Tight junction protein occludin is an internalization factor for SARS-CoV-2 infection and mediates virus cell-to-cell transmission.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads efficiently by spike-mediated, direct cell-to-cell transmission. However, the underlying mechanism is poorly understood. Herein, we demonstrate that the tight junction protein occludin (OCLN) is critical to this process. SARS-CoV-2 infection alters OCLN distribution and expression and causes syncytium formation that leads to viral spread. OCLN knockdown fails to alter SARS-CoV-2 binding but significantly lowers internalization, syncytium formation, and transmission. OCLN overexpression also has no effect on virus binding but enhances virus internalization, cell-to-cell transmission, and replication. OCLN directly interacts with the SARS-CoV-2 spike, and the endosomal entry pathway is involved in OCLN-mediated cell-to-cell fusion rather than in the cell surface entry pathway. All SARS-CoV-2 strains tested (prototypic, alpha, beta, gamma, delta, kappa, and omicron) are dependent on OCLN for cell-to-cell transmission, although the extent of syncytium formation differs between strains. We conclude that SARS-CoV-2 utilizes OCLN as an internalization factor for cell-to-cell transmission.

High-resolution ribosome profiling reveals translational selectivity for transcripts in bovine preimplantation embryo development.

High-resolution ribosome fractionation and low-input ribosome profiling of bovine oocytes and preimplantation embryos has enabled us to define the translational landscapes of early embryo development at an unprecedented level. We analyzed the transcriptome and the polysome- and non-polysome-bound RNA profiles of bovine oocytes (germinal vesicle and metaphase II stages) and early embryos at the two-cell, eight-cell, morula and blastocyst stages, and revealed four modes of translational selectivity: (1) selective translation of non-abundant mRNAs; (2) active, but modest translation of a selection of highly expressed mRNAs; (3) translationally suppressed abundant to moderately abundant mRNAs; and (4) mRNAs associated specifically with monosomes. A strong translational selection of low-abundance transcripts involved in metabolic pathways and lysosomes was found throughout bovine embryonic development. Notably, genes involved in mitochondrial function were prioritized for translation. We found that translation largely reflected transcription in oocytes and two-cell embryos, but observed a marked shift in the translational control in eight-cell embryos that was associated with the main phase of embryonic genome activation. Subsequently, transcription and translation become more synchronized in morulae and blastocysts. Taken together, these data reveal a unique spatiotemporal translational regulation that accompanies bovine preimplantation development.

Extracellular vesicles from mouse trophoblast cells: Effects on neural progenitor cells and potential participants in the placenta-brain axis†.

The fetal brain of the mouse is thought to be dependent upon the placenta as a source of serotonin (5-hydroxytryptamine; 5-HT) and other factors. How factors reach the developing brain remains uncertain but are postulated here to be part of the cargo carried by placental extracellular vesicles (EV). We have analyzed the protein, catecholamine, and small RNA content of EV from mouse trophoblast stem cells (TSC) and TSC differentiated into parietal trophoblast giant cells (pTGC), potential primary purveyors of 5-HT. Current studies examined how exposure of mouse neural progenitor cells (NPC) to EV from either TSC or pTGC affect their transcriptome profiles. The EV from trophoblast cells contained relatively high amounts of 5-HT, as well as dopamine and norepinephrine, but there were no significant differences between EV derived from pTGC and from TSC. Content of miRNA and small nucleolar (sno)RNA, however, did differ according to EV source, and snoRNA were upregulated in EV from pTGC. The primary inferred targets of the microRNA (miRNA) from both pTGC and TSC were mRNA enriched in the fetal brain. NPC readily internalized EV, leading to changes in their transcriptome profiles. Transcripts regulated were mainly ones enriched in neural tissues. The transcripts in EV-treated NPC that demonstrated a likely complementarity with miRNA in EV were mainly up- rather than downregulated, with functions linked to neuronal processes. Our results are consistent with placenta-derived EV providing direct support for fetal brain development and being an integral part of the placenta-brain axis.

