Altered histone modification landscapes underpin defects in uterine stromal cell decidualization in aging females.

Decidualization describes the transformation of the uterine stroma in response to an implanting embryo, a process critical for supporting the development of the early embryo, for ensuring normal placentation and ultimately for a healthy reproductive outcome. Maternal age has been found to impede the progression of decidualization, heightening the risk of reproductive problems. Here, we set out to comprehensively characterize this deficit by performing transcriptomic and epigenomic profiling approaches specifically in the uterine stromal cell (UtSC) compartment of young and aged female mice. We find that UtSCs from aged females are globally far less responsive to the decidualization stimulus triggered by exposure to the steroid hormones estrogen and progesterone. Despite an overall transcriptional hyperactivation of genes that are differentially expressed as a function of maternal age, the hormonally-regulated genes specifically fail to be activated in aged UtSCs. Moreover, even in their unstimulated "ground" state, UtSCs from aged females are epigenetically distinct, as determined by genomic enrichment profiling for the active and repressive histone marks H3K4me3 and H3K9me3, respectively. We find that many hormone-inducible genes exhibit a profound lack of promoter-associated H3K4me3 in aged UtSCs, implying that a significant enrichment of active histone marks prior to gene stimulation is required to enable the elicitation of a rapid transcriptional response. With this combination of criteria, our data highlight specific deficits in epigenetic marking and gene expression of ion channels and vascular markers. These results point to fundamental defects in muscle-related and perivascular niche functions of the uterine stroma with advanced maternal age.

Prevalence of Moral Injury, Burnout, Anxiety, and Depression in Healthcare Workers 2 Years in to the COVID-19 Pandemic.

ABSTRACT: COVID-19 has led to marked increases in healthcare worker distress. Studies of these phenomena are often limited to a particular element of distress or a specific subset of healthcare workers. We administered the Moral Injury Symptom Scale for Healthcare Professionals, Copenhagen Burnout Inventory, Patient Health Questionnaire-9, and Generalized Anxiety Disorder-7 via online survey to 17,000 employees of a large academic medical center between December 2021 and February 2022. A total of 1945 participants completed the survey. Across all roles, the prevalence of moral injury, burnout, depression, and anxiety were 40.9%, 35.3%-60.6%, 25.4%, and 24.8%, respectively. Furthermore, 8.1% had been bothered by thoughts that they would be better off dead or of hurting themselves for "several days" or more frequently. Healthcare workers across all roles and practice settings are experiencing unsustainable levels of distress, with 1 in 12 regularly experiencing thoughts of self-harm.

Medicine shouldn't be this hard: The intersection of physician moral injury and patient healthcare experience in pediatric complex care.

Dr. Jay Neufeld's story in If I Betray These Words is a detailed account of one physician's catastrophic journey through moral injury when caring for pediatric patients with complex medical conditions [1]. Many clinicians may recognize Jay's journey in their own experiences, but what deserves parallel consideration are the journeys of patients and families when they are accompanied by physicians at risk of moral injury. This case study illustrates the tight link between drivers of physician moral injury and patients' negative healthcare experiences. These include (1) decisions directed by health insurance regulations and prior authorizations; (2) the electronic medical record (EMR); and (3) healthcare systems focused on revenue generation.

The Trophoblast Compartment Helps Maintain Embryonic Pluripotency and Delays Differentiation towards Cardiomyocytes.

Normal developmental progression relies on close interactions between the embryonic and extraembryonic lineages in the pre- and peri-gastrulation stage conceptus. For example, mouse epiblast-derived FGF and NODAL signals are required to maintain a stem-like state in trophoblast cells of the extraembryonic ectoderm, while visceral endoderm signals are pivotal to pattern the anterior region of the epiblast. These developmental stages also coincide with the specification of the first heart precursors. Here, we established a robust differentiation protocol of mouse embryonic stem cells (ESCs) into cardiomyocyte-containing embryoid bodies that we used to test the impact of trophoblast on this key developmental process. Using trophoblast stem cells (TSCs) to produce trophoblast-conditioned medium (TCM), we show that TCM profoundly slows down the cardiomyocyte differentiation dynamics and specifically delays the emergence of cardiac mesoderm progenitors. TCM also strongly promotes the retention of pluripotency transcription factors, thereby sustaining the stem cell state of ESCs. By applying TCM from various mutant TSCs, we further show that those mutations that cause a trophoblast-mediated effect on early heart development in vivo alter the normal cardiomyocyte differentiation trajectory. Our approaches provide a meaningful deconstruction of the intricate crosstalk between the embryonic and the extraembryonic compartments. They demonstrate that trophoblast helps prolong a pluripotent state in embryonic cells and delays early differentiative processes, likely through production of leukemia inhibitory factor (LIF). These data expand our knowledge of the multifaceted signaling interactions among distinct compartments of the early conceptus that ensure normal embryogenesis, insights that will be of significance for the field of synthetic embryo research.

