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Background DNA methylation (DNAm) profiling has emerged as a powerful tool for characterizing the placental methylome. However, previous studies have focused primarily on whole placental tissue, which is a mixture of epigenetically distinct cell populations. Here, we present the first methylome-wide analysis of first trimester ( n  = 9) and term ( n  = 19) human placental samples of four cell populations: trophoblasts, Hofbauer cells, endothelial cells, and stromal cells, using the Illumina EPIC methylation array, which quantifies DNAm at > 850,000 CpGs. Results The most distinct DNAm profiles were those of placental trophoblasts, which are central to many pregnancy-essential functions, and Hofbauer cells, which are a rare fetal-derived macrophage population. Cell-specific DNAm occurs at functionally-relevant genes, including genes associated with placental development and preeclampsia. Known placental-specific methylation marks, such as those associated with genomic imprinting, repetitive element hypomethylation, and placental partially methylated domains, were found to be more pronounced in trophoblasts and often absent in Hofbauer cells. Lastly, we characterize the cell composition and cell-specific DNAm dynamics across gestation. Conclusions Our results provide a comprehensive analysis of DNAm in human placental cell types from first trimester and term pregnancies. This data will serve as a useful DNAm reference for future placental studies, and we provide access to this data via download from GEO (GSE159526), through interactive exploration from the web browser ( https://robinsonlab.shinyapps.io/Placental_Methylome_Browser/ ), and through the R package planet , which allows estimation of cell composition directly from placental DNAm data.

Bone is one of the most important organs in the human body. It provides structure, function, and protection for other vital organs; therefore, bone maintenance and homeostasis are critical processes. As humans age, their bone mineral density decreases, which leads to diseases like osteoporosis. This disease affects one in two women and one in five men aged 50 and over. As the aging population increases, the interest and significance of studying this debilitating bone disease becomes more relevant. Current therapeutic products for osteoporosis have many side effects and can be taken for a limited number of years. Most therapeutic products only focus on decreasing bone resorption, not increasing bone formation. Bone morphogenetic protein 2 is an essential growth factor that drives osteoblast differentiation and activity and is essential for bone formation. However, usage in the clinic is unsuccessful due to several side effects. Recently, a signaling disparity in bone marrow stromal cells within the bone morphogenetic protein pathway that led to decreased bone morphogenetic protein 2 responsiveness was identified in patients diagnosed with osteoporosis. However, it is unclear how other cell populations, especially osteoblasts, which are key players in bone remodeling, are affected and whether the bone morphogenetic protein pathway is affected during osteoporosis. Our research group designed a novel peptide, casein kinase 2.3, that acts downstream of the bone morphogenetic receptor type Ia and increases bone mineralization in murine cells and primary bovine osteoblasts. The aim of the study presented here was to compare the responsiveness of osteoblasts to bone morphogenetic protein 2 and casein kinase 2.3, especially in patients diagnosed with osteoporosis. Mature osteoblasts were extracted from patients diagnosed with osteoporosis or osteoarthritis from Christiana Care Hospital in Newark, Delaware. They were stimulated with either bone morphogenetic protein 2 or casein kinase 2.3, and their effect on osteoblast activity was determined. The osteoporotic patients showed no mineralization response to bone morphogenetic protein 2 stimulation, while the osteoarthritis patients significantly responded to bone morphogenetic protein 2 stimulation. Furthermore, markers for osteoblast activity were increased by casein kinase 2.3, which was in sharp contrast to bone morphogenetic protein 2. This further supports a major bone morphogenetic protein signaling disparity in both the elderly and those suffering with osteoporosis. Both patient types did significantly respond to casein kinase 2.3. Further analysis of the bone morphogenetic protein pathway could lead to new therapeutic products for osteoporosis.