Adaptation and content validation of measure yourself medical outcomes profile (MYMOP) for 7-11 year-old children.

BACKGROUND: The Measure Yourself Medical Outcome Profile (MYMOP) is an individualised tool designed for adults but used with children without any evidence of validation in this population. Individualised instruments are patient-specific rather than disease-specific and therefore can be applied across various health conditions. This study sought to adapt, and content validate the MYMOP for application in 7-11 year old children. METHODS: There were two main phases of the four iterations: expert consultation (three rounds) and interviews with child-parent pairs at the Outpatient clinics of a Children's Hospital. Thematic analysis was undertaken using an inductive, interpretative approach. RESULTS: Four paediatricians completed the first survey, five paediatricians participated in the focus group, and four paediatric health-related quality of life (HRQOL) research experts completed the second survey. Several changes were recommended to the MYMOP by the expert groups. Twenty-five children (17 general medicine, and 8 diabetes/endocrine clinic) aged 7-11 years completed the draft paediatric MYMOP (P-MYMOP) and were interviewed. Results demonstrated that the majority of participants were able to identify their own problems and activity limitations, and all participants understood the 7-point faces scale. Most parents and children perceived that the P-MYMOP would be useful to complete before clinic appointments. CONCLUSIONS: The P-MYMOP is the first content-validated generic individualised HRQOL measure for children 7-11 years old. Given that validation is an iterative process, further research to assess its feasibility, reliability, and construct validity is required.

FGF2, LIF, and IGF1 (FLI) supplementation during human in vitro maturation enhances markers of gamete competence.

STUDY QUESTION: Does a chemically defined maturation medium supplemented with FGF2, LIF, and IGF1 (FLI) improve in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) obtained from children, adolescents, and young adults undergoing ovarian tissue cryopreservation (OTC)? SUMMARY ANSWER: Although FLI supplementation did not increase the incidence of oocyte meiotic maturation during human IVM, it significantly improved quality outcomes, including increased cumulus cell expansion and mitogen-activated protein kinase (MAPK) expression as well as enhanced transzonal projection retraction. WHAT IS KNOWN ALREADY: During OTC, COCs, and denuded oocytes from small antral follicles are released into the processing media. Recovery and IVM of these COCs is emerging as a complementary technique to maximize the fertility preservation potential of the tissue. However, the success of IVM is low, especially in the pediatric population. Supplementation of IVM medium with FLI quadruples the efficiency of pig production through improved oocyte maturation, but whether a similar benefit occurs in humans has not been investigated. STUDY DESIGN, SIZE, DURATION: This study enrolled 75 participants between January 2018 and December 2021 undergoing clinical fertility preservation through the Fertility & Hormone Preservation & Restoration Program at the Ann & Robert H. Lurie Children's Hospital of Chicago. Participants donated OTC media, accumulated during tissue processing, for research. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants who underwent OTC and include a pediatric population that encompassed children, adolescents, and young adults ≤22 years old. All participant COCs and denuded oocytes were recovered from media following ovarian tissue processing. IVM was then performed in either a standard medium (oocyte maturation medium) or one supplemented with FLI (FGF2; 40 ng/ml, LIF; 20 ng/ml, and IGF1; 20 ng/ml). IVM outcomes included meiotic progression, cumulus cell expansion, transzonal projection retraction, and detection of MAPK protein expression. MAIN RESULTS AND THE ROLE OF CHANCE: The median age of participants was 6.3 years, with 65% of them classified as prepubertal by Tanner staging. Approximately 60% of participants had been exposed to chemotherapy and/or radiation prior to OTC. On average 4.7 ± 1 COCs and/or denuded oocytes per participant were recovered from the OTC media. COCs (N = 41) and denuded oocytes (N = 29) were used for IVM (42 h) in a standard or FLI-supplemented maturation medium. The incidence of meiotic maturation was similar between cohorts (COCs: 25.0% vs 28.6% metaphase II arrested eggs in Control vs FLI; denuded oocytes: 0% vs 5.3% in Control vs FLI). However, cumulus cell expansion was 1.9-fold greater in COCs matured in FLI-containing medium relative to Controls and transzonal projection retraction was more pronounced (2.45 ± 0.50 vs 1.16 ± 0.78 projections in Control vs FLIat 16 h). Additionally, MAPK expression was significantly higher in cumulus cells obtained from COCs matured in FLI medium for 16-18 h (chemiluminescence corrected area 621,678 vs 2,019,575 a.u., P = 0.03). LIMITATIONS, REASONS FOR CAUTION: Our samples are from human participants who exhibited heterogeneity with respect to age, diagnosis, and previous treatment history. Future studies with larger sample sizes, including adult participants, are warranted to determine the mechanism by which FLI induces MAPK expression and activation. Moreover, studies that evaluate the developmental competence of eggs derived from FLI treatment, including assessment of embryos as outcome measures, will be required prior to clinical translation. WIDER IMPLICATIONS OF THE FINDINGS: FLI supplementation may have a conserved beneficial effect on IVM for children, adolescents, and young adults spanning the agricultural setting to clinical fertility preservation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Department of Obstetrics and Gynecology startup funds (F.E.D.), Department of Surgery Faculty Practice Plan Grant and the Fertility & Hormone Preservation & Restoration Program at the Ann & Robert H. Lurie Children's Hospital of Chicago (M.M.L. and E.E.R.). M.M.L. is a Gesualdo Foundation Research Scholar. Y.Y.'s research is supported by the internal research funds provided by Colorado Center of Reproductive Medicine. Y.Y., L.D.S., R.M.R., and R.S.P. have a patent pending for FLI. The remaining authors have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

