Serum Hydroxysteroid (17beta) dehydrogenase 1 concentration in pregnant women correlates with pregnancy-associated plasma protein a but does not serve as an independent marker for preeclampsia.

Hydroxysteroid (17beta) dehydrogenase 1 (HSD17B1) is a steroid synthetic enzyme expressed in ovarian granulosa cells and placental syncytiotrophoblasts. Here, HSD17B1 serum concentration was measured with a validated immuno assay during pregnancy at three time points (12-14, 18-20 and 26-28 weeks of gestation). The concentration increased 2.5-fold (p < 0.0001) and 1.7-fold (p = 0.0019) during the follow-up period for control women and women who later developed preeclampsia (PE), respectively, and a significant difference was observed at weeks 26-28 (p = 0.0266). HSD17B1 concentration at all the three time points positively correlated with serum PAPPA measured at the first time point (first time point r = 0.38, p = 1.1x10-10; second time point r = 0.27, p = 5.9x10-6 and third timepoint r = 0.26, p = 2.3x10-5). No correlation was observed between HSD17B1 and placental growth factor (PLGF). Serum HSD17B1, furthermore, negatively correlated with the mother's weight and body mass index (BMI), mirroring the pattern observed for PAPPA. The univariable logistic regression identified a weak association between HSD17B1 at 26-28 weeks and later development of PE (P = 0.04). Also, the best multivariable model obtained using penalized logistic regression with stable iterative variable selection at 26-28 weeks included HSD17B1, together with PLGF, PAPPA and the mother's BMI. While the area under the ROC curve of the model was higher than that of the adjusted PLGF, the difference was not statistically significant. In summary, the serum concentration of HSD17B1 correlated with PAPPA, another protein expressed in syncytiotrophoblasts, and with mother's weight and BMI but could not be considered as an independent marker for PE.

Cell type markers indicate distinct contributions of decidual stromal cells and natural killer cells in preeclampsia.

In brief: Preeclampsia is a common serious disorder that can occur during pregnancy. This study uses integrative analysis of preeclampsia transcriptomes and single-cell transcriptomes to predict cell type-specific contributions to preeclampsia. Abstract: Preeclampsia is a devastating pregnancy disorder and a major cause of maternal and perinatal mortality. By combining previous transcriptomic results on preeclampsia with single-cell sequencing data, we here predict distinct and partly unanticipated contributions of decidual stromal cells and uterine natural killer cells in early- and late-onset preeclampsia.

Overexpression of Human Estrogen Biosynthetic Enzyme Hydroxysteroid (17beta) Dehydrogenase Type 1 Induces Adenomyosis-like Phenotype in Transgenic Mice.

Hydroxysteroid (17beta) dehydrogenase type 1 (HSD17B1) is an enzyme that converts estrone to estradiol, while adenomyosis is an estrogen-dependent disease with poorly understood pathophysiology. In the present study, we show that mice universally over-expressing human estrogen biosynthetic enzyme HSD17B1 (HSD17B1TG mice) present with adenomyosis phenotype, characterized by histological and molecular evaluation. The first adenomyotic changes with endometrial glands partially or fully infiltrated into the myometrium appeared at the age of 5.5 months in HSD17B1TG females and became more prominent with increasing age. Preceding the phenotype, increased myometrial smooth muscle actin positivity and increased amount of glandular myofibroblast cells were observed in HSD17B1TG uteri. This was accompanied by transcriptomic upregulation of inflammatory and estrogen signaling pathways. Further, the genes upregulated in the HSD17B1TG uterus were enriched with genes previously observed to be induced in the human adenomyotic uterus, including several genes of the NFKB pathway. A 6-week-long HSD17B1 inhibitor treatment reduced the occurrence of the adenomyotic changes by 5-fold, whereas no effect was observed in the vehicle-treated HSD17B1TG mice, suggesting that estrogen is the main upstream regulator of adenomyosis-induced uterine signaling pathways. HSD17B1 is considered as a promising drug target to inhibit estrogen-dependent growth of endometrial disorders. The present data indicate that HSD17B1 over-expression in TG mice results in adenomyotic changes reversed by HSD17B1 inhibitor treatment and HSD17B1 is, thus, a potential novel drug target for adenomyosis.

