SenNet recommendations for detecting senescent cells in different tissues.

Once considered a tissue culture-specific phenomenon, cellular senescence has now been linked to various biological processes with both beneficial and detrimental roles in humans, rodents and other species. Much of our understanding of senescent cell biology still originates from tissue culture studies, where each cell in the culture is driven to an irreversible cell cycle arrest. By contrast, in tissues, these cells are relatively rare and difficult to characterize, and it is now established that fully differentiated, postmitotic cells can also acquire a senescence phenotype. The SenNet Biomarkers Working Group was formed to provide recommendations for the use of cellular senescence markers to identify and characterize senescent cells in tissues. Here, we provide recommendations for detecting senescent cells in different tissues based on a comprehensive analysis of existing literature reporting senescence markers in 14 tissues in mice and humans. We discuss some of the recent advances in detecting and characterizing cellular senescence, including molecular senescence signatures and morphological features, and the use of circulating markers. We aim for this work to be a valuable resource for both seasoned investigators in senescence-related studies and newcomers to the field.

Maternal SARS-CoV-2 infection in pregnancy disrupts gene expression in Hofbauer cells with limited impact on cytotrophoblasts.

BACKGROUND: Hofbauer cells (HBCs) and cytotrophoblasts (CTBs) are major cell populations in placenta. The indirect impact of maternal SARS-CoV-2 disease on these cells that are not directly infected has not been extensively studied. Herein, we profiled gene expression in HBCs and CTBs isolated from placentae of recovered pregnant subjects infected with SARS-CoV-2 during all trimesters of pregnancy, placentae from subjects with active infection, SARS-CoV-2 vaccinated subjects, and those who were unexposed to the virus. METHODS: Placentae were collected within 4 h post-delivery and membrane-free tissues were enzymatically digested for the isolation of HBCs and CTBs. RNA extracted from HBCs and CTBs were sequenced using 150bp paired-end reads. Differentially expressed genes (DEGs) were identified by DESeq2 package in R and enriched in GO Biological Processes, KEGG Pathway, Reactome Gene Sets, Hallmark Gene Sets, and Canonical Pathways. Protein-protein interactions among the DEGs were modelled using STRING and BioGrid. RESULTS: Pregnant subjects (n = 30) were recruited and categorized into six groups: infected with SARS-CoV-2 in i) the first (1T, n = 4), ii) second (2T, n = 5), iii) third (3T, n = 5) trimester, iv) tested positive at delivery (Delivery, n = 5), v) never infected (Control, n = 6), and vi) fully mRNA-vaccinated by delivery (Vaccinated, n = 5). Compared to the Control group, gene expression analysis showed that HBCs from infected subjects had significantly altered gene expression profiles, with the 2T group having the highest number of DEGs (1,696), followed by 3T and 1T groups (1,656 and 958 DEGs, respectively). These DEGs were enriched for pathways involved in immune regulation for host defense, including production of cytokines, chemokines, antimicrobial proteins, ribosomal assembly, neutrophil degranulation inflammation, morphogenesis, and cell migration/adhesion. Protein-protein interaction analysis mapped these DEGs with oxidative phosphorylation, translation, extracellular matrix organization, and type I interferon signaling. Only 95, 23, and 8 DEGs were identified in CTBs of 1T, 2T, and 3T groups, respectively. Similarly, 11 and 3 DEGs were identified in CTBs and HBCs of vaccinated subjects, respectively. Reassuringly, mRNA vaccination did not induce an inflammatory response in placental cells. CONCLUSIONS: Our studies demonstrate a significant impact of indirect SARS-CoV-2 infection on gene expression of inner mesenchymal HBCs, with limited effect on lining CTB cells isolated from pregnant subjects infected and recovered from SARS-CoV-2. The pathways associated with these DEGs identify potential targets for therapeutic intervention.

Ultrasound guidance to replicate transuterine BioGlue injection in the fetal hydrocephalus sheep model.

INTRODUCTION: Congenital hydrocephalus often results in irreversible and severe damage to the brain despite postnatal interventions. The potential for prenatal intervention to mitigate these deleterious effects underscores the importance of a suitable animal model. We aimed assess the results of an ultrasound guided transuterine approach to replicate the BioGlue injection fetal hydrocephalus model. METHODS: Pregnant ewes were anesthetized at 95 days of gestation and BioGlue was injected into the fetal cisterna magna under ultrasound guidance through the uterus. Ventriculomegaly was assessed by MRI and histology. RESULTS: Nine pregnant ewes were included in the study, and their fetuses were divided into the the BioGlue intervention group (n=9 fetuses) or the control group (n=7 fetuses) who were not injected. Although hydrocephalus was noted in 5 of 9 fetuses in the intervention group, the ability to induce hydrocephalus went from 0% to 100% in the last 3 fetuses following technical modifications. None of the controls developed hydrocephalus. Fetal brains with hydrocephalus demonstrated increased IBA1+ compared to control animals. CONCLUSIONS: While technical challenges were noted, the ultrasound guided transuterine approach to replicate the BioGlue fetal hydrocephalus model in sheep showed consistent and reproducible results. This model offers the advantage of directly visualizing the location of the needle tip and injection of the BioGlue. This technique offers an alternative for testing novel approaches for prenatal congenital hydrocephalus treatment.