The placenta: epigenetic insights into trophoblast developmental models of a generation-bridging organ with long-lasting impact on lifelong health.

The placenta is a unique organ system that functionally combines both maternal and fetal cell types with distinct lineage origins. Normal placentation is critical for developmental progression and reproductive success. Although the placenta is best known for its nutrient supply function to the fetus, genetic experiments in mice highlight that the placenta is also pivotal for directing the proper formation of specific fetal organs. These roles underscore the importance of the placenta for pregnancy outcome and lifelong health span, which makes it essential to better understand the molecular processes governing placental development and function and to find adequate models to study it. In this review, we provide an overview of placental development and highlight the instructional role of the epigenome in dictating cell fate decisions specifically in the placental trophoblast cell lineage. We then focus on recent advances in exploring stem cell and organoid models reflecting the feto-maternal interface in mice and humans that provide much-improved tools to study events in early development. We discuss stem cells derived from the placenta as well as those artificially induced to resemble the placenta, and how they can be combined with embryonic stem cells and with endometrial cell types of the uterus to reconstitute the early implantation site. We then allude to the exciting prospects of how these models can be harnessed in biomedicine to enhance our understanding of the pathological underpinnings of pregnancy complications in a patient-specific manner, and ultimately to facilitate therapeutic approaches of tissue- and organ-based regenerative medicine.

Quantifying the relationship between cell proliferation and morphology during development of the face.

Morphogenesis requires highly coordinated, complex interactions between cellular processes: proliferation, migration, and apoptosis, along with physical tissue interactions. How these cellular and tissue dynamics drive morphogenesis remains elusive. Three dimensional (3D) microscopic imaging poses great promise, and generates elegant images. However, generating even moderate through-put quantified images is challenging for many reasons. As a result, the association between morphogenesis and cellular processes in 3D developing tissues has not been fully explored. To address this critical gap, we have developed an imaging and image analysis pipeline to enable 3D quantification of cellular dynamics along with 3D morphology for the same individual embryo. Specifically, we focus on how 3D distribution of proliferation relates to morphogenesis during mouse facial development. Our method involves imaging with light-sheet microscopy, automated segmentation of cells and tissues using machine learning-based tools, and quantification of external morphology via geometric morphometrics. Applying this framework, we show that changes in proliferation are tightly correlated to changes in morphology over the course of facial morphogenesis. These analyses illustrate the potential of this pipeline to investigate mechanistic relationships between cellular dynamics and morphogenesis during embryonic development.

Defects in placental syncytiotrophoblast cells are a common cause of developmental heart disease.

Placental abnormalities have been sporadically implicated as a source of developmental heart defects. Yet it remains unknown how often the placenta is at the root of congenital heart defects (CHDs), and what the cellular mechanisms are that underpin this connection. Here, we selected three mouse mutant lines, Atp11a, Smg9 and Ssr2, that presented with placental and heart defects in a recent phenotyping screen, resulting in embryonic lethality. To dissect phenotype causality, we generated embryo- and trophoblast-specific conditional knockouts for each of these lines. This was facilitated by the establishment of a new transgenic mouse, Sox2-Flp, that enables the efficient generation of trophoblast-specific conditional knockouts. We demonstrate a strictly trophoblast-driven cause of the CHD and embryonic lethality in one of the three lines (Atp11a) and a significant contribution of the placenta to the embryonic phenotypes in another line (Smg9). Importantly, our data reveal defects in the maternal blood-facing syncytiotrophoblast layer as a shared pathology in placentally induced CHD models. This study highlights the placenta as a significant source of developmental heart disorders, insights that will transform our understanding of the vast number of unexplained congenital heart defects.

Mechanisms and function of de novo DNA methylation in placental development reveals an essential role for DNMT3B.