High real-world effectiveness of 12-week elbasvir/grazoprevir without resistance testing in the treatment of patients with HCV genotype 1a infection in Norway.

BACKGROUND AND AIMS: The recommended treatment duration of hepatitis C virus (HCV) genotype 1a (GT1a) infection with elbasvir/grazoprevir (EBR/GZR) in the presence of a high baseline viral load and resistance associated substitutions (RAS) is 16 weeks with ribavirin added. The objective of this study was to evaluate the real-world effectiveness of 12 weeks of EBR/GZR without ribavirin and regardless of baseline viral load and RAS testing. METHOD: This retrospective, observational cohort study was performed at five Norwegian hospitals that did not systematically utilize RAS testing. All adult patients with chronic HCV GT1a and compensated liver disease who had received 12 weeks of EBR/GZR without ribavirin and baseline RAS testing, were included. The primary endpoint was sustained virologic response at week 12 (SVR12), or if not available, at week 4 (SVR4). RESULTS: We included 433 patients and attained SVR data on 388. The mean age was 45.7 years (22-73 years). 67.2% were male. HIV co-infection was present in 3.8% (16/424) and cirrhosis in 4% (17/424). The viral load was >800 000 IU/mL in 55.0% (235/427) of patients. Overall SVR was achieved in 97.2% (377/388). SVR was achieved in 98.3% (169/172) of those with viral load ≤800 000 IU/mL and in 96.2% (202/210) of those with viral load >800 000 IU/mL. CONCLUSION: We observed high SVR rates among patients with HCV GT1a infection treated with EBR/GZR for 12 weeks without ribavirin, with no regard to baseline viral load and no RAS testing.

Diastereoselective synthesis of cyclic tetrapeptide pseudoxylallemycin A illuminates the impact of base during macrolactamization.