Spatial proteomics reveals phenotypic and functional differences in T cell and macrophage subsets during villitis of unknown etiology.

Villitis of unknown etiology (VUE) is a prevalent inflammatory pathology of the placenta characterized by infiltration of maternal T cells and accumulation of fetal macrophages into chorionic villi. VUE is associated with a variety of adverse clinical outcomes, including fetal growth restriction and fetal demise. Evaluation of the phenotypic and functional differences between two immune cell types associated with this pathology, namely T cells and macrophages, was completed to gain a deeper understanding of the immuno-pathogenesis of VUE. GeoMx Digital Spatial Profiling was performed on placental tissue from 4 high grade VUE cases and 4 controls with no underlying pathology. Placental tissues were fluorescently labeled with CD3 and CD68 antibodies and oligo-conjugated antibodies against 48 protein targets. Overall, T cells in VUE exhibited upregulated markers of activation, memory, and antigen experience compared to controls and were altered based on placental location (villi vs. decidua). Additionally, villous macrophages in VUE upregulated costimulatory and major histocompatibility complex class I and II molecules compared to controls and macrophage subtypes in the decidua. Data herein provides new mechanistic insights into T cell and macrophage biology in VUE which contribute to this abnormal immune response to pregnancy.

Exosomal microRNAs in breast cancer: towards theranostic applications.

Breast cancer is one of the top two reproductive cancers responsible for high rates of morbidity and mortality among women globally. Despite the advancements in the treatment of breast cancer, its early diagnosis remains a challenge. Recent evidence indicates that despite the adroit use of numerous strategies to facilitate rapid and precision-oriented screening of breast cancer at the community level through the use of mammograms, Fine-needle aspiration cytology (FNAC) and biomarker tracking, no strategy has been unequivocally accepted as a gold standard for facilitating rapid screening for disease. This necessitates the need to identify novel strategies for the detection and triage of breast cancer lesions at higher rates of specificity, and sensitivity, whilst taking into account the epidemiologic and social-demographic features of the patients. Recent shreds of evidence indicate that exosomes could be a robust source of biomaterial for the rapid screening of breast cancer due to their high stability and their presence in body fluids. Increasing evidence indicates that the Exosomal microRNAs- play a significant role in modifying the tumour microenvironment of breast cancers, thereby potentially aiding in the proliferation, invasion and metastasis of breast cancer. In this review, we summarize the role of ExomiRs in the tumour microenvironment in breast cancer. These ExomiRs can also be used as candidate biomarkers for facilitating rapid screening and triaging of breast cancer patients for clinical intervention.

Endocrine disrupting chemicals: gestational diabetes and beyond.

Gestational Diabetes Mellitus (GDM) has been on the rise for the last two decades along with the growing incidence of obesity. The ubiquitous use of Endocrine-Disrupting Chemicals (EDCs) worldwide has been associated with this increase in GDM incidence. Epigenetic modifications such as DNA methylation, histone acetylation, and methylation have been associated with prenatal exposure to EDCs. EDC exposure can also drive a sustained disruption of the hypothalamus-pituitary-thyroid axis and various other signaling pathways such as thyroid signaling, PPARγ signaling, PI3K-AKT signaling. This disruption leads to impaired glucose metabolism, insulin resistance as well as ß-cell dysfunction, which culminate into GDM. Persistent EDC exposure in pregnant women also increases adipogenesis, which results in gestational weight gain. Importantly, pregnant mothers transfer these EDCs to the fetus via the placenta, thus leading to other pregnancy-associated complications such as intrauterine growth restriction (IUGR), and large for gestational age neonates. Furthermore, this early EDC exposure of the fetus increases the susceptibility of the infant to metabolic diseases in early life. The transgenerational impact of EDCs is also associated with higher vascular tone, cognitive aberrations, and enhanced susceptibility to lifestyle disorders including reproductive health anomalies. The review focuses on the impact of environmental toxins in inducing epigenetic alterations and increasing the susceptibility to metabolic diseases during pregnancy needs to be extensively studied such that interventions can be developed to break this vicious cycle. Furthermore, the use of EDC-associated ExomiRs from the serum of patients can help in the early diagnosis of GDM, thereby leading to triaging of patients based on increasing risk factor of the clinicopathological condition.

Evaluation of the maternal systemic immune system during frozen euploid embryo transfer according to cycle outcome.