Transcriptomic responses to hypoxia in endometrial and decidual stromal cells.

Human reproductive success depends on a properly decidualized uterine endometrium that allows implantation and the formation of the placenta. At the core of the decidualization process are endometrial stromal fibroblasts (ESF) that differentiate to decidual stromal cells (DSC). As variations in oxygen levels are functionally relevant in endometrium both upon menstruation and during placentation, we assessed the transcriptomic responses to hypoxia in ESF and DSC. In both cell types, hypoxia-upregulated genes in classical hypoxia pathways such as glycolysis and the epithelial mesenchymal transition. In DSC, hypoxia restored an ESF-like transcriptional state for a subset of transcription factors that are known targets of the progesterone receptor, suggesting that hypoxia partially interferes with progesterone signaling. In both cell types, hypoxia modified transcription of several inflammatory transcription factors that are known regulators of decidualization, including decreased transcription of STATs and increased transcription of CEBPs. We observed that hypoxia-upregulated genes in ESF and DSC had a significant overlap with genes previously detected to be upregulated in endometriotic stromal cells. Promoter analysis of the genes in this overlap suggested the hypoxia-upregulated Jun/Fos and CEBP transcription factors as potential drivers of endometriosis-associated transcription. Using immunohistochemistry, we observed increased expression of JUND and CEBPD in endometriosis lesions compared to healthy endometria. Overall, the findings suggest that hypoxic stress establishes distinct transcriptional states in ESF and DSC and that hypoxia influences the expression of genes that contribute to the core gene regulation of endometriotic stromal cells.

Revisiting redox-active antioxidant defenses in response to hypoxic challenge in both hypoxia-tolerant and hypoxia-sensitive fish species.

It is not known whether changes in antioxidant levels always occur in fish in response to the oxidative stress that usually accompanies a hypoxic challenge. The studies of antioxidant responses to hypoxia in fish have mostly focused on very anoxia-tolerant species and indicate that there is an enhancement of antioxidant defenses. Here we present new data on redox-active antioxidants from three species, which range in their tolerance to hypoxia: the epaulette shark, threespine stickleback, and rainbow trout, together with a compilation of results from other studies that have measured oxidative stress parameters in hypoxia-exposed fish. The results suggest that in general, fish do not show an increase in redox-active antioxidant defense in response to oxidative stress associated with hypoxia. Rather, the changes in antioxidant defenses during hypoxia are very much species- and tissue-specific and are not linked to the level of hypoxia tolerance of the fish species.

Developmental transcription of genes putatively associated with growth in two sturgeon species of different growth rate.

In the present study, we surveyed developmental changes in the transcription of growth hormone (gh), insulin-like growth factor-I (igf-I), ghrelin (ghrl) and vascular endothelial growth factor (vegf) genes in the largest freshwater fish, European sturgeon (Beluga, Huso huso) and compared the same parameters to that of its phylogenically close moderate-sized species, Persian sturgeon (Acipenser persicus). The transcripts of gh, igf-I, ghrl and vegf were detected at all developmental time-points of Persian sturgeon and Beluga from embryos to juvenile fish. Changes in normalized gh, igf-I, ghrl and vegf transcription by using the geometric average of genes encoding ribosomal protein L6 (RPL6) and elongation factor (EF1A) over the time of development of Persian sturgeon and Beluga were statistically significant (P<0.05). Our results showed that the mRNA expression levels of both igf-I and ghrl were low during early larval development and then increased significantly to the late larval time-points when larvae started exogenous feeding. In both Beluga and Persian sturgeon, after a low mRNA expression during the embryonic stage, the transcript levels of vegf displayed an increasing trend during yolk-sac fry, consistent with organogenesis. The vegf level remained constantly high in the time of exogenous feeding. The highest detection of gh transcripts coincided with the end of the embryonic stage (hatching time) in Persian sturgeon and 3 days-post-hatching (dph) in Beluga. In Persian sturgeon, the gh transcript started to decrease to the rest of the developmental time-points, whereas in Beluga gh transcript had a marked second increase from the time of exogenous feeding (20-dph). This Beluga specific increase in gh transcription may be associated with the marked growth rate and extraordinary size of this fish species.