Therapeutic agents with unique molecular structures and new mechanisms of action are needed to confront the phenomenon of multidrug resistance among bacteria. Pseudoxylallemycins, cyclic tetrapeptide (CTP) natural products, have exhibited modest antibiotic activity, but their synthesis has proven challenging. Inherent ring strain in CTPs decreases the rate of cyclization in lieu of polymerization and racemization pathways, which has resulted in previous syntheses describing mixtures of diastereomers containing predominantly an undesired epimer. We have optimized the cyclization step of pseudoxylallemycin A to favor production of the natural diastereomer; notably, variation of the base, temperature, and solvent with peptide coupling reagent propylphosphonic anhydride (T3P) afforded exquisite selectivity for the natural product in as high as 97 : 3 DR, and our conditions can provide the natural product in up to 32% overall yield through 8 steps. Employing weaker bases than those typically used in peptide coupling reactions led to the greatest improvement in diastereoselectivity, and these studies demonstrated that the identity of the amine base has enormous impact on the rate of C-terminal epimerization when T3P is used, a variable usually considered of lesser consequence when combined with typical amide coupling reagents. Toward fully characterizing pseudoxylallemycin stereoisomers, variable temperature NMR was described as a tool to more clearly analyze CTPs that exhibit multiple conformational states. These synthetic and spectroscopic insights were applied toward synthesizing several natural product analogues, and their antibacterial activity was examined using microdilution assays.

The role of BMP4 signaling in trophoblast emergence from pluripotency.

The Bone Morphogenetic Protein (BMP) signaling pathway has established roles in early embryonic morphogenesis, particularly in the epiblast. More recently, however, it has also been implicated in development of extraembryonic lineages, including trophectoderm (TE), in both mouse and human. In this review, we will provide an overview of this signaling pathway, with a focus on BMP4, and its role in emergence and development of TE in both early mouse and human embryogenesis. Subsequently, we will build on these in vivo data and discuss the utility of BMP4-based protocols for in vitro conversion of primed vs. naïve pluripotent stem cells (PSC) into trophoblast, and specifically into trophoblast stem cells (TSC). PSC-derived TSC could provide an abundant, reproducible, and ethically acceptable source of cells for modeling placental development.

Bisphenol A and bisphenol S disruptions of the mouse placenta and potential effects on the placenta-brain axis.

Placental trophoblast cells are potentially at risk from circulating endocrine-disrupting chemicals, such as bisphenol A (BPA). To understand how BPA and the reputedly more inert bisphenol S (BPS) affect the placenta, C57BL6J mouse dams were fed 200 µg/kg body weight BPA or BPS daily for 2 wk and then bred. They continued to receive these chemicals until embryonic day 12.5, whereupon placental samples were collected and compared with unexposed controls. BPA and BPS altered the expression of an identical set of 13 genes. Both exposures led to a decrease in the area occupied by spongiotrophoblast relative to trophoblast giant cells (GCs) within the junctional zone, markedly reduced placental serotonin (5-HT) concentrations, and lowered 5-HT GC immunoreactivity. Concentrations of dopamine and 5-hydroxyindoleacetic acid, the main metabolite of serotonin, were increased. GC dopamine immunoreactivity was increased in BPA- and BPS-exposed placentas. A strong positive correlation between 5-HT+ GCs and reductions in spongiotrophoblast to GC area suggests that this neurotransmitter is essential for maintaining cells within the junctional zone. In contrast, a negative correlation existed between dopamine+ GCs and reductions in spongiotrophoblast to GC area ratio. These outcomes lead to the following conclusions. First, BPS exposure causes almost identical placental effects as BPA. Second, a major target of BPA/BPS is either spongiotrophoblast or GCs within the junctional zone. Third, imbalances in neurotransmitter-positive GCs and an observed decrease in docosahexaenoic acid and estradiol, also occurring in response to BPA/BPS exposure, likely affect the placental-brain axis of the developing mouse fetus.

Effects of Integrated Vector Management in the Control of Malaria Infection: An Intervention Study in a Malaria Endemic Community in Nigeria.

