit. We observed error rates from bias of over 85% in some samples, while technical variation was very low at less than 5% for most bacteria. The effects of DNA extraction and PCR amplification for our protocols were much larger than those due to sequencing and classification. The processing steps affected different bacteria in different ways, resulting in amplified and suppressed observed proportions of a community. When predictive models were applied to clinical samples from a subject, the predicted microbiome profiles were better reflections of the physiology and diagnosis of the subject at the visits than the observed community compositions.
Bias in 16S studies due to DNA extraction and PCR amplification will continue to require attention despite further advances in sequencing technology. Analysis of mock communities can help assess bias and facilitate the interpretation of results from environmental samples.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Neutrophils extensively infiltrate maternal blood vessels in preeclampsia. This could explain why multiple organs are affected in this enigmatic disorder. Lipid peroxides produced by the placenta are ...probably the first factors that activate neutrophils as they circulate through the intervillous space, but then a second factor specific to pregnancy comes into play, protease-activated receptor 1. The only time neutrophils express protease-activated receptor 1 is during pregnancy. This means that neutrophils can be activated by a mechanism specific to pregnancy, that is, by proteases. Two proteases that are elevated in preeclampsia and activate protease-activated receptor 1 are matrix metalloproteinase-1 and neutrophil elastase. There is an 8-fold increase in vascular protease-activated receptor 1 expression in women with preeclampsia, and protease-activated receptor 1 is also expressed on the placenta, a pregnancy-specific tissue.
The question arises if the pregnancy-specific expression of protease-activated receptor 1 is essential to the pathophysiology of preeclampsia. Protease activation of protease-activated receptor 1 in neutrophils of women with normal pregnancies causes activation of RhoA kinase. RhoA kinase phosphorylates nuclear factor-kappa B causing its translocation from the cytosol into the nucleus, increasing the expression of inflammatory genes. This signaling pathway is blocked by inhibition of either protease-activated receptor 1 or RhoA kinase activity. In contrast, neutrophils obtained from preeclamptic women are already activated, with nuclear factor-kappa B localized in the nucleus. Surprisingly, inhibition of either protease-activated receptor 1 or RhoA kinase results in an efflux of nuclear factor-kappa B from the nucleus back into the cytoplasm. Cyclooxygenase-2 seems to be a downstream mediator between protease-activated receptor 1 and RhoA kinase because aspirin inhibits the nuclear translocation of nuclear factor-kappa B and inhibits neutrophil production of superoxide, thromboxane, and tumor necrosis factor alpha.
Currently, low-dose aspirin is the standard of care to prevent preeclampsia in high-risk women. Generally, the actions of low-dose aspirin are attributed to selective inhibition of maternal platelet thromboxane production. However, a recent study showed that beneficial effects extend to the placenta, where aspirin corrected the imbalance of increased thromboxane and reduced prostacyclin and oxidative stress. Selective inhibition of placental thromboxane is possible because thromboxane and prostacyclin are compartmentalized. Thromboxane is produced by trophoblast cells and prostacyclin by endothelial cells, so as aspirin crosses the placenta, its levels decline, sparing prostacyclin. Placental oxidative stress is attenuated because cyclooxygenase-2 inhibition decreases the generation of reactive oxygen species to decrease the formation of isoprostanes.
The clinical manifestations of preeclampsia can be explained by protease activation of protease-activated receptor 1 in different tissues. In neutrophils, it can account for their activation and inflammatory response. In vascular tissue, protease-activated receptor 1 activation leads to enhanced vascular reactivity to angiotensin II to cause hypertension. In the placenta, it leads to oxidative stress, increased soluble fms-like tyrosine kinase, and thromboxane production. Activation of protease-activated receptor 1 on endothelial cells causes contraction, leading to edema and proteinuria, and activation on platelets leads to coagulation abnormalities. As proteases that activate protease-activated receptor 1 are elevated in the circulation of women with preeclampsia, consideration should be given to the inhibition of protease-activated receptor 1 as a treatment. Recently, The Food and Drug Administration (FDA) approved a protease-activated receptor 1 inhibitor, creating an opportunity to test whether protease-activated receptor 1 inhibition can prevent and/or treat preeclampsia, but a standard dose of aspirin might be just as effective by blocking its downstream actions.
PDF
