Cell Reports Medicine

Successful embryo implantation requires complex interactions between the embryo and the endometrium. Improvements in embryo testing have led to increased success rates for embryo transfer following in vitro fertilization, however, even with euploid embryos under ideal conditions, failure occurs in more than 30% of cases. Methods for diagnosing and improving endometrial function are currently lacking. Here we developed a functional test ("Simbryo FX") for the ability of the endometrium to support embryo implantation. We collected endometrial biopsy samples from over 100 patients, and used these to grow endometrial organoids. We interacted these organoids with blastoids, pluripotent stem cell based models of the human blastocyst, and measured the hCG produced by the blastoids as well as the degree to which the blastoids invaded the organoids. We found that both of these measures correlated with clinical outcomes, and that combining them allowed us to predict the likelihood of failure in the next embryo transfer with high specificity. Thus, interacting patient-derived endometrial organoids with blastoids represents a promising approach to evaluating endometrial function among patients preparing for embryo transfer.

Severe preeclampsia (sPE) is a major cause of maternal and fetal morbidity worldwide, yet its placental molecular heterogeneity remains poorly defined by current clinical diagnosis. To resolve the molecular architecture of sPE, here we integrated DNA methylation and proteomic profiling from a multi-ethnical cohort of 444 placentas from the Hawaiian Biorepository (HiBR), including 169 sPE cases, matched preterm controls and full-term controls. To address cellular heterogeneity in bulk placental tissue, we developed HOMED (Hierarchically Optimized Methylation Deconvolution), a single-cell-guided hierarchical framework for inferring placental cell-type composition from DNA methylation data. HOMED-adjusted integrative analyses identified extensive subtype-specific alterations involving hypoxia, angiogenesis, immune activation, trophoblast differentiation and metabolic remodeling. Molecular stratification revealed two reproducible sPE subtypes with divergent placental aging trajectories. One subtype exhibited a pre-mature placental state marked by accelerated placental aging, whereas the other displayed slower accelerated placental aging but a substantially increased risk of small-for-gestational-age birth (P = 0.028). These subtypes were independently replicated across six external cohorts and further supported by proteomic signatures achieving a classification accuracy of 0.88. Integrative epigenomic and proteomic analyses linked the growth-restricted subtype to hypoxia-associated glycolytic remodeling, suggesting distinct pathogenic mechanisms underlying clinically diagnosed sPE. Together, our findings redefine severe preeclampsia as a biologically heterogeneous placental disorder composed of molecularly distinct subtypes with divergent aging trajectories and fetal growth outcomes, providing a framework for mechanism-based stratification and precision obstetric medicine.

Alpha-gal Syndrome (AGS) is a potentially life-threatening allergy caused by an IgE-mediated immune response to galactose--1,3-galactose (alpha-gal), a carbohydrate epitope present in most mammalian meats. Currently, strict avoidance of mammalian meat remains the primary management strategy for affected individuals, and alpha-gal-free beef is not commercially available. Here, we leverage cultivated meat as a biotechnology plat-form to address this unmet clinical need by engineering alpha-gal-free bovine muscle cells. Using CRISPR/Cas9 genome editing, we disrupted GGTA1, the gene encoding 1,3-galactosyltransferase, in immortalized bovine satellite cells (iBSCs). High-efficiency editing produced clonal GGTA1 knockout iBSCs harboring a homozygous frameshift mutation. Flow cytometry and immunofluorescence confirmed loss of the alpha-gal epitope, while bulk RNA-seq indicated minimal disruption of global gene expression and preserved myogenic differentiation capacity. Importantly, lysates from GGTA1 knockout iBSCs elicited substantially reduced basophil activation in assays using plasma from a patient with AGS, indicating reduced basophil activation consistent with reduced allergenic potential. Together, these findings establish a proof of concept for engineering AGS-compatible cultivated meat and demonstrate the potential of cultivated meat technologies to address human health challenges. HIGHLIGHTS{circ} CRISPR/Cas9-mediated disruption of GGTA1 eliminated alpha-gal from bovine satellite cells {circ}GGTA1 knockout cells retained myogenic identity and differentiation capacity {circ}GGTA1 knockout reduced basophil activation in an alpha-gal syndrome immune assay {circ}Genome-edited bovine cells provide a proof of concept for AGS-compatible cultivated meat

