In 2019-2022, we enrolled men who have sex with men who did not engage in receptive anal sex but had other anorectal exposures (e.g., rimming). Of 29 men who tested positive for rectal chlamydia by nucleic acid amplification testing, only 10 (34.5%) were viable by culture or viability PCR.

STUDY QUESTION:Do particular pairs of individuals modeling sexual pairs exhibit differences in the induction of semen-mediated long-lasting tolerance in support of pregnancy? SUMMARY ANSWER:Yes, factors from both sexes contribute to the variability in the induction of long-lived regulatory T cells (Tregs) in response to semen, with factors related to the recipient seeming to slightly outweigh the factors related to the semen donor. WHAT IS KNOWN ALREADY:Semen contains multiple immunomodulatory factors that can influence reproductive tract immunity in the recipient, promote tolerance to antigens from the semen donor (designated henceforth as 'paternal'), and ultimately support successful pregnancy. These factors include the soluble fraction containing prostaglandins and cytokines, as well as a very high concentration of extracellular vesicles (EVs). Both soluble and vesicular fractions of semen have been shown to promote tolerogenic phenotypes and migration in antigen-presenting cells (APCs), and to induce Tregs. This study was undertaken to understand the variation in tolerance induction between pairs of individuals modeling sexually active couples. STUDY DESIGN SIZE DURATION:We designed an in vitro pairing study using random semen donors (N = 11) and peripheral mononuclear cells (PBMCs) from random self-identified female recipients (N = 3). Semen was fractionated into vesicle-enriched (semen extracellular vesicles; SEVs) and soluble (vesicle-depleted semen plasma; VDSP) fractions. Monocyte-derived dendritic cells (MoDCs) from each of the recipients were exposed to semen fractions from each donor, then cocultured with autologous naïve T cells to assess tolerogenic responses, specifically, generation of Tregs (FOXP3+ CD25Hi CD127Lo CD4+ T cells) and their phenotypic differences (TIGIT+, PD-1+ CTLA-4+, or AREG+ Tregs). These cells were then screened for T cell receptor-dependent activation by activation-induced marker (AIM) expression assay. These responses were compared across all pairs of semen donors and recipient cells. In addition, semen fractions were screened for candidate mediators of tolerance, such as human leukocyte antigen G (HLA-G) and adenosine-generating enzymes. PARTICIPANTS/MATERIALS SETTING METHODS:This study used an in vitro human cell culture approach in which primary blood-derived immune cells were exposed to semen-derived vesicular and non-vesicular fractions. Blood samples were obtained from healthy self-identified female donors, and semen samples were obtained from healthy participants who provided initial screening specimens for a contraceptive study. Semen-derived vesicular and non-vesicular fractions were characterized by western-blot analysis for markers associated with immune tolerance. Primary immune cells were exposed to these semen fractions, and the major endpoints included changes in Treg phenotypes and subset frequencies, as well as AIM expression. These outcomes were assessed by flow cytometry. No animal models were used. MAIN RESULTS AND THE ROLE OF CHANCE:HLA-G content in semen (SEV and VDSP) varied markedly across donors, ranging from undetectable to high concentrations. Similarly, semen's capacity to generate adenosine from ATP differed by ∼4-fold among donors. Induction of Tregs by SEV and VDSP from 11 semen donors was highly variable, from 2.8% to 63.4% of CD4+ T cell population. Semen fractions from a few donors consistently elicited lower tolerogenic responses across all recipients. However, when we considered the combined effects of both semen fractions, none of the semen donors failed to elicit a measurable tolerogenic response. On the other hand, one recipient was a consistently low responder to tolerance induction. Thus, both recipient immune cells' features and semen-intrinsic properties are significant determinants of tolerogenic responsiveness. Semen re-exposure enhanced Treg AIM expression. The strongest effect was observed when Tregs were exposed to VDSP-loaded MoDCs. In contrast, SEV-loaded MoDCs caused a much weaker response. LARGE SCALE DATA:N/A. LIMITATIONS REASONS FOR CAUTION:One limitation of our study is that we used semen and circulating recipient cells from random donors not selected for any clinical characteristics, such as history of infertility. In addition, we did not correlate HLA-G content and adenosine-generating enzyme expression levels with the Treg and AIM readouts. We show, via Treg activation, that semen induces tolerance to its antigens, but we do not demonstrate which specific antigens are recognized. We posit that the variation in semen-induced tolerance observed between couples plays a role in reproductive outcomes; however, larger studies in clinically characterized cohorts and using cells from the tissue microenvironment will be required to establish this link. WIDER IMPLICATIONS OF THE FINDINGS:Our findings demonstrate that semen-induced tolerance is highly variable and may be more strongly shaped by recipient factors than by semen donor factors. In addition, the generation of Tregs with enhanced activation to previously encountered semen supports the concepts of trained immunity and antigen-specific tolerance that arise cumulatively following exposure to semen from the partner. These Tregs are poised to expand and support pregnancies with fetuses expressing paternal alloantigens. FUNDING:This work was supported by the following grants: National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01AI153342) to L.V., National Institutes of Health/National Institute on Drug Abuse (R01DA040386) to F.H., National Institutes of Health/National Center for Advancing Translational Sciences (KL2TR002317) to G.G.G. DISCLOSURES:The authors have no conflicts of interest to declare.