BACKGROUND/AIM: Malaria is a vector borne disease with high morbidity and mortality in endemic regions. In view to eliminating the disease, integrated vector and environmental hygiene practices have been advocated. There is paucity of studies on the effect of vector control measures on asymptomatic malaria infection which has been observed to be a reflection of malaria transmission. METHODS: Longitudinal community-based intervention study carried out from October to December 2017. Study participants were 477 individuals living in 100 households selected by snow-balling sampling methods. Pre-intervention period included training of all heads of households on vector control methods. During the intervention period, each household received waste bins, two long lasting insecticide bed nets and had wire screen on their doors and windows; every household member was screened for malaria (antigen) using the pf rapid diagnostic test kits. Each household were monitored to ensure they comply with the environmental hygiene practices they were taught. Post-intervention malaria infection was obtained at 8 week being end of the intervention period. RESULTS: Of the 100 households selected, 54.0% were from the lower social class, 45.0% middle class and only 1.0% upper class. Mean age [±] of the heads of the households was 37.1 ± 11.0 (range 16-68) years. There were 477 individuals recruited in the study from the 100 households; 234 (49.0%) females and 243 (51.0%) males; median age was 20.0 (range 1-100) years. Prevalence of malaria infection using mRDT during pre-intervention was 16.8% and an incidence of 1.3% post-intervention. There was 92.0% reduction in asymptomatic malaria infection showing marked reduction in malaria transmission in the study locale. CONCLUSION: Some integrated vector control measures such as use of insecticide-treated net and sanitation were found effective methods for reducing malaria infection and transmission in endemic region.

CONTEXTE/OBJECTIF: Le paludisme est une maladie à transmission vectorielle avec une morbidité et une mortalité élevées dans les régions endémiques. En vue d'éliminer la maladie, des pratiques d'hygiène intégrée des vecteurs et de l'environnement ont été préconisées. Il existe peu d'études sur l'effet des mesures de lutte antivectorielle sur l'infection palustre asymptomatique, qui s'est avérée être le reflet de la transmission du paludisme. MÉTHODES: Étude longitudinale d'intervention communautaire réalisée d'octobre à décembre 2017. Les participants à l'étude étaient 477 personnes vivant dans 100 ménages sélectionnés par des méthodes d'échantillonnage en boule de neige. La période de pré-intervention comprenait la formation de tous les chefs de ménage sur les méthodes de lutte antivectorielle. Au cours de la période d'intervention, chaque ménage a reçu des poubelles, deux moustiquaires à insecticide longue durée et avait des grillages sur leurs portes et fenêtres ; chaque membre du ménage a été dépisté pour le paludisme (antigène) à l'aide des kits de test de diagnostic rapide pf. Chaque ménage a été suivi pour s'assurer qu'il respecte les pratiques d'hygiène environnementale qui lui ont été enseignées. L'infection antipaludique post-intervention a été obtenue à 8 semaines, fin de la période d'intervention. RÉSULTATS: Sur les 100 ménages sélectionnés, 54,0% appartenaient à la classe sociale inférieure, 45,0% à la classe moyenne et seulement 1,0% à la classe supérieure. L'âge moyen [±] des chefs de ménage était de 37,1 ± 11,0 (fourchette de 16 à 68) ans. Il y avait 477 personnes recrutées dans l'étude à partir des 100 ménages ; 234 (49,0 %) femmes et 243 (51,0 %) hommes ; l'âge médian était de 20,0 (intervalle de 1 à 100) ans. La prévalence de l'infection du paludisme à l'aide de mRDT pendant la pré-intervention était de 16,8 % et l'incidence de 1,3 % après l'intervention. Il y avait une réduction de 92,0 % de l'infection asymptomatique du paludisme, montrant une réduction marquée de la transmission du paludisme dans le lieu de l'étude. CONCLUSION: Certaines mesures intégrées de lutte antivectorielle telles que l'utilisation de moustiquaires imprégnées d'insecticide et l'assainissement se sont révélées être des méthodes efficaces pour réduire l'infection et la transmission du paludisme dans les régions endémiques. Mots clés: Endémique, Hygiène Environnementale, Ménages, Intervention, Paludisme, Transmission, Vecteur.

Asymmetric expression of proteins in the granules of the placentomal Binucleate cells in Giraffa camelopardalis†.