Glycolysis and Heavy Menstrual Bleeding.

Menstrual cycle is a major determinant in female reproductive health. In a recent report, Mao et al. (2022) associated deficient glycolysis with heavy menstrual bleeding. This commentary summarizes these recent findings and the importance of glycolysis and decidualization in endometrial function. It will also discuss if in the light of the recent findings menstrual bleeding is better conceived as a primary endometrial disorder inherent to endometrium or as a secondary endometrial disorder caused by other endometrial conditions.

Transcriptional responses to hypoxia are enhanced by recurrent hypoxia (hypoxic preconditioning) in the epaulette shark.

All animals require molecular oxygen for aerobic energy production, and oxygen availability has played a particularly important role in the evolution of aquatic animals. This study investigates how previous exposure to hypoxia (preconditioning) primes protective transcriptional responses in a hypoxia-tolerant vertebrate species, the epaulette shark (Hemiscyllium ocellatum). The epaulette shark is a basal cartilaginous fish that in its natural environment experiences cyclic hypoxic periods. We evaluated whether the transcription of a set of crucial prosurvival genes is affected differently by a single short-term (2 h) exposure to sublethal hypoxia compared with eight such successive hypoxia exposures (hypoxia preconditioning). We discovered that hypoxia preconditioning amplifies transcriptional responses compared with animals that experienced a single hypoxic bout. In the heart we observed that hypoxic preconditioning, but not a single hypoxic exposure, resulted in higher transcript levels of genes that regulate oxygen and energy homeostasis, including those of hypoxia-inducible factor-1 alpha, adenosine signaling pathway components, and genes affecting circulation [prostaglandin synthetase 2 (cox-2) and natriuretic peptide C]. This suggests that in a single short-term hypoxic bout, the responses to low oxygen are regulated at the level of pre-existing proteins or translational and posttranslational machinery, whereas transcriptional responses are induced in experiments that parallel the natural environmental cycles of oxygen availability. These findings have general implications for understanding how vertebrates regulate protective gene expression upon physiological stress.

Molecular evolution of the metazoan PHD-HIF oxygen-sensing system.

Metazoans rely on aerobic energy production, which requires an adequate oxygen supply. During reduced oxygen supply (hypoxia), the most profound changes in gene expression are mediated by transcription factors known as hypoxia-inducible factors (HIFs). HIF alpha proteins are commonly posttranslationally regulated by prolyl-4-hydroxylase (PHD) enzymes, which are direct "sensors" of cellular oxygen levels. We examined the molecular evolution of the metazoan PHD-HIF oxygen-sensing system by constructing complete phylogenies for PHD and HIF alpha genes and used computational tools to characterize the molecular changes underlying the functional divergence of PHD and HIF alpha duplicates. The presence of PHDs in metazoan genomes predates the emergence of HIF alphas. Our analysis revealed an unexpected diversity of PHD genes and HIF alpha sequence characteristics in invertebrates, suggesting that the simple oxygen-sensing systems of Caenorhabditis and Drosophila may not be typical of other invertebrate bilaterians. We studied the early vertebrate evolution of the system by sequencing these genes in early-diverging cartilaginous fishes, elasmobranchs. Cartilaginous fishes appear to have three paralogs of both PHD and HIF alpha. The novel sequences were used as outgroups for a detailed molecular analysis of PHD and HIF alpha duplicates in a major air-breathing vertebrate lineage, the mammals, and a major water-breathing vertebrate lineage, the teleosts. In PHDs, functionally divergent amino acid sites were detected near the HIF alpha-binding channel and beta2beta3 loop that defines its substrate specificity. In HIF alphas, more functional divergence was found in teleosts than in mammals, especially in the HIF-1 alpha PAS domain and HIF-2 alpha oxygen-dependent degradation (ODD) domains, which interact with PHDs. Overall, in the vertebrates, elevated substitution rates in the HIF-2 alpha N-terminal ODD domain, together with a functional divergence associated with the known differences in PHD2 versus PHD1/3 substrate specificity, have contributed to the tighter oxygen-sensitive regulation of HIF-1 alpha than that of HIF-2 alpha.