DNA methylation is a repressive epigenetic modification that is essential for development, exemplified by the embryonic and perinatal lethality observed in mice lacking de novo DNA methyltransferases (DNMTs). Here we characterise the role for DNMT3A, 3B and 3L in gene regulation and development of the mouse placenta. We find that each DNMT establishes unique aspects of the placental methylome through targeting to distinct chromatin features. Loss of Dnmt3b results in de-repression of germline genes in trophoblast lineages and impaired formation of the maternal-foetal interface in the placental labyrinth. Using Sox2-Cre to delete Dnmt3b in the embryo, leaving expression intact in placental cells, the placental phenotype was rescued and, consequently, the embryonic lethality, as Dnmt3b null embryos could now survive to birth. We conclude that de novo DNA methylation by DNMT3B during embryogenesis is principally required to regulate placental development and function, which in turn is critical for embryo survival.

The Norms and Corporatization of Medicine Influence Physician Moral Distress in the United States.

PhenomenonMoral distress, which occurs when someone's moral integrity is seriously compromised because they feel unable to act in accordance with their core values and obligations, is an increasingly important concern for physicians. Due in part to limited understanding of the root causes of moral distress, little is known about which approaches are most beneficial for mitigating physicians' distress. Our objective was to describe system-level factors in United States (U.S.) healthcare that contribute to moral distress among pediatric hospitalist attendings and pediatric residents.ApproachIn this qualitative study, we conducted one-on-one semi-structured interviews with pediatric hospitalist attendings and pediatric residents from 4 university-affiliated, freestanding children's hospitals in the U.S. between August 2019 and February 2020. Data were coded with an iteratively developed codebook, categorized into themes, and then synthesized.FindingsWe interviewed 22 hospitalists and 18 residents. Participants described in detail how the culture of medicine created a context that cultivated moral distress. Norms of medical education and the practice of medicine created conflicts between residents' strong sense of professional responsibility to serve the best interests of their patients and the expectations of a hierarchical system of decision-making. The corporatization of the U.S. healthcare system created administrative and financial pressures that conflicted with the moral responsibility felt by both residents and hospitalists to provide the care that their patients and families needed.InsightsThese findings highlight the critical role of systemic sources of moral distress. These findings suggest that system-level interventions must supplement existing interventions that target individual health care providers. Preventing and managing moral distress will require a broad approach that addresses systemic drivers, such as the corporatization of medicine, which are entrenched in the culture of medicine.

What Should Clinicians Do When Health Services Are Improperly Billed in Their Names?

The Centers for Medicare and Medicaid Services mandates physicians' responsibility for making sure that reimbursement for services physicians provide to patients is accurate and appropriate. Yet the shift of physician practice ownership to various employment models has amplified a dilemma. Physicians working as employees for some US health care companies might not know about services billed in their name, much less be able to review or contest when, which, to whom, or at what costs services were billed. Although such practices violate legal standards, many employed physicians are now accountable without transparency or agency. This commentary on a case considers this set of problems in contemporary billing and reimbursement structure and practice.

Pathways of DNA Demethylation.

The regulation of the genome relies on the overlying epigenome to instruct, define, and restrict the activities of cellular differentiation and growth integral to embryonic development, as well as defining the key activities of terminally differentiated cell types. These instructions are positioned as readers, writers, and erasers in their functional roles. Among the sizeable repertoire of epigenetic instructions, DNA methylation is perhaps the best understood process. In mammals, multiple cycles of reprogramming, the addition and removal of DNA methylation coupled with modulation of chromatin post-translational modifications (PMTs), constitute critical phases when the developing embryo must negotiate lineage specification and commitment events which serve to canalise development. During these reprogramming events the DNA methylation instruction is often removed, thereby allowing a change in developmental restriction, resulting in a return to a more plastic and pluripotent state. Thus, in germline reprogramming, DNA demethylation is essential in order to give rise to fully functional gametes which are inherited across generations and poised to restore totipotency. A similar return to a less differentiated state can also be achieved experimentally. DNA methylation constitutes one of the significant barriers to erroneous induced pluripotency, and loss of DNA methylation is a prerequisite for the generation of induced pluripotent stem cells (iPSCs). Taking fully differentiated cells, such as skin fibroblast cells or peripheral blood cells, and turning back the developmental clock by generating iPSCs constituted a technological breakthrough in 2006, offering unprecedented promise in precision regenerative medicine. In this chapter, I will explore mechanistic possibilities for DNA demethylation in the context of natural and experimentally induced epigenetic reprogramming. The balance of the maintenance of DNA methylation as a heritable mark together with its potential for timely removal is essential for lifelong health and may be key in our understanding of aging and the potential to limit or reverse that process.