The human placenta is the composite of multiple cell types, each which contributes uniquely to placental function. Small non-coding RNAs (sncRNAs) are regulators of gene expression and can be cell-specific. The sncRNA transcriptome of individual placental cell types has not yet been investigated due to difficulties in their procurement and isolation. Using a custom sequencing method, we explored the expression of seven sncRNA species (miRNA, piRNA, rRNA, scaRNA, snRNA, snoRNA, tRNA) from whole chorionic villi and four major sample-matched FACS-sorted cell type (cytotrophoblast, stromal, endothelial, Hofbauer) samples from 9 first trimester and 17 term placentas. After normalization for technical variables, samples clustered primarily by cell type lineage. No sncRNAs were uniquely expressed by cell type, however, mean expression differed by cell type for 115 sncRNAs. Known placentally-expressed sncRNAs showed differing expression by cell type and trimester. Expression of few sncRNAs varied by sex. Lastly, sample-matched sncRNA expression and DNA methylation correlation was not significant, although high correlation (> R 2  ± 0.6) was observed for some sncRNA-CpG pairs. This study represents the first exploration of the sncRNA transcriptome of bulk placental villi and placental cell types, informing about the expression and regulatory patterns underlying human placental development.

Congenital heart disease (CHD) is often associated with fetal growth abnormalities. During the first trimester of pregnancy, the heart and placenta develop concurrently, and share key developmental pathways. It is hypothesized that defective morphogenesis of either organ is synergistically linked. However, many studies determined to understand the mechanisms behind CHD overlook the contribution of the placenta. In this study, we aimed to identify commonly expressed genes between first trimester heart and placenta cells using two publicly available single cell sequencing databases. Using a systematic computational approach, we identified 328 commonly expressed genes between heart and placenta endothelial cells and enrichment in pathways including Vasculature Development (GO:0001944, FDR 2.90E−30), and Angiogenesis (GO:0001525, FDR 1.18E−27). We also found, in comparison with fetal heart endothelial cells, 197 commonly expressed genes with placenta extravillous trophoblasts, 128 with cytotrophoblasts and 80 with syncytiotrophoblasts, and included genes such as FLT1 , GATA2 , ENG and CDH5 . Finally, comparison of first trimester cardiomyocytes and placenta cytotrophoblasts revealed 53 commonly expressed genes and enrichment in biological processes integral to cellular function including Cellular Respiration (GO:0045333; FDR 5.05E−08), Ion Transport (GO:0006811; FDR 2.08E−02), and Oxidation–Reduction Process (GO:0055114; FDR 1.58E−07). Overall, our results identify specific genes and cellular pathways common between first trimester fetal heart and placenta cells which if disrupted may concurrently contribute to the developmental perturbations resulting in CHD.

Background Sex differences in the function and morphology of the human placenta can lead to sex differences in pregnancy outcomes. X chromosome inactivation (XCI) is the primary mechanism for dosage compensation between the sexes, and is strongly associated with X-chromosome promoter DNA methylation (DNAme) in somatic cells. However, in the placenta, low X-chromosome promoter DNAme has been reported. The placenta is a complex organ consisting of cells of different developmental origins, but the sex differences in DNAme by specific cell types have not been investigated. Methods We examined sex-influenced DNAme from 18 to 19 samples each of endothelial, stromal, cytotrophoblast and Hofbauer cells, sorted from term placentas, as well as matched whole chorionic villi. We also compared these profiles with data from 65 endothelial cell samples from placental chorionic plate arteries and veins (XX = 16, XY = 13) and umbilical cord veins (XX = 22, XY = 14). All data were derived from Illumina Infinium HumanMethylation450 or EPIC DNAme arrays. Sex-stratified analyses of the X/Y and autosomal DNAme were undertaken to identify DNAme differences associated with sex chromosome complement. Results The DNAme distribution on both the X and Y chromosomes differed by cell type. These differences clustered according to the differing developmental origins from extraembryonic mesoderm (endothelial/stromal), trophectoderm (cytotrophoblast) and epiblast (Hofbauer cells), with Hofbauer cells sharing a similar distribution with blood and umbilical endothelial cells. Interestingly, the typical XCI-associated DNAme at promoter CpG islands (CGI) on the X-chromosome of XX cells was absent for endothelial/stromal cells and present only at low levels in trophoblasts, suggesting that de novo establishment of promoter-CGI DNAme on the X-chromosome may differ by cell type. Conclusion The lack of X-linked promoter DNAme in extraembryonic mesoderm-derived cells (endothelial/stromal) is consistent with a distinct developmental origin of these populations relative to the other placental and umbilical cell types. Autosomal DNAme also showed cell-type differences in alignment with cellular relationships observed for sex chromosomes. This work suggests the effects of sex chromosome complement on pregnancy outcomes may differ by placental cell type. Highlights X and Y- chromosome DNA methylation differs by placental cell type. DNA methylation patterns are consistent with the distinct developmental origins of different placental cell types. DNA methylation typically present at X-linked promoters in somatic XX cells was absent in villous endothelial and stromal cells. Endothelial cells in the small vessels of placenta differ strikingly in DNAme from those in umbilical endothelium. Summary The human placenta is a complex organ comprised of diverse cells of different origins, and its function and structure exhibit sex differences associated with pregnancy outcomes. We examined the DNA methylation (DNAme) of placental cells in autosomes and sex chromosomes separately to investigate the impact of sex on the DNAme of placental cells. We used 94 placental cell samples (XX = 50, XY = 44) including 18–19 samples each of endothelial, stromal, Hofbauer cells, cytotrophoblasts and whole chorionic villi, and compared these with public data from 65 endothelial cell samples derived from placental plate artery and vein (XX = 16, XY = 13) and umbilical vein (XX = 22, XY = 14). The DNAme distribution of the X-chromosome differed by cell type in a manner that reflected differing developmental origins. Female (XX) placental endothelial/stromal cells showed distinct DNAme distributions from cytotrophoblast, and both differed from that of Hofbauer cells, which shared a similar DNAme distribution with blood. Interestingly, the typical DNAme associated with X chromosome inactivation was absent or low in endothelial/stromal cells and cytotrophoblasts, suggesting that the DNAme patterns may differ by cell types and their origins. Y-chromosome and autosomal DNAme also showed cell-type differences consistent with a common developmental origin of endothelial and stromal cells distinct from other placental cell types. This work provides insight into the influence of sex and cellular developmental origin on DNAme of mature human placental cell types.