Dirichlet process mixture models for single-cell RNA-seq clustering.

Clustering of cells based on gene expression is one of the major steps in single-cell RNA-sequencing (scRNA-seq) data analysis. One key challenge in cluster analysis is the unknown number of clusters and, for this issue, there is still no comprehensive solution. To enhance the process of defining meaningful cluster resolution, we compare Bayesian latent Dirichlet allocation (LDA) method to its non-parametric counterpart, hierarchical Dirichlet process (HDP) in the context of clustering scRNA-seq data. A potential main advantage of HDP is that it does not require the number of clusters as an input parameter from the user. While LDA has been used in single-cell data analysis, it has not been compared in detail with HDP. Here, we compare the cell clustering performance of LDA and HDP using four scRNA-seq datasets (immune cells, kidney, pancreas and decidua/placenta), with a specific focus on cluster numbers. Using both intrinsic (DB-index) and extrinsic (ARI) cluster quality measures, we show that the performance of LDA and HDP is dataset dependent. We describe a case where HDP produced a more appropriate clustering compared to the best performer from a series of LDA clusterings with different numbers of clusters. However, we also observed cases where the best performing LDA cluster numbers appropriately capture the main biological features while HDP tended to inflate the number of clusters. Overall, our study highlights the importance of carefully assessing the number of clusters when analyzing scRNA-seq data.

COVID-19-specific transcriptomic signature detectable in blood across multiple cohorts.

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading across the world despite vast global vaccination efforts. Consequently, many studies have looked for potential human host factors and immune mechanisms associated with the disease. However, most studies have focused on comparing COVID-19 patients to healthy controls, while fewer have elucidated the specific host factors distinguishing COVID-19 from other infections. To discover genes specifically related to COVID-19, we reanalyzed transcriptome data from nine independent cohort studies, covering multiple infections, including COVID-19, influenza, seasonal coronaviruses, and bacterial pneumonia. The identified COVID-19-specific signature consisted of 149 genes, involving many signals previously associated with the disease, such as induction of a strong immunoglobulin response and hemostasis, as well as dysregulation of cell cycle-related processes. Additionally, potential new gene candidates related to COVID-19 were discovered. To facilitate exploration of the signature with respect to disease severity, disease progression, and different cell types, we also offer an online tool for easy visualization of the selected genes across multiple datasets at both bulk and single-cell levels.

Histone H3K4me3 breadth in hypoxia reveals endometrial core functions and stress adaptation linked to endometriosis.

Trimethylation of histone H3 at lysine 4 (H3K4me3) is a marker of active promoters. Broad H3K4me3 promoter domains have been associated with cell type identity, but H3K4me3 dynamics upon cellular stress have not been well characterized. We assessed this by exposing endometrial stromal cells to hypoxia, which is a major cellular stress condition. We observed that hypoxia modifies the existing H3K4me3 marks and that promoter H3K4me3 breadth rather than height correlates with transcription. Broad H3K4me3 domains mark genes for endometrial core functions and are maintained or selectively extended upon hypoxia. Hypoxic extension of H3K4me3 breadth associates with stress adaptation genes relevant for the survival of endometrial cells including transcription factor KLF4, for which we found increased protein expression in the stroma of endometriosis lesions. These results substantiate the view on broad H3K4me3 as a marker of cell identity genes and reveal participation of H3K4me3 extension in cellular stress adaptation.