Padi2/3 Deficiency Alters the Epigenomic Landscape and Causes Premature Differentiation of Mouse Trophoblast Stem Cells.

Histone citrullination is a relatively poorly studied epigenetic modification that involves the irreversible conversion of arginine residues into citrulline. It is conferred by a small family of enzymes known as protein arginine deiminases (PADIs). PADI function supports the pluripotent state of embryonic stem cells, but in other contexts, also promotes efficient cellular differentiation. In the current study, we sought to gain deeper insights into the possible roles of PADIs in mouse trophoblast stem cells (TSCs). We show that Padi2 and Padi3 are the most highly expressed PADI family members in TSCs and are rapidly down-regulated upon differentiation. Padi2/3 double knockout (DKO) TSCs express lower levels of stem cell transcription factors CDX2 and SOX2 and are prone to differentiate into extremely large trophoblast giant cells, an effect that may be mediated by centrosome duplication defects. Interestingly, Padi2/3 DKO TSCs display alterations to their epigenomic landscape, with fewer H3K9me3-marked chromocentric foci and globally reduced 5-methylcytosine levels. DNA methylation profiling identifies that this effect is specifically evident at CpG islands of critical trophoblast genes, such as Gata3, Peg3, Socs3 and Hand1. As a consequence of the hypomethylated state, these factors are up-regulated in Padi2/3 DKO TSCs, driving their premature differentiation. Our data uncover a critical epigenetic role for PADI2/3 in safeguarding the stem cell state of TSCs by modulating the DNA methylation landscape to restrict precocious trophoblast differentiation.

MusMorph, a database of standardized mouse morphology data for morphometric meta-analyses.

Complex morphological traits are the product of many genes with transient or lasting developmental effects that interact in anatomical context. Mouse models are a key resource for disentangling such effects, because they offer myriad tools for manipulating the genome in a controlled environment. Unfortunately, phenotypic data are often obtained using laboratory-specific protocols, resulting in self-contained datasets that are difficult to relate to one another for larger scale analyses. To enable meta-analyses of morphological variation, particularly in the craniofacial complex and brain, we created MusMorph, a database of standardized mouse morphology data spanning numerous genotypes and developmental stages, including E10.5, E11.5, E14.5, E15.5, E18.5, and adulthood. To standardize data collection, we implemented an atlas-based phenotyping pipeline that combines techniques from image registration, deep learning, and morphometrics. Alongside stage-specific atlases, we provide aligned micro-computed tomography images, dense anatomical landmarks, and segmentations (if available) for each specimen (N = 10,056). Our workflow is open-source to encourage transparency and reproducible data collection. The MusMorph data and scripts are available on FaceBase ( www.facebase.org , https://doi.org/10.25550/3-HXMC ) and GitHub ( https://github.com/jaydevine/MusMorph ).

What Law Enforcement Can Learn From Health Care About Moral Injury.

A career as a public servant carries risk of moral injury. Law enforcement and health care personnel must subordinate some personal values in service to the public. Transgression and betrayal are primary mechanisms of moral injury, which this article explains. Identifying which value system (eg, personal, professional, or social) is affected by moral injury can inform efforts to mitigate it, help support personnel who have experienced it, and preserve public servants' connection to the broader missions of their professions.

Advanced Maternal Age Differentially Affects Embryonic Tissues with the Most Severe Impact on the Developing Brain.

Advanced maternal age (AMA) poses the single greatest risk to a successful pregnancy. Apart from the impact of AMA on oocyte fitness, aged female mice often display defects in normal placentation. Placental defects in turn are tightly correlated with brain and cardiovascular abnormalities. It therefore follows that placenta, brain and heart development may be particularly susceptible to the impact of AMA. In the current study, we compared global transcriptomes of placentas, brains, hearts, and facial prominences from mid-gestation mouse conceptuses developed in young control (7-13 wks) and aging (43-50 wks) females. We find that AMA increases transcriptional heterogeneity in all tissues, but particularly in fetal brain. Importantly, even overtly normally developed embryos from older females display dramatic expression changes in neurodevelopmental genes. These transcriptomic alterations in the brain are likely induced by defects in placental development. Using trophoblast stem cells (TSCs) as a model, we show that exposure to aging uterine stromal cell-conditioned medium interferes with normal TSC proliferation and causes precocious differentiation, recapitulating many of the defects observed in placentas from aged females. These data highlight the increased risk of AMA on reproductive outcome, with neurodevelopment being the most sensitive to such early perturbations and with potential for lifelong impact.