Background The influence of genetics on variation in DNA methylation (DNAme) is well documented. Yet confounding from population stratification is often unaccounted for in DNAme association studies. Existing approaches to address confounding by population stratification using DNAme data may not generalize to populations or tissues outside those in which they were developed. To aid future placental DNAme studies in assessing population stratification, we developed an ethnicity classifier, PlaNET (Placental DNAme Elastic Net Ethnicity Tool), using five cohorts with Infinium Human Methylation 450k BeadChip array (HM450k) data from placental samples that is also compatible with the newer EPIC platform. Results Data from 509 placental samples were used to develop PlaNET and show that it accurately predicts (accuracy = 0.938, kappa = 0.823) major classes of self-reported ethnicity/race (African: n  = 58, Asian: n  = 53, Caucasian: n  = 389), and produces ethnicity probabilities that are highly correlated with genetic ancestry inferred from genome-wide SNP arrays (> 2.5 million SNP) and ancestry informative markers ( n  = 50 SNPs). PlaNET’s ethnicity classification relies on 1860 HM450K microarray sites, and over half of these were linked to nearby genetic polymorphisms ( n  = 955). Our placental-optimized method outperforms existing approaches in assessing population stratification in placental samples from individuals of Asian, African, and Caucasian ethnicities. Conclusion PlaNET provides an improved approach to address population stratification in placental DNAme association studies. The method can be applied to predict ethnicity as a discrete or continuous variable and will be especially useful when self-reported ethnicity information is missing and genotyping markers are unavailable.

The success of regulatory T cell (Treg) therapies depends on the source of Treg and the quality of the Treg manufacturing product that maintains Treg identity. Commonly used methods to identify Treg, including assessment of FOXP3 expression and demethylation of the Treg-specific demethylated region (TSDR), may not be sufficient on their own to ensure that Treg cell therapy drug products have an optimal identity and phenotype prior to infusion into patients. To address this critical need, we developed a robust framework to molecularly characterize Treg products using next-generation sequencing. By systematically profiling Treg and effector T cells (Teff) pre- and post-expansion, we defined the molecular fingerprints for expanded Treg products. We employed a non-parametric algorithm to score Treg manufacturing products for their cell identity and expansion fingerprints. The identity fingerprint reflects Treg cell identity by effectively distinguishing Treg from Teff cells irrespective of their activation status, with 100% sensitivity and specificity, while the expansion fingerprint discriminates expanded versus endogenous Treg or Teff cells. We also showed that the identity fingerprint predicts Treg stability in in vitro settings and can be used to illustrate differences in drug products generated using distinct strategies. We further applied fingerprinting to bulk RNA sequencing (RNA-seq) data from endogenous and expanded Treg cells in a Phase 2 clinical trial for type 1 diabetes (T1D), demonstrating its ability to capture Treg identity and expansion in an independent study. This Treg fingerprinting method provides a powerful tool to molecularly characterize Treg products, potentially enabling correlative analysis with the safety and efficacy outcomes of Treg-based cell therapies.