Mature granulated trophoblast binucleate cells (BNC) have been found in all ruminant placentas examined histologically so far. BNC are normally fairly evenly distributed throughout the fetal villus and all their granules contain a similar variety of hormones and pregnancy associated glycoproteins (PAGs). Only the Giraffe is reported to show a different BNC protein expression, this paper is designed to investigate that. Gold labelled Lectin histochemistry and protein immunocytochemistry were used on deplasticised 1 µm sections of a wide variety of ruminant placentomes with a wide range of antibodies and lectins. In the Giraffe placentomes, even though the lectin histochemistry shows an even distribution of BNC throughout the trophoblast of the placental villi, the protein expression in the BNC granules is limited to the BNC either in the apex or the base of the villi. Placental lactogens and Prolactin (PRL) are present only in basally situated BNC: PAGs only in the apical BNC. PRL is only found in the Giraffe BNC which react with many fewer of the wide range of antibodies used here to investigate the uniformity of protein expression in ruminant BNC. The possible relevance of these differences to ruminant function and evolution is considered to provide a further example of the versatility of the BNC system.

Incidence of thrombotic complications related to weight-based dosing of activated prothrombin complex concentrate (aPCC) for reversal of apixaban and rivaroxaban in obese patients.

Factor eight inhibitor bypassing activity (aPCC) is recommended as a non-specific reversal agent for direct oral anticoagulants (DOACs) according to the 2017 American College of Cardiology (ACC) guidelines for reversal of anticoagulation. Factor eight inhibitor bypassing activity carries a black box warning for thrombotic events such as stroke, pulmonary embolism, deep vein thrombosis, and myocardial infarction, particularly at high doses. This was a retrospective, single-center, cohort investigation that included patients who received a weight-based dose of aPCC for reversal of apixaban and rivaroxaban between January 1, 2015, and December 31, 2020. Patients were grouped by BMI as obese (BMI ≥ 30 kg/m2) or non-obese (BMI < 30 kg/m2) for analysis. The primary outcome of this investigation was the occurrence of thrombotic complications [venous thromboembolism (VTE), myocardial infarction, stroke] documented in the medical record at any point during hospitalization after administration of aPCC. Secondary outcomes included bleeding complications, in-hospital mortality, ICU and hospital length of stay. Patients in the obese group were younger [76.4 years (SD +/- 11.3 years) vs. 69.6 years (SD +/- 12.4 years); p < 0.0001] and a higher proportion had a diagnosis of diabetes mellitus prior to admission [37 (19.2%) vs. 35 (36.8%); p = 0.0011]. There was no difference in the primary outcome of thrombotic events between non-obese and obese patients [12 (6.2%) vs. 5 (5.3%); p = 0.75], or for any of the secondary outcomes of bleeding, in-hospital mortality or length of stay. This investigation did not reveal a difference in rates of thrombosis or bleeding events between obese and non-obese patients who received aPCC for reversal of apixaban and rivaroxaban.

Association between opioid use disorder and blunted heart rate variability among opioid-treated chronic pain patients.

Given the severity of the ongoing opioid epidemic, it is essential to understand the mechanisms of risk for development and maintenance of opioid use disorder (OUD). The aim of the current large-scale psychophysiological investigation was to test whether patients with OUD had lower resting-state high-frequency heart rate variability (HF-HRV) than those without OUD, controlling for sociodemographic and clinical confounds. Additionally, we tested whether HF-HRV was associated with opioid craving in this population. Participants in this cross-sectional study were 490 chronic pain patients (50.4% female) treated with long-term opioid therapy. OUD diagnosis was determined by psychiatric interview. HF-HRV was measured at resting baseline. We computed the association between OUD and resting-state HF-HRV, controlling for age, gender, race, pain severity, emotional distress and opioid dose. Opioid craving was measured with visual analogue scales to assess whether HF-HRV was associated with craving. Results showed that resting HF-HRV was significantly lower for patients with OUD than for those without OUD (p < 0.001, d = 0.36), indicating deficits in autonomic flexibility. OUD diagnosis (p = 0.002) and OUD severity (p = 0.03) were associated with lower HF-HRV in regression models accounting for a range of confounders. Additionally, lower HF-HRV was significantly (but weakly) correlated with heightened opioid craving (r = -0.166, p < 0.001). Overall, findings suggest that resting-state HF-HRV may serve as a valid biomarker of addiction among people on long-term opioid therapy.