The successful development of several COVID-19 vaccines has substantially reduced morbidity and mortality in regions of the world where the vaccines have been deployed. However, in the wake of the emergence of viral variants, able to evade vaccine induced neutralizing antibodies, real world vaccine efficacy has begun to show differences across the mRNA platforms, suggesting that subtle variation in immune responses induced by the BNT162b2 and mRNA1273 vaccines may provide differential protection. Given our emerging appreciation for the importance of additional antibody functions, beyond neutralization, here we profiled the postboost binding and functional capacity of the humoral response induced by the BNT162b2 and mRNA-1273 in a cohort of hospital staff. Both vaccines induced robust humoral immune responses to WT SARS-CoV-2 and VOCs. However, differences emerged across epitopespecific responses, with higher RBD- and NTD-specific IgA, as well as functional antibodies (ADNP and ADNK) in mRNA-1273 vaccine recipients. Additionally, RBD-specific antibody depletion highlighted the different roles of non-RBD-specific antibody effector function induced across the mRNA vaccines, providing novel insights into potential differences in protective immunity generated across these vaccines in the setting of newly emerging VOCs.

To better understand how pregnancy impacts humoral immunity, we conducted an in-depth longitudinal analysis of the kinetics and characteristics of vaccine responses in a prospective cohort of pregnant and non-pregnant women. Humoral immune responses observed among pregnant participants who received the mRNA-delivered SARS-CoV-2 vaccination, including their effector functions, were in some cases marginally lower than those among non-pregnant controls, while prior infection was associated with some potentiation in humoral responses. Importantly, vaccine-induced antibodies were efficiently transferred across the placenta, providing the fetus with passive immunity and underscoring the dual benefit of maternal vaccination for both mother and neonate against COVID-19. Delayed induction of spike-specific IgG4 following the primary two-dose vaccination series was observed in vaccine recipients, independent of pregnancy status. In a subset (n=6) of pregnant women whose spike-specific serum IgG repertoires were extensively profiled at the clonotypic level over time as part of another study, we proteomically identified secreted IgG clonotypes that had class-switched to IgG4. Matching of these clonotypes detected as IgG4 to those defined as SARS-CoV-2 spike-specific revealed that, while a minority of total clonotypes, they were elicited early in the immunization series and tended to be more highly mutated, more prevalent, and more persistent than clonotypes in the serological repertoire that were not detected as IgG4. Consistent with the increase in secreted vaccine-specific IgG4 over time, but its poorer placental transfer, these clonotypes were detected at greater levels in maternal but not cord blood at the time of delivery as compared to 28 days post the second vaccine dose. These findings indicate some impact in the kinetics, characteristics, and functions of the humoral response that may be associated with pregnancy-related immune modulation. Conservation of the late class-switch recombination to IgG4 that has previously been associated with mRNA-based SARS-CoV-2 vaccines raises questions about how different immunological states and vaccine components influence short- and long-term characteristics of the humoral immune response.

Adverse pregnancy outcomes, such as sporadic and recurrent miscarriages and stillbirths, are significant medical concerns, impacting up to 15% of clinically recognised pregnancies. These outcomes are highly complex and multifactorial, with up to 50% of cases classified as idiopathic, highlighting a substantial gap in our understanding of their biological basis. Along with external risk factors, polygenic variability contributes to idiopathic pregnancy loss, suggesting that large-scale genetic studies could offer insights into its mechanisms, reveal novel drug targets, and lead to new treatments. This study assesses current knowledge from genome-wide association studies (GWAS) using genotyping arrays, whole-genome imputation, and sequencing for variant discovery, emphasising genetic predisposition to adverse pregnancy outcomes. We summarise existing efforts identifying 30 genetic loci associated with pregnancy loss and related endophenotypes, integrating them into a polygenic score (PGS) and conducting a phenome-wide PGS association analysis of 280 ICD-10 outcomes in nearly 500,000 UK Biobank participants. We report associations between pregnancy loss PGS and an increased risk for diaphragmatic hernia (OR[95%CI]=1.02[1.01-1.03], P=9.15x10-), eosinophilic esophagitis (OR[95%CI]=1.05[1.03-1.06], P=1.44x10-), and asthma with exacerbation (OR[95%CI]=1.02[1.01-1.03], P=1.71x10-), significant after correction for multiple testing and suggesting new mechanistic pathophysiology in pregnancy loss susceptibility. Additionally, Mendelian Randomisation (MR) studies identified higher BMI and smoking as risk factors for pregnancy loss, while the roles of caffeine and alcohol intake, maternal age, and family history of miscarriage warrant further investigation through adequately powered MR analyses. Well-designed and comprehensive GWAS studies, particularly across diverse ancestry groups, are urgently needed for idiopathic recurrent pregnancy loss. Such studies should overcome issues with identification of women suffering for this condition and related pregnancy losses to support better care and timely interventions, aiming for healthy live birth outcomes.