Computational strategies for single-cell multi-omics integration.

Single-cell omics technologies are currently solving biological and medical problems that earlier have remained elusive, such as discovery of new cell types, cellular differentiation trajectories and communication networks across cells and tissues. Current advances especially in single-cell multi-omics hold high potential for breakthroughs by integration of multiple different omics layers. To pair with the recent biotechnological developments, many computational approaches to process and analyze single-cell multi-omics data have been proposed. In this review, we first introduce recent developments in single-cell multi-omics in general and then focus on the available data integration strategies. The integration approaches are divided into three categories: early, intermediate, and late data integration. For each category, we describe the underlying conceptual principles and main characteristics, as well as provide examples of currently available tools and how they have been applied to analyze single-cell multi-omics data. Finally, we explore the challenges and prospective future directions of single-cell multi-omics data integration, including examples of adopting multi-view analysis approaches used in other disciplines to single-cell multi-omics.

Differential ATAC-seq and ChIP-seq peak detection using ROTS.

Changes in cellular chromatin states fine-tune transcriptional output and ultimately lead to phenotypic changes. Here we propose a novel application of our reproducibility-optimized test statistics (ROTS) to detect differential chromatin states (ATAC-seq) or differential chromatin modification states (ChIP-seq) between conditions. We compare the performance of ROTS to existing and widely used methods for ATAC-seq and ChIP-seq data using both synthetic and real datasets. Our results show that ROTS outperformed other commonly used methods when analyzing ATAC-seq data. ROTS also displayed the most accurate detection of small differences when modeling with synthetic data. We observed that two-step methods that require the use of a separate peak caller often more accurately called enrichment borders, whereas one-step methods without a separate peak calling step were more versatile in calling sub-peaks. The top ranked differential regions detected by the methods had marked correlation with transcriptional differences of the closest genes. Overall, our study provides evidence that ROTS is a useful addition to the available differential peak detection methods to study chromatin and performs especially well when applied to study differential chromatin states in ATAC-seq data.

Elasmobranch qPCR reference genes: a case study of hypoxia preconditioned epaulette sharks.

BACKGROUND: Elasmobranch fishes are an ancient group of vertebrates which have high potential as model species for research into evolutionary physiology and genomics. However, no comparative studies have established suitable reference genes for quantitative PCR (qPCR) in elasmobranchs for any physiological conditions. Oxygen availability has been a major force shaping the physiological evolution of vertebrates, especially fishes. Here we examined the suitability of 9 reference candidates from various functional categories after a single hypoxic insult or after hypoxia preconditioning in epaulette shark (Hemiscyllium ocellatum). RESULTS: Epaulette sharks were caught and exposed to hypoxia. Tissues were collected from 10 controls, 10 individuals with single hypoxic insult and 10 individuals with hypoxia preconditioning (8 hypoxic insults, 12 hours apart). We produced sequence information for reference gene candidates and monitored mRNA expression levels in four tissues: cerebellum, heart, gill and eye. The stability of the genes was examined with analysis of variance, geNorm and NormFinder. The best ranking genes in our study were eukaryotic translation elongation factor 1 beta (eef1b), ubiquitin (ubq) and polymerase (RNA) II (DNA directed) polypeptide F (polr2f). The performance of the ribosomal protein L6 (rpl6) was tissue-dependent. Notably, in one tissue the analysis of variance indicated statistically significant differences between treatments for genes that were ranked as the most stable candidates by reference gene software. CONCLUSIONS: Our results indicate that eef1b and ubq are generally the most suitable reference genes for the conditions and tissues in the present epaulette shark studies. These genes could also be potential reference gene candidates for other physiological studies examining stress in elasmobranchs. The results emphasise the importance of inter-group variation in reference gene evaluation.