Introduction:The regulation of immune responses to promote tolerance to the fetus is critical for successful pregnancy. An understudied aspect of this process is the initiation of regulation pre-conception via exposure to semen. Our study aimed to understand how semen impacts recipient dendritic cells (DCs) and their subsequent role in shaping CD4 T cell differentiation. Methods:Monocyte-derived DCs (MoDCs) were exposed to semen extracellular vesicles (SEV) or vesicle-depleted semen plasma (VDSP). Phenotypic and functional markers were analyzed using flow cytometry. We also exposed epithelial sheets from vaginal tissue to SEV and VDSP, and measured the number and marker expression of emigrating cells. Finally, we tested how SEV- or VDSP-exposed DCs altered CD4 T cell differentiation by co-culturing exposed MoDCs or tissue emigrated cells with autologous naïve CD4 T cells. Results:MoDCs exhibited a significant increase of CD141, CD1a, CD38, and ILT4 expression when exposed to SEV or VDSP. A unique feature of semen-treated MoDCs was expression of indoleamine 2,3-dioxygenase (IDO), a potent contributor to the induction of regulatory T cells (Tregs). SEV but not VDSP significantly increased the emigration of intraepithelial DCs. Additionally, SEV significantly enhanced the expression of multiple immunoregulatory markers in the emigrated DCs. After co-culture, we observed significantly more FOXP3+ Tregs expressing high levels of TIGIT in the groups that were initially exposed to SEV. Discussion:These findings indicate that exposure to SEV induces a tolerogenic program in DCs that can direct differentiation of a unique memory Treg subset, primed for expansion and presumably destined to support a successful pregnancy.

PROBLEM:The decidua is the interface between the uterus and the fetus. Studying decidual cells can reveal how healthy pregnancies are supported and mechanisms of pregnancy complications. There are two methods of obtaining decidual tissue following delivery. The placental bed can be suctioned following C-section deliveries, or a thin layer of decidual tissue can be dissected from the placenta. This study aimed to compare immune cell populations obtained using the two methods. METHOD OF STUDY:From individuals with scheduled C-sections, we collected peripheral blood, decidua via vacuum suction of the placental bed, and decidua via dissection of the uterine-facing side of the placenta. Samples were analyzed using a 22-color full-spectrum flow cytometry panel to identify immune cell subsets and functional markers. RESULTS:The cellular composition of both decidual tissue collection methods were more similar to each other than to peripheral blood. Decidua collected via vacuum suction (Suc. decidua) had more live CD45+ cells. Decidua collected via dissection of the uterine-facing side of the placenta (Plac. decidua) had significantly higher expression of Helios in CD4+ cells, suggesting more fetal T cells. Both types of decidual samples contained similar levels of Tr1-like regulatory T lymphocytes expressing LAG3 and CD49b, whereas peripheral blood did not have this cell type. CONCLUSION:Collecting decidual tissue using either method resulted in largely similar immune cell populations, suggesting studies are largely comparable regardless of whether samples were collected via suction or placental dissection. This will allow for greater flexibility in sample collection methods.

Non-neutralizing functions of antibodies, including phagocytosis, may play a role in Chlamydia trachomatis (CT) infection, but these functions have not been studied and assays are lacking. We utilized a flow-cytometry-based assay to determine whether serum samples from a well-characterized cohort of CT-infected and naive control individuals enhanced phagocytosis via Fc-receptor-expressing THP-1 cells, and whether this activity correlated with antibody titers. Fc-receptor-mediated phagocytosis was detected only in CT+ donors. Phagocytosis generally did not correlate well with antibody titer. In addition, we found that complement from both CT+ and negative individuals enhanced phagocytosis of CT into primary neutrophils. These results suggest that anti-CT antibodies can have functions that are not reflected by titer. This method could be used to quantitively measure Fc-receptor-mediated function of anti-CT antibodies or complement activity and could reveal new immune correlates of protection.