Preterm birth (PTB) is a leading cause of perinatal and infant mortality worldwide. PTB can be induced by systemic maternal or intra-uterine infection or by sterile intra-amniotic inflammation driven by alarmins such as interleukin-1 (IL-1). We reported earlier that Broad-Spectrum Chemokine Inhibitor (BSCI) prevented PTB in murine and non-human primate models of infection-mediated preterm labor. Here, we investigated whether BSCI can prevent PTB in pregnant C57BL/6 mice following ultrasound-guided intra-amniotic injection of IL-1 (400 ng per sac) on gestational day (GD)16.5. Half the mice received BSCI (10 mg/kg, intravenous daily) beginning GD15.5 and through to term. The impact of IL-1 alone or IL-1 plus BSCI was assessed on (i) injection-to-delivery interval, fetal survival, placental and neonatal weight; (ii) cytokine and chemokine levels in maternal plasma and amniotic fluid (by Luminex assay) and inflammatory gene expression in maternal and fetal tissues (by Real-Time RT-PCR); (iii) global multi-omic profiling of myometrial tissues, including gene expression (RNA sequencing), chromatin accessibility and regulatory landscape (ATAC-seq), and protein abundance profiling by tandem mass spectrometry (TMT-MS proteomics); (iv) uterine leukocyte infiltration (by immunofluorescence with automated quantification). Pre-treatment with BSCI i) prevented IL-1-induced PTB; (ii) significantly attenuated cytokine and chemokine signals in maternal plasma, myometrium, decidua, and placenta, and amniotic fluid; (iii) suppressed myometrial contraction-associated genes, including Nfkb1, Ptgs2, Akr1c18, and Gja1; (iv) prevented broad IL-1-induced changes in myometrial gene expression, chromatin accessibility, and proteomic extracellular matrix (ECM) structural remodeling; (v) reduced uterine F4/80+ macrophage counts and changed M1-M2 balance. BSCI-treated dams that delivered at term had live pups with normal placental and fetal weight. Taken together, BSCI reduced the incidence of IL-1-mediated PTB and maintained uterine quiescence by suppressing uterine inflammation and global changes in labor gene expression and chromatin accessibility. BSCI represents a promising therapeutic approach for PTB prevention in high-risk pregnant women.

Ovarian cancer is a heterogeneous group of tumors in both cell type and natural history. While outcomes are generally favorable when detected early, the most common subtype, high-grade serous carcinoma (HGSOC), typically presents at an advanced stage and portends less favorable prognoses. Its aggressive nature has thwarted early detection efforts through conventional detection methods such as serum CA125 and ultrasound screening and thus inspired the investigation of novel biomarkers. Here, we report the systematic development of an extracellular-vesicle (EV)-based test to detect early-stage HGSOC. Our study is based on emerging insights into HGSOC biology, notably that it arises from precursor lesions within the fallopian tube before traveling to ovarian and/or peritoneal surfaces. To identify HGSOC marker candidates, we established murine fallopian tube (mFT) cells with oncogenic mutations in Brca1/2, Tp53, and Pten genes, and performed proteomic analyses on mFT EVs. The identified markers were then evaluated with an orthotopic HGSOC animal model. In serially-drawn blood samples of tumor-bearing mice, mFT-EV markers increased with tumor initiation, supporting their potential use in early cancer detection. A pilot human clinical study (n = 51) further narrowed EV markers to five candidates, EpCAM, CD24, VCAN, HE4, and TNC. Combined expression of these markers achieved high OvCa diagnostic accuracy (cancer vs. non-cancer) with a sensitivity of 0.89 and specificity of 0.93. The same five markers were also effective in a three-group classification: non-cancer, early-stage (I & II) HGSOC, and late-stage (III & IV) HGSOC. In particular, they differentiated early-stage HGSOC from the rest with a specificity of 0.91. Minimally invasive and repeatable, this EV-based testing could be a versatile and serial tool for informing patient care and monitoring women at high risk for ovarian cancer.