The effectiveness of an Australian community suicide prevention networks program in preventing suicide: a controlled longitudinal study.

BACKGROUND: Suicide is a major issue affecting communities around the world. Community-based suicide prevention approaches can tailor activities at a local level and are recognised as a key component of national suicide prevention strategies. Despite this, research exploring their effects on completed suicides is rare. This study examined the effect of a national program of community suicide prevention networks on suicide rates in catchment areas across Australia. METHODS: Australian suicide data from the National Coronial Information System for 2001-2017 were mapped to geographic catchment areas of community suicide prevention networks and matched control areas with similar characteristics. The effect of network establishment on suicide rates was evaluated using longitudinal models including fixed effects for site type (network or control), time, season, and intervention (network establishment), with site included as a random intercept. RESULTS: Sixty suicide prevention networks were included, servicing areas with a population of 3.5 million. Networks varied in when they were established, ranging from 2007 to 2016. Across the time-period, suicide rates per 100,000 per quarter averaged 3.73 (SD = 5.35). A significant reduction in the suicide rate of 7.0% was found after establishment of networks (IRR = 0.93, 95% CI 0.87 to 0.99, p = .025). CONCLUSION: This study found evidence of an average reduction in suicide rates following the establishment of suicide prevention networks in Australian communities. These findings support the effectiveness of empowering local communities to take action to prevent suicide.

Dynamics of trophoblast differentiation in peri-implantation-stage human embryos.

Single-cell RNA sequencing of cells from cultured human blastocysts has enabled us to define the transcriptomic landscape of placental trophoblast (TB) that surrounds the epiblast and associated embryonic tissues during the enigmatic day 8 (D8) to D12 peri-implantation period before the villous placenta forms. We analyzed the transcriptomes of 3 early placental cell types, cytoTB (CTB), syncytioTB (STB), and migratoryTB (MTB), picked manually from cultured embryos dissociated with trypsin and were able to follow sublineages that emerged from proliferating CTB at the periphery of the conceptus. A unique form of CTB with some features of STB was detectable at D8, while mature STB was at its zenith at D10. A form of MTB with a mixed MTB/CTB phenotype arose around D10. By D12, STB generation was in decline, CTB had entered a new phase of proliferation, and mature MTB cells had begun to move from the main body of the conceptus. Notably, the MTB transcriptome at D12 indicated enrichment of transcripts associated with IFN signaling, migration, and invasion and up-regulation of HLA-C, HLA-E, and HLA-G. The STB, which is distinct from the STB of later villous STB, had a phenotype consistent with intense protein export and placental hormone production, as well as migration and invasion. The studies show that TB associated with human embryos is in rapid developmental flux during peri-implantation period when it must invade, signal robustly to the mother to ensure that the pregnancy continues, and make first contact with the maternal immune system.

Early onset preeclampsia in a model for human placental trophoblast.

We describe a model for early onset preeclampsia (EOPE) that uses induced pluripotent stem cells (iPSCs) generated from umbilical cords of EOPE and control (CTL) pregnancies. These iPSCs were then converted to placental trophoblast (TB) representative of early pregnancy. Marker gene analysis indicated that both sets of cells differentiated at comparable rates. The cells were tested for parameters disturbed in EOPE, including invasive potential. Under 5% O2, CTL TB and EOPE TB lines did not differ, but, under hyperoxia (20% O2), invasiveness of EOPE TB was reduced. RNA sequencing analysis disclosed no consistent differences in expression of individual genes between EOPE TB and CTL TB under 20% O2, but, a weighted correlation network analysis revealed two gene modules (CTL4 and CTL9) that, in CTL TB, were significantly linked to extent of TB invasion. CTL9, which was positively correlated with 20% O2 (P = 0.02) and negatively correlated with invasion (P = 0.03), was enriched for gene ontology terms relating to cell adhesion and migration, angiogenesis, preeclampsia, and stress. Two EOPE TB modules, EOPE1 and EOPE2, also correlated positively and negatively, respectively, with 20% O2 conditions, but only weakly with invasion; they largely contained the same sets of genes present in modules CTL4 and CTL9. Our experiments suggest that, in EOPE, the initial step precipitating disease is a reduced capacity of placental TB to invade caused by a dysregulation of O2 response mechanisms and that EOPE is a syndrome, in which unbalanced expression of various combinations of genes affecting TB invasion provoke disease onset.