BACKGROUND:Chlamydia trachomatis (CT) acquired orally may survive passage through the gastrointestinal tract and establish an infection in the rectum, but how often this occurs is unknown. METHODS:In 2019 to 2022, we enrolled individuals assigned male at birth who reported sex with men and denied receptive anal sex in the past 2 years. Participants enrolled at the Seattle Sexual Health Clinic or online. Participants completed a behavioral survey and self-collected rectal swabs for CT nucleic acid amplification testing (NAAT) and culture and viability polymerase chain reaction (vPCR). We defined oral exposures as fellatio, cunnilingus, and oral-anal (i.e., rimming). RESULTS:We enrolled 275 men; 60 (22%) reported only oral exposures in the past 12 months. Of these, 5 (8.3%) tested positive for rectal CT by NAAT; 1 (2%) had viable CT detected (culture positive; vPCR positive). Another 43 participants reported only oral exposures in the past 2 months, but rectal exposures 3 to 12 months ago. Of these, 4 (9%) tested NAAT positive for rectal CT; 1 had viable CT detected (culture negative; vPCR positive). CONCLUSIONS:Passage of CT from the mouth to the rectum occurs but is most often nucleic acid remnants rather than viable bacteria. Nonetheless, it seems possible to establish a viable rectal CT infection via oral exposures.

Evidence suggests cholesterol accumulation in pro-inflammatory endothelial cells (EC) contributes to triggering atherogenesis and driving atherosclerosis progression. Therefore, inhibiting miR-33a-5p within inflamed endothelium may prevent and treat atherosclerosis by enhancing apoAI-mediated cholesterol efflux by upregulating ABCA1. However, it is not entirely elucidated whether inhibition of miR-33a-5p in pro-inflammatory EC is capable of increasing ABCA1-dependent cholesterol efflux. In our study, we initially transfected LPS-challenged, immortalized mouse aortic EC (iMAEC) with either pAntimiR33a5p plasmid DNA or the control plasmid, pScr. We detected significant increases in both ABCA1 protein expression and apoAI-mediated cholesterol efflux in iMAEC transfected with pAntimiR33a5p when compared to iMAEC transfected with pScr. We subsequently used polymersomes targeting inflamed endothelium to deliver either pAntimiR33a5p or pScr to cultured iMAEC and showed that the polymersomes were selective in targeting pro-inflammatory iMAEC. Moreover, when we exposed LPS-challenged iMAEC to these polymersomes, we observed a significant decrease in miR-33a-5p expression in iMAEC incubated with polymersomes containing pAntimR33a5p versus control iMAEC. We also detected non-significant increases in both ABCA1 protein and apoAI-mediated cholesterol in iMAEC exposed to polymersomes containing pAntimR33a5p when compared to control iMAEC. Based on our results, inhibiting miR-33a-5p in pro-inflammatory EC exhibits atheroprotective effects, and so precisely delivering anti-miR-33a-5p to these cells is a promising anti-atherogenic strategy.

Background The current testing approach to diagnose Chlamydia trachomatis (CT) infection relies on nucleic acid amplification tests (NAATs). These tests are highly sensitive, but do not distinguish between active infection and residual bacterial nucleic acid which may remain after resolution of infection, or via cross-contamination. Better methods to assess the viability of CT detected in clinical samples would be useful in determining the relevance of CT detection in a variety of clinical settings. The goal of this study was to test viability PCR (vPCR) as a method to distinguish viable bacteria from non-viable CT. Methods The vPCR relies on a propidium monoazide dye (PMAxx), which intercalates into accessible DNA from dead organisms and prevents their detection in a PCR assay for the CT ompA gene. We used digital PCR to quantify absolute genome copy numbers from samples. We validated the vPCR approach using laboratory stocks of CT with known viability. Then, we tested total DNA, viable CT DNA, and culture results from 18 clinical vaginal specimens and 25 rectal clinical specimens, all of which had tested positive by NAAT. Results In laboratory stocks of CT, vPCR using defined ratios of heat-killed to live bacteria tracked closely with expected results. In vaginal clinical specimens, vPCR and total DNA results were correlated, though total DNA genomes outnumbered viable genomes by 2.2–52.6-fold more copies. As expected, vPCR detected more total genomes than culture results. Both vPCR and total DNA correlated with culture results (Spearman correlation R = 0.8425 for total DNA and 0.8056 for vPCR). Ten rectal NAAT positive specimens were negative by total DNA PCR, vPCR, and were negative or inconclusive by culture. Of the 6 rectal specimens that were culture positive, all were total DNA and vPCR positive. vPCR additionally detected viable bacterial DNA in 8 specimens which were NAAT + and culture negative, though levels were very low (mean 1,357 copies/ml) Conclusions vPCR is a fast and easy method to assess viability in clinical specimens and is more correlated with culture results than total DNA PCR. Inconsistent ratios between total DNA and vPCR results suggest that the amount of dead bacteria varies considerably in clinical specimens. Results from rectal specimens suggest that many NAAT positive specimens do not in fact represent live replicating bacteria, and likely result in significant overuse of unnecessary antibiotics.