It remains elusive why some triple-negative breast cancer (TNBC) patients respond poorly to existing therapies while others respond well. Our retrospective analysis of historical gene expression datasets reveals that increased expression of immunosuppressive cytokine S100A8/A9 in early-stage tumors is robustly associated with subsequent disease progression in TNBC. Although it has recently gained recognition as a potential anticancer target, S100A8/A9 has not been integrated into clinical study designs evaluating molecularly targeted therapies. Our small molecule screen has identified PIM kinase inhibitors as capable of decreasing S100A8/A9 expression in multiple cell types, including TNBC and immunosuppressive myeloid cells. Furthermore, combining PIM inhibition and immune checkpoint blockade induces significant antitumor responses, especially in otherwise resistant S100A8/A9-high PD-1/PD-L1-positive tumors. Importantly, serum S100A8/A9 levels mirror those of tumor S100A8/A9 in a syngeneic mouse model of TNBC. Thus, our data suggest that S100A8/A9 could be a predictive and pharmacodynamic biomarker in clinical trials evaluating combination therapy targeting PIM and immune checkpoints in TNBC and encourage the development of S100A8/A9-based liquid biopsy tests.

Following SARS-CoV-2 infection, [~]10-35% of COVID-19 patients experience long COVID (LC), in which often debilitating symptoms persist for at least three months. Elucidating the biologic underpinnings of LC could identify therapeutic opportunities. We utilized machine learning methods on biologic analytes and patient reported outcome surveys provided over 12 months after hospital discharge from >500 hospitalized COVID-19 patients in the IMPACC cohort to identify a multi-omics "recovery factor". IMPACC participants who experienced LC had lower recovery factor scores compared to participants without LC. Biologic characterization revealed increased levels of plasma proteins associated with inflammation, elevated transcriptional signatures of heme metabolism, and decreased androgenic steroids in LC patients. The recovery factor was also associated with altered circulating immune cell frequencies. Notably, recovery factor scores were predictive of LC occurrence in patients as early as hospital admission, irrespective of acute disease severity. Thus, the recovery factor identifies patients at risk of LC early after SARS-CoV-2 infection and reveals LC biomarkers and potential treatment targets.

Head and neck squamous cell carcinoma (HNSCC) exhibits profound heterogeneity in clinical presentation, treatment response, and immune landscape. While prior classification systems have identified molecular and immune subtypes in this disease, their applicability to real-world clinical settings remains restricted to small, homogeneous cohorts and limited by lack of multimodal data integration and interpretation. We performed integrated multi-omics analysis including transcriptomic, genomic (copy number, single-nucleotide variants) on 1,149 tumors from 1,102 HNSCC patients across treatment settings. Using the Similarity Network Fusion (SNF) algorithm, we defined immune subtype clusters (ISCs) based on the full immune gene landscape. These clusters were characterized using mutational, transcriptional, and immune cell enrichment analyses, and mapped to hypoxia and traditional subtypes. Associations with clinical outcomes, including progression-free survival, were evaluated across first-line and post-metastatic treatment settings. Four distinct immune subtype clusters (ISC1-ISC4) were identified: ISC1: immune-cold and EMT-enriched; ISC2: immune activated; ISC3: mixed immune-regulatory and stromal-enriched phenotype; and ISC4: immunosuppressed. Distinct treatment response patterns were observed across subtypes in subjects treated with checkpoint inhibitors, chemotherapy, and combination regimens. 44 Patients with matched pre/post treatment tumors revealed treatment-associated transitions between immune subtypes: checkpoint inhibitor treatment enriched for immune activation, while chemotherapy treatment enriched for immunosuppressive signaling pathways. This study provides a clinically relevant immune subtyping framework for HNSCC based on real-world, multi-omics data. These subtypes reflect dynamic tumor-immune states and associated with treatment response and survival, supporting their use in guiding immune-based therapy in HNSCC.