Infertility affects 15 % of couples in the US, and many turn to assisted reproductive technologies, including in vitro fertilization and subsequent frozen embryo transfer (FET) to become pregnant. This study aimed to perform a broad assessment of the maternal immune system to determine if there are systemic differences on the day of FET in cycles that result in a live birth compared to those that do not. Women undergoing FET of euploid embryos were recruited and blood was collected on the day of FET as well as at early timepoints in pregnancy. Sixty immune and angiogenic proteins were measured in plasma, and gene expression of 92 immune-response related genes were evaluated in peripheral blood mononuclear cells (PBMCs). We found plasma concentrations of interleukin-13 (IL-13) and macrophage derived chemokine (MDC) were significantly lower on the day of FET in cycles that resulted in a live birth. We also found genes encoding C-C chemokine receptor type 5 (CCR5), CD8 subunit alpha (CD8A) and SMAD family member 3 (SMAD3) were upregulated in PBMCs on the day of FET in cycles that resulted in live birth. Measurements of immune mediators from maternal blood could serve as prognostic markers during FET to guide clinical decision making and further our understanding of implantation failure.

Comparison of a modified staging system with 8th edition AJCC criteria in a North American cohort of pT2/pT3 HPV-negative penile squamous cell carcinoma.

The staging for pT2/pT3 penile squamous cell carcinoma (pSCC) has undergone major changes. Some authors proposed criteria wherein the distinction between pT2/pT3 was made using the same histopathological variables that are currently utilized to differentiate pT1a/pT1b. In this single-institution, North American study, we focused on (HPV-negative) pT2/3 pSCCs (i.e., tumors invading corpus spongiosum/corpus cavernosum), and compared the prognostic ability of the following systems: (i) AJCC (8th edition) criteria; (ii) modified staging criteria proposed by Sali et al. (Am J Surg Pathol. 2020; 44:1112-7). In the proposed system, pT2 tumors were defined as those devoid of lymphovascular invasion (LVI) or perineural invasion (PNI), and were not poorly differentiated; whereas pT3 showed one or more of the following: LVI, PNI, and/or grade 3. 48 pT2/pT3 cases were included (AJCC, pT2: 27 and pT3: 21; Proposed, pT2: 22 and pT3: 26). The disease-free survival (DFS) and progression-free survival (PFS) did not differ between pT2 and pT3, following the current AJCC definitions (p = 0.19 and p = 0.10, respectively). When the pT2/3 stages were reconstructed using the modified criteria, however, a statistically significant difference was present in both DFS and PFS between pT2 and pT3 (p = 0.004 and p = 0.003, respectively). The proposed staging system has the potential to improve the prognostication of pT2/pT3 tumors in pSCC. Each of these histopathologic variables has been shown to have a significant association with outcomes in pSCC, which is an advantage. Further studies are needed to demonstrate the utility of this modified staging system in patient populations from other geographic regions.

High-risk human papilloma virus status & outcomes for penile squamous cell carcinoma: A single institution experience.

OBJECTIVES: There is a paucity of data on North American cohorts of patients with penile squamous cell carcinoma (pSCC). Herein, we aimed to assess the sensitivity of various modalities to identify human papillomavirus (HPV) status, determine the prevalence of high-risk HPV-positivity, and evaluate the prognostic impact of relevant clinicopathologic variables. METHODS: Patients with pSCC (n = 121) consecutively treated with partial/total penectomy (2000-2022) at a single institution were included. HPV status (based on immunohistochemistry [IHC], in situ hybridization [ISH], and panviral metagenomic sequencing [PMS]), histologic features, and outcomes were reviewed. Outcome events included death due to disease and progression. RESULTS: The majority of patients were white (105/121, 86.8%). Thirty-seven (30.6%) were high-risk HPV-positive, and morphologic evaluation had a sensitivity of 97.3% (95% confidence interval [CI], 86.2-99.5) for predicting high-risk HPV status compared to IHC/ISH/PMS. Disease progression was more common among high-risk HPV-negative compared to high-risk HPV-positive patients (HR 2.74, CI 1.12-8.23, P = 0.03). Moreover, among high-risk HPV-negative patients, those with moderate-poorly differentiated tumors had increased disease-specific mortality (32.6%, CI 17.1-48.1) compared to those with well-differentiated tumors (0%). Among high-risk HPV-positive patients, those with basaloid morphology had lower disease-specific mortality (0% vs 14.4%, CI 0.0-33.1). CONCLUSIONS: We demonstrate high-risk HPV-positivity in approximately one-third of patients with pSCC. Morphologic evaluation alone had a high sensitivity in correctly determining HPV status. Our results suggest that high-risk HPV status and morphologic features (differentiation in high-risk HPV-negative, and basaloid subtype in high-risk HPV-positive pSCC) may have prognostic value.