In addition to their role in protein translation, tRNAs can be cleaved into shorter, biologically active fragments called tRNA fragments (tRFs). Specific tRFs from spermatocytes can propagate metabolic disorders in second generations of mice. Thus, tRFs in germline cells are a mechanism of epigenetic inheritance. It has also been shown that stress and toxins can cause alterations in tRF patterns. We were therefore interested in whether injecting illicit drugs, a major stressor, impacts tRFs in germline cells. We sequenced RNA from spermatocytes and from semen-derived exosomes from people who inject illicit drugs (PWID) and from non-drug using controls, both groups of unknown fertility status. All PWID injected opioids daily, but most also used other illicit drugs. The tRF cleavage products from Gly-GCC tRNA were markedly different between spermatocytes from PWID compared to controls. Over 90% of reads in controls mapped to shorter Gly-GCC tRFs, while in PWID only 45% did. In contrast, only 4.1% of reads in controls mapped to a longer tRFs versus 45.6% in PWID. The long/short tRF ratio was significantly higher in PWID than controls (0.23 versus 0.16, P = 0.0128). We also report differential expression of a group of small nucleolar RNAs (snoRNAs) in semen-derived exosomes, including, among others, ACA14a, U19, and U3-3. Thus, PWID exhibited an altered cleavage pattern of tRNA-Gly-GCC in spermatocytes and an altered cargo of snoRNAs in semen-derived exosomes. Participants were not exclusively using opioids and were not matched with controls in terms of diet, chronic disease, or other stressors, so our finding are not conclusively linked to opioid use. However, all individuals in the PWID group did inject heroin daily. Our study indicates a potential for opioid injection and/or its associated multi-drug use habits and lifestyle changes to influence epigenetic inheritance.

Single-molecule localization microscopy (SMLM) allows super-resolution imaging, mapping, counting, and sizing of biological nanostructures such as cell organelles and extracellular vesicles (EVs), but sizing structures smaller than ∼100 nm can be inaccurate due to single-molecule localization error caused by distortion of the point spread function and limited photon number. Here we demonstrate a method to correct localization error when sizing vesicles and other spherical nanoparticles with SMLM and compare sizing results using two vesicle labeling schemes. We use mean approximation theory to derive a simple equation using full width at half-maximum (FWHM) for correcting particle sizes measured by two-dimensional SMLM, validate the method by sizing streptavidin-coated polystyrene nanobeads with the SMLM technique dSTORM with and without error correction, using transmission electron microscopy (TEM) for comparison, and then apply the method to sizing small seminal EVs. Nanobead sizes measured by dSTORM became increasingly less accurate (larger than TEM values) for beads smaller than 50 nm. The error-correction method reduced the size difference versus TEM from 15% without error correction to 7% with error correction for 40 nm beads, from 44% to 9% for 30 nm beads, and from 66% to 15% for 20 nm beads. Seminal EVs were labeled with a lipophilic membrane dye (MemBright 700) and with an Alexa Fluor 488-anti-CD63 antibody conjugate, and were sized separately using both dyes by dSTORM. Error-corrected exosome diameters were smaller than uncorrected values: 72 nm vs 79 nm mean diameter with membrane dyes; 84 nm vs 97 nm with the antibody-conjugated dyes. The mean error-corrected diameter was 12 nm smaller when using the membrane dye than when using the antibody-conjugated dye likely due to the large size of the antibody. Thus, both the error-correction method and the compact membrane labeling scheme reduce overestimation of vesicle size by SMLM. This error-correction method has a low computational cost as it does not require correction of individual blinking events, and it is compatible with all SMLM techniques (e.g., PALM, STORM, and DNA-PAINT).