In response to the need for a safe, efficacious vaccine that elicits vigorous T cell as well as humoral protection against SARS-CoV-2 infection, we have developed a dual-antigen COVID-19 vaccine comprising both the viral spike (S) protein modified to increase cell-surface expression (S-Fusion) and nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to enhance MHC class I and II presentation and T-cell responses. The antigens are delivered using a human adenovirus serotype 5 (hAd5) platform with E1, E2b, and E3 regions deleted that has been shown previously in cancer vaccine studies to be safe and effective in the presence of pre-existing hAd5 immunity. The findings reported here are focused on human T-cell responses due to the likelihood that such responses will sustain efficacy against emerging variants, a hypothesis supported by our in silico prediction of T-cell epitope HLA binding for both the first-wave SARS-CoV-2 A strain and the B.1.351 strain K417N, E484K, and N501Y spike and T201I N variants. We demonstrate the hAd5 S-Fusion + N-ETSD vaccine antigens expressed by previously SARS-CoV-2-infected patient dendritic cells elicit Th1 dominant activation of autologous patient T cells, indicating the vaccine antigens have the potential to elicit immune responses in previously infected patients. For participants in our open-label Phase 1b study of the vaccine (NCT04591717; https://clinicaltrials.gov/ct2/show/NCT04591717), the magnitude of Th-1 dominant S- and N-specific T-cell responses after a single prime subcutaneous injection were comparable to T-cell responses from previously infected patients. Furthermore, vaccinated participant T-cell responses to S were similar for A strain S and a series of spike variant peptides, including S variants in the B.1.1.7 and B.1.351 strains. The findings that this dual-antigen vaccine elicits SARS-CoV-2-relevant T-cell responses and that such cell-mediated protection is likely to be sustained against emerging variants supports the testing of this vaccine as a universal booster that would enhance and broaden existing immune protection conferred by currently approved S-based vaccines.

Responses to single agent immunotherapies have remained modest in high-grade serous ovarian cancer (HGSC), suggesting the need for combination treatments. Identifying clinically effective immunotherapy combinations (IC) requires pre-clinical testing using models representing the patient-specific immune microenvironment. Here, we established a functional immuno-oncology platform for high-throughput and functional testing of IC using HGSC patient-derived immunocompetent cultures (iPDCs) established on patient-derived omentum gel matrix. We employed genomic and single-cell analysis to assess the intricate and functional characteristics of the iPDCs combined with tumor and immune cell-specific cytotoxic responses. Corroborating the clinical response to Poly (ADP-ribose) polymerase inhibitors (PARPi), iPDCs showed homologous recombination deficiency (HRD) - specific response to PARPi. Importantly, drug responses from iPDCs of chemotherapy and PARPi refractory patients corresponded with patient outcomes and aligned with distinct pathway activities from single-cell RNA sequencing analysis. Furthermore, iPDCs from HRD tumors showed response to anti-PD1 antibody as measured by decrease in tumor cells combined with augmented T cell activation. High-throughput drug testing followed by single cell-imaging from iPDCs revealed patient-specific responses to combination of ataxia telangiectasia and Rad3-related inhibitor (ATRi) with DNA damaging agents or immunotherapies. Integration of cytotoxic responses with immune cell states uncovered patient-specific immune activation with the combination of ATRi and a novel immunotherapy targeting Autotaxin (ATX), and this response was significantly associated with a tumor-cell replication stress biomarker in single-cell analysis of tCycIF highly multiplexed imaging. In conclusion, iPDCs provide a platform for high-throughput screening and functional testing of immuno-oncology agents for precision oncology in HGSC.

Immune checkpoint inhibitor-induced myotoxicity (ICI-M) comprises myocarditis, myositis, and myasthenia gravis-like syndrome, demanding rapid recognition and therapy. Using immunophenotyping and transcriptomics analysis from blood and muscle, we identified distinct CD38hi and KIR+ CD8 T cells in ICI-M. Abatacept rescued patients and altered the composition and clonality of these cells. Dynamics of CD38hi and KIR+ CD8 T cells effectively supported therapeutic monitoring, offering personalized treatment in life-threatening irAEs.