RepViz: a replicate-driven R tool for visualizing genomic regions.

OBJECTIVE: Visualization of sequencing data is an integral part of genomic data analysis. Although there are several tools to visualize sequencing data on genomic regions, they do not offer user-friendly ways to view simultaneously different groups of replicates. To address this need, we developed a tool that allows efficient viewing of both intra- and intergroup variation of sequencing counts on a genomic region, as well as their comparison to the output of user selected analysis methods, such as peak calling. RESULTS: We present an R package RepViz for replicate-driven visualization of genomic regions. With ChIP-seq and ATAC-seq data we demonstrate its potential to aid visual inspection involved in the evaluation of normalization, outlier behavior, detected features from differential peak calling analysis, and combined analysis of multiple data types. RepViz is readily available on Bioconductor ( https://www.bioconductor.org/packages/devel/bioc/html/RepViz.html ) and on Github ( https://github.com/elolab/RepViz ).

Decidualization of Human Endometrial Stromal Fibroblasts is a Multiphasic Process Involving Distinct Transcriptional Programs.

Decidual stromal cells differentiate from endometrial stromal fibroblasts (ESFs) under the influence of progesterone and cyclic adenosine monophosphate (cAMP) and are essential for implantation and the maintenance of pregnancy. They evolved in the stem lineage of placental (eutherian) mammals coincidental with the evolution of implantation. Here we use the well-established in vitro decidualization protocol to compare early (3 days) and late (8 days) gene transcription patterns in immortalized human ESF. We document extensive, dynamic changes in the early and late decidual cell transcriptomes. The data suggest the existence of an early signal transducer and activator of transcription (STAT) pathway dominated state and a later nuclear factor κB (NFKB) pathway regulated state. Transcription factor expression in both phases is characterized by putative or known progesterone receptor ( PGR) target genes, suggesting that both phases are under progesterone control. Decidualization leads to proliferative quiescence, which is reversible by progesterone withdrawal after 3 days but to a lesser extent after 8 days of decidualization. In contrast, progesterone withdrawal induces cell death at comparable levels after short or long exposure to progestins and cAMP. We conclude that decidualization is characterized by a biphasic gene expression dynamic that likely corresponds to different phases in the establishment of the fetal-maternal interface.

Variable patterns in the molecular evolution of the hypoxia-inducible factor-1 alpha (HIF-1alpha) gene in teleost fishes and mammals.

The hypoxia-inducible factor-1 alpha (HIF-1alpha) protein is the major regulator of oxygen-dependent gene expression and a member of the bHLH-PAS family of transcription factors. In this study we compared and contrasted the rate and mode of HIF-1alpha molecular evolution between teleost fishes and mammals, as well as within teleost fishes and mammals. Various likelihood methods for estimating codon substitutions were used to detect different modes of selection. Although the overall evolutionary rate in teleost HIF-1alpha was at least twice as fast as in mammalian HIF-1alpha, the crucial interaction domains were observed to be under stringent negative selection in all vertebrates. Relaxed negative selection on less crucial regions of teleost HIF-1alpha compared to mammalian HIF-1alpha was detected, but no evidence for positive selection that was supported by all methods was found. We suggest that the relaxed selective constraints in teleost HIF-1alpha may be associated with the variable environmental oxygen levels to which teleosts have been exposed during their evolutionary history. However, in teleosts the positions with partial support for positive selection were not found in the vicinity of the HIF-1alpha domains which confer the oxygen sensitivity, but in the bHLH-PAS domain responsible for DNA binding and dimerization. The pattern of selection in the bHLH-PAS domain has some similarities with the patterns observed in the adaptive evolution of the homeodomain of Hox genes and may be typical in the molecular evolution of transcription factors.

Evolution: Oxygen and early animals.

The biology of sponges provides clues about how early animals may have dealt with low levels of oxygen.