Atherosclerosis is driven by intimal arterial macrophages accumulating cholesterol. Atherosclerosis also predominantly occurs in areas consisting of proinflammatory arterial endothelial cells. At time of writing, there are no available clinical treatments that precisely remove excess cholesterol from lipid-laden intimal arterial macrophages. Delivery of anti-miR-33a-5p to macrophages has been shown to increase apoAI-mediated cholesterol efflux via ABCA1 upregulation but delivering transgenes to intimal arterial macrophages is challenging due to endothelial cell barrier integrity. In this study, we aimed to test whether lipoparticles targeting proinflammatory endothelial cells can participate in endothelial cell-derived exosome exploitation to facilitate exosome-mediated transgene delivery to macrophages. We constructed lipoparticles that precisely target the proinflammatory endothelium and contain a plasmid that expresses XMOTIF-tagged anti-miR-33a-5p (LP-pXMoAntimiR33a5p), as XMOTIF-tagged small RNA demonstrates the capacity to be selectively shuttled into exosomes. The cultured cells used in our study were immortalized mouse aortic endothelial cells (iMAECs) and RAW 264.7 macrophages. From our results, we observed a significant decrease in miR-33a-5p expression in macrophages treated with exosomes released basolaterally by LPS-challenged iMAECs incubated with LP-pXMoAntimiR33a5p when compared to control macrophages. This decrease in miR-33a-5p expression in the treated macrophages caused ABCA1 upregulation as determined by a significant increase in ABCA1 protein expression in the treated macrophages when compared to the macrophage control group. The increase in ABCA1 protein also simulated ABCA1-dependent cholesterol efflux in treated macrophages-as we observed a significant increase in apoAI-mediated cholesterol efflux-when compared to the control group of macrophages. Based on these findings, strategies that involve combining proinflammatory-targeting lipoparticles and exploitation of endothelial cell-derived exosomes appear to be promising approaches for delivering atheroprotective transgenes to lipid-laden arterial intimal macrophages.

Studying the placenta can provide information about the mechanistic pathways of pregnancy disease. However, analyzing placental tissues and manipulating placental function in real-time during pregnancy is not feasible. The ex vivo placental perfusion model allows observing important aspects of the physiology and pathology of the placenta, while maintaining its viability and functional integrity, and without causing harm to mother or fetus. In this review, we describe and compare setups for this technically complex model and summarize outcomes from various published studies. We hope that our review will encourage wider use of ex vivo placental perfusion, which in turn would generate more knowledge to improve pregnancy outcomes.

The extracellular RNA communication consortium (ERCC) is an NIH-funded program aiming to promote the development of new technologies, resources, and knowledge about exRNAs and their carriers. After Phase 1 (2013-2018), Phase 2 of the program (ERCC2, 2019-2023) aims to fill critical gaps in knowledge and technology to enable rigorous and reproducible methods for separation and characterization of both bulk populations of exRNA carriers and single EVs. ERCC2 investigators are also developing new bioinformatic pipelines to promote data integration through the exRNA atlas database. ERCC2 has established several Working Groups (Resource Sharing, Reagent Development, Data Analysis and Coordination, Technology Development, nomenclature, and Scientific Outreach) to promote collaboration between ERCC2 members and the broader scientific community. We expect that ERCC2's current and future achievements will significantly improve our understanding of exRNA biology and the development of accurate and efficient exRNA-based diagnostic, prognostic, and theranostic biomarker assays.

Chronic kidney disease is a progressive, incurable condition that involves a gradual loss of kidney function. While there are no non-invasive biomarkers available to determine whether individuals are susceptible to developing chronic kidney disease, small RNAs within urinary exosomes have recently emerged as a potential candidate to use for assessing renal function. Ultracentrifugation is the gold standard for urinary exosome isolation. However, extravesicular small RNA contamination can occur when isolating exosomes from biological fluids using ultracentrifugation, which may lead to misidentifying the presence of certain small RNA species in human urinary exosomes. Therefore, we characterized human urinary exosomal preparations isolated by ultracentrifugation alone, or via ultracentrifugation followed by size exclusion chromatography (SEC) column-purification. Using nanoparticle tracking analysis, we identified SEC fractions containing robust amounts of exosome-sized particles, that we further characterized using immunoblotting. When compared to exosomal preparations isolated by ultracentrifugation only, SEC fractionated exosomal preparations showed higher levels of the exosome-positive marker CD81. Moreover, while the exosome-negative marker calnexin was undetectable in SEC fractionated exosomal preparations, we did observe calnexin detection in the exosomal preparations isolated by ultracentrifugation alone, which implies contamination in these preparations. Lastly, we imaged SEC fractionated exosomal preparations using transmission electron microscopy to confirm these preparations contained human urinary exosomes. Our results indicate that combining ultracentrifugation and SEC column-purification exosome isolation strategies is a powerful approach for collecting contaminant-free human urinary exosomes and should be considered when exosomes devoid of contamination are needed for downstream applications.