Fecal virome transfer (FVT) shows promise in reducing necrotizing enterocolitis (NEC), likely due to donor bacteriophages preventing the gut dysbiosis preceding disease. However, concurrent transfer of eukaryotic viruses may carry a risk of infection for the recipient. To increase safety, we investigated chemostat propagation as a method to eliminate eukaryotic viruses from donor feces while maintaining a diverse and reproducible bacteriophage community. Donor feces was collected from healthy suckling piglets and inoculated into a fermenter containing growth media supplemented with lactose and milk oligosaccharides (MOs). During continuous medium exchange (20% volume/h), dilution significantly reduced eukaryotic viruses. Viral richness was concurrently reduced although still preserving a stable community of 200-250 bacteriophages. Inclusion of MOs in the medium ensured higher bacterial richness and a bacterial community closer resembling donor feces. Fecal Lactobacillaceae bacteria were lost during cultivation but partially replaced by members of the Bacteroidota phylum in MO-supplemented cultures, accompanied by phages predicted to have Parabacteroides as host. After cultivation, virus-like particles (VLPs) were isolated, and their ability to reduce NEC incidence tested in vivo. Preterm piglets were delivered by cesarean section and received either the lactose- or MO-propagated viromes by oral route (n = 14-15/group). These were compared with groups receiving the same dose of donor fecal virome (1010 VLPs/kg) or vehicle control. The piglets were subsequently fed infant formula for 96 hours followed by euthanasia and tissue sampling. Both chemostat-propagated viromes effectively mitigated diarrhea compared to the donor virome. The donor virome partially engrafted in recipients and led to higher levels of Lactobacillaceae bacteria and Lactobacillaceae targeting phages. However, these signatures were lost in recipients of chemostat-propagated viromes, and only minor microbiome effects and no NEC prevention were observed. To conclude, we provide in vivo proof-of-concept for chemostat propagation of fecal viruses as a means to deplete eukaryotic viruses and in turn reduce side effects in newborn virome recipients. However, chemostat culture conditions need further optimization to preserve the donor phageome.

The lack of functional precision models that recapitulate the pathology and structure/function relationship of advanced ovarian cancer (OC) within an appropriate anatomic setting constitutes a hurdle on the path to developing more reliable therapies and matching those therapies with the right patients. Here, we developed and characterized an Organotypic Mesentery Membrane Culture (OMMC) model as a novel ex-vivo platform where freshly resected human patient OC tumor tissue or established cell lines are seeded directly atop living intact rat mesenteric membranes, rapidly engraft, and enable functional assessment of treatment response to FDA-approved standard care of treatment as single and combination drug therapies within just five days. This study showed successful survival of dissected mesentery tissue, survival and engraftment of tumor cells and patient tumor tissue seeded on OMMCs, mesentery-tumor cell interaction, and quantification of tumor response to treatment and off-target toxicity. Summarized "drug sensitivity scores", using a multi-parametric algorithm, were also calculated for each patients treatment response, enabling us to suggest the most effective therapeutic option. Finally, we compared drug sensitivity results from patient tumor tissue on OMMCs to matched outcomes of individual patients in the clinic and identified positive correlations in drug sensitivity, beginning to validate the functionality of OMMCs as a functional predictor of treatment response. Summary sentenceWe have successfully developed and characterized a novel ex-vivo platform for personalized treatment of metastatic ovarian cancer.

Preterm birth is the leading cause of infant mortality resulting in over one million neonatal deaths annually. Maternal urinary tract infection (UTI) during pregnancy increases risk for preterm birth; however, biological processes mediating UTI-associated preterm birth are not well-described. We established a murine maternal UTI model in which challenge with uropathogenic E. coli resulted in preterm birth in about half of dams. Dams experiencing preterm birth displayed excessive bladder inflammation and altered uteroplacental T cell polarization compared to non-laboring infected dams, with no differences in bacterial burdens. Additional factors associated with preterm birth included higher proportions of male fetuses and lower maternal serum IL-10. Furthermore, exogenous maternal IL-10 treatment absolved UTI-associated preterm birth but contributed to fetal growth restriction in this model. Using urine samples from a cohort of human pregnancies with or without UTI, we correlated urinary cytokines with birth outcomes and urine culture status. These analyses yielded a non-invasive, highly predictive three-model system for evaluating preterm birth risk implicating cytokines IL-10, IL-15, IL-1{beta}, and IL-1RA. Our unique bimodal murine model coupled with patient samples provides a platform to investigate immunological and microbial factors governing UTI-associated preterm birth, revealing novel therapeutic opportunities to predict or prevent preterm birth.