Background Human placental DNA methylation (DNAme) data is a valuable resource for studying sex differences during gestation, as DNAme profiles after delivery reflect the cumulative effects of gene expression patterns and exposures across gestation. Here, we present an analysis of sex differences in autosomal DNAme in the uncomplicated term placenta ( n = 343) using the Illumina 450K array. Results At a false discovery rate < 0.05 and a mean sex difference in DNAme beta value of > 0.10, we identified 162 autosomal CpG sites that were differentially methylated by sex and replicated in an independent cohort of samples ( n = 293). Several of these differentially methylated CpG sites were part of larger correlated regions of sex differential DNAme. Although global DNAme levels did not differ by sex, the majority of significantly differentially methylated CpGs were more highly methylated in male placentae, the opposite of what is seen in differential methylation analyses of somatic tissues. Patterns of autosomal DNAme at these 162 CpGs were significantly associated with maternal age (in males) and newborn birthweight standard deviation (in females). Conclusions Our results provide a comprehensive analysis of sex differences in autosomal DNAme in the term human placenta. We report a list of high-confidence autosomal sex-associated differentially methylated CpGs and identify several key features of these loci that suggest their relevance to sex differences observed in normative and complicated pregnancies.

Steroid cell tumors not otherwise specified are one of the rare virilizing ovarian tumors. Most of the tumors are benign. This case report illustrates the challenge in managing steroid cell tumor not otherwise specified, which starts from determining its malignant potential, surveillance, and adjuvant treatment option. Steroid cell tumors not otherwise specified are one of the rare virilizing ovarian tumors. Most of the tumors are benign. This case report illustrates the challenge in managing steroid cell tumor not otherwise specified, which starts from determining its malignant potential, surveillance, and adjuvant treatment option.

Metabolic acidosis is common in chronic kidney disease (CKD). However, when the severity of acidosis is disproportionate to renal dysfunction, exogenous causes must be excluded. Topical carbonic anhydrase inhibitors (CAIs) used for glaucoma are commonly perceived as locally acting; in patients with normal renal function their plasma free-drug concentrations are far below those required for systemic effects. In CKD, however, reduced clearance and reduced renal acid-excretory reserve can unmask systemic toxicity. Individual recognition of the syndrome remains uncommon at the bedside, particularly when it presents in the context of acute-on-chronic kidney injury, where its laboratory features can be obscured by acute kidney injury(AKI)-related acidosis. A 73-year-old Asian male with stage 3 CKD and glaucoma presented with progressive dyspnea, anorexia, and a 3-kg weight loss one month after starting fixed-combination brinzolamide 1% / timolol 0.5% eye drops. Laboratory evaluation revealed severe metabolic acidosis (pH 7.29; serum bicarbonate 8.9 mmol/L, from a pre-exposure baseline of 20.3 mmol/L) and acute-on-chronic kidney injury (creatinine 3.9 mg/dL versus baseline ~ 2.0 mg/dL). The disturbance was mixed: a delta-delta ratio of 0.42 (calculated using a reference normal serum bicarbonate of 24 mmol/L) indicated a substantial non-anion-gap (hyperchloremic) component superimposed on a smaller high-anion-gap component, with hypokalemia (K 3.3 mmol/L), hyperchloremia (Cl 107 mmol/L), positive urine anion gap (+ 33.4 mmol/L), and a fractional excretion of bicarbonate of 4.3% measured during established acidosis (without bicarbonate loading). Other causes of metabolic acidosis were systematically excluded. Causality assessment by the Naranjo Adverse Drug Reaction Probability Scale yielded a score of 7 (probable). Discontinuation of the ophthalmic solution and alkali therapy resulted in the resolution of acidosis and recovery of renal function to baseline within one week, sustained at one-year follow-up. Topical CAIs can undergo significant systemic absorption, bypassing first-pass metabolism, and precipitate life-threatening acidosis in patients with reduced renal reserve. Clinicians must maintain a high index of suspicion for ophthalmic medications as hidden causes of mixed acid-base disorders in patients with CKD.