Is SARS-CoV-2 Infection a Risk Factor for Early Pregnancy Loss? ACE2 and TMPRSS2 Coexpression and Persistent Replicative Infection in Primitive Trophoblast.

BACKGROUND: SARS-CoV-2 infection in term placenta is rare. However, growing evidence suggests that susceptibility of the human placenta to infection may vary by gestational age and pathogen. For several viral infections, susceptibility appears to be greatest during early gestation. Peri-implantation placental infections that result in pre-clinical pregnancy loss would typically go undetected. Little is known about the effects of SARS-CoV-2 on the peri-implantation human placenta since this time in pregnancy can only be modeled in vitro. METHODS: We used a human embryonic stem cell (hESC)-derived model of peri-implantation placental development to assess patterns of ACE2 and TMPRSS2 transcription and protein expression in primitive trophoblast. We then infected the same trophoblast cell model with a clinical isolate of SARS-CoV-2 and documented infection dynamics. RESULTS: ACE2 and TMPRSS2 were transcribed and translated in hESC-derived trophoblast, with preferential expression in syncytialized cells. These same cells supported replicative and persistent infection by SARS-CoV-2, while non-syncytialized trophoblast cells in the same cultures did not. CONCLUSIONS: Co-expression of ACE2 and TMPRSS2 in hESC-derived trophoblast and the robust and replicative infection limited to syncytiotrophoblast equivalents support the hypothesis that increased viral susceptibility may be a defining characteristic of primitive trophoblast.

Neither gonadotropin nor cumulus cell expansion is needed for the maturation of competent porcine oocytes in vitro†.

In-vitro maturation (IVM) of oocytes from immature females is widely used in assisted reproductive technologies. Here we illustrate that cumulus cell (CC) expansion, once considered a key indicator of oocyte quality, is not needed for oocytes to mature to the metaphase II (MII) stage and to gain nuclear and cytoplasmic competence to produce offspring. Juvenile pig oocytes were matured in four different media: (1) Basal (-gonadotropins (GN) - FLI); (2) -GN + FLI (supplement of FGF2, LIF, and IGF1); (3) +GN - FLI; and (4) +GN + FLI. There was no difference in maturation to MII or progression to the blastocyst stage after fertilization of oocytes that had been matured in -GN + FLI medium and oocytes matured in +GN + FLI medium. Only slight CC expansion occurred in the two media lacking GN compared with the two where GN was present. The cumulus-oocytes-complexes (COC) matured in +GN + FLI exhibited the greatest expansion. We conclude that FLI has a dual role. It is directly responsible for oocyte competence, a process where GN are not required, and, when GN are present, it has a downstream role in enhancing CC expansion. Our study also shows that elevated phosphorylated MAPK may not be a necessary correlate of oocyte maturation and that the greater utilization of glucose by COC observed in +GN + FLI medium probably plays a more significant role to meet the biosynthetic needs of the CC to expand than to attain oocyte developmental competence. Gene expression analyses have not been informative in providing a mechanism to explain how FLI medium enhances oocyte competence without promoting CC expansion.

Modeling human peri-implantation placental development and function†.

It is very difficult to gain a better understanding of the events in human pregnancy that occur during and just after implantation because such pregnancies are not yet clinically detectable. Animal models of human placentation are inadequate. In vitro models that utilize immortalized cell lines and cells derived from trophoblast cancers have multiple limitations. Primary cell and tissue cultures often have limited lifespans and cannot be obtained from the peri-implantation period. We present here two contemporary models of human peri-implantation placental development: extended blastocyst culture and stem-cell derived trophoblast culture. We discuss current research efforts that employ these models and how such models might be used in the future to study the "black box" stage of human pregnancy.