Subfertility and recurrent pregnancy loss (RPL) affect a significant proportion of couples worldwide. Genetic causes can be seen in up to 30% of these individuals but require multiple genetic tests, which often impede a comprehensive work up. Newer genomic technologies, such as PacBio HiFi long read sequencing (LRS) can detect most subclasses of variations (such as structural rearrangement, monogenic disorders) through one single test. In this multicenter study, we enrolled couples with unexplained subfertility and/or RPL and performed HiFi LRS to determine the underlying genetic etiology. Participants were recruited using a standardized inclusion/ exclusion criteria to rule out other known causes of subfertility and/or RPL. 96 individuals were recruited across the 5 sites. Average age of participants was 36 years (range 30-46 years). Among the 84 individuals who completed sequencing, 4.8% were identified with a likely genetic diagnosis and variants of uncertain significance were identified in another 14.2% of individuals. One individual was identified with an ACMG secondary finding, and while multiple carriers for recessive genetic disorders were identified, none of the couples were identified to be at increased risk. This study highlights the utility of performing genomic sequencing in couples with unexplained subfertility and/or RPL, with 1 in 10 couples harboring a clinically significant variant. In addition, use of HiFi LRS allowed for characterization of different subclasses of genomic variations through a single test. Future studies, including exploring the cost effectiveness and resource utilization of LRS as first line test, will help in optimizing care for such couples. TWEETABLE STATEMENTA single long-read genome sequencing test can consolidate multiple genetic investigations and uncover clinically relevant causes in couples with unexplained subfertility and recurrent pregnancy loss. AT A GLANCEO_LIWhy was this study conducted? O_LIMany couples with subfertility and recurrent pregnancy loss remain undiagnosed after multiple conventional genetic tests C_LIO_LIExisting workflows require sequential testing and may miss complex genomic variants C_LI C_LIO_LIWhat are the key findings? O_LILong-read genome sequencing identified clinically relevant variants in [~]1 in 10 couples with unexplained subfertility or recurrent pregnancy loss C_LIO_LIA single assay enabled detection of multiple variant types, including structural and sequence variants C_LI C_LIO_LIWhat does this study add to what is already known? O_LIDemonstrates feasibility of a unified genomic testing approach in a real-world multicenter cohort C_LIO_LISupports a potential shift from fragmented testing toward a single comprehensive genomic workflow C_LI C_LI

Interferons (IFNs) are antiviral cytokines induced very early after SARS-CoV-2 infection and are crucial for viral clearance, shaping immunity, and preventing the development of severe COVID-19. We previously demonstrated that a single injection of peginterferon-lambda1 (PEG-IFN-{lambda}) accelerated viral clearance in COVID-19 patients. To determine if the rapid viral decline was mediated by enhanced immunity, we assessed in vivo responses to PEG-IFN-{lambda} by single cell RNA sequencing and measured SARS-CoV-2-specific T cell and antibody responses between placebo and PEG-IFN-{lambda}-treated patients. PEG-IFN-{lambda} treatment induced interferon stimulated genes in peripheral immune cells expressing IFNLR1, with plasmacytoid dendritic cells having the greatest response, followed by B cells. PEG-IFN-{lambda} did not significantly affect SARS-CoV-2-specific antibody levels in plasma or the magnitude or functionality of virus-specific T cells. However, we identified a delayed T cell response in older adults, suggesting that PEG-IFN-{lambda} can overcome the delay in adaptive immunity to accelerate viral clearance in patients most at risk for severe disease. Taken together, PEG-IFN-{lambda} offers an early COVID-19 treatment option for outpatients to boost innate antiviral defenses without dampening peripheral SARS-CoV-2 adaptive immunity

mRNA-lipid nanoparticle (LNP) vaccines are detectable in human blood after vaccination, but platform-specific differences in systemic persistence and transcript integrity remain poorly defined. We analyzed serial blood samples from 73 participants receiving Moderna mRNA-1273 (three formulations), Pfizer/BioNTech BNT162b2, or an investigational receptor-binding domain (RBD) mRNA vaccine (three different doses). Using droplet digital polymerase chain reaction (ddPCR) assays, we quantified total and long-range linked ("intact") vaccine mRNA, and we measured vaccine-specific ionizable lipids by liquid chromatography-mass spectrometry (LC-MS). Across platforms, mRNA decay was fastest for mRNA-1273, intermediate for BNT162b2, and slowest for the RBD vaccine, with ionizable lipid decay following the same rank order. Notably, intact spike mRNA declined two-fold faster after mRNA-1273 than BNT162b2 vaccination. Kinetics modelling revealed platform-dependent coupling of mRNA and lipid kinetics: intact mRNA tracked closely with SM-102 for mRNA-1273, whereas ALC-0315 persisted longer than intact mRNA for BNT162b2. A ten-fragment linkage ddPCR panel spanning the spike transcript showed lower linkage toward 3'-proximal regions that mirrored the administered mRNA-1273 formulation. Together, these data establish a quantitative framework for benchmarking mRNA-LNP platform kinetics and transcript integrity in humans.