Deep learning-based prediction of one-year mortality in Finland is an accurate but unfair aging marker.

Short-term mortality risk, which is indicative of individual frailty, serves as a marker for aging. Previous age clocks focused on predicting either chronological age or longer-term mortality. Aging clocks predicting short-term mortality are lacking and their algorithmic fairness remains unexamined. We developed a deep learning model to predict 1-year mortality using nationwide longitudinal data from the Finnish population (FinRegistry; n = 5.4 million), incorporating more than 8,000 features spanning up to 50 years. We achieved an area under the curve (AUC) of 0.944, outperforming a baseline model that included only age and sex (AUC = 0.897). The model generalized well to different causes of death (AUC > 0.800 for 45 of 50 causes), including coronavirus disease 2019, which was absent in the training data. Performance varied among demographics, with young females exhibiting the best and older males the worst results. Extensive prediction fairness analyses highlighted disparities among disadvantaged groups, posing challenges to equitable integration into public health interventions. Our model accurately identified short-term mortality risk, potentially serving as a population-wide aging marker.

Risk factors for severe respiratory syncytial virus infection during the first year of life: development and validation of a clinical prediction model.

BACKGROUND: Novel immunisation methods against respiratory syncytial virus (RSV) are emerging, but knowledge of risk factors for severe RSV disease is insufficient for optimal targeting of interventions against them. Our aims were to identify predictors for RSV hospital admission from registry-based data and to develop and validate a clinical prediction model to guide RSV immunoprophylaxis for infants younger than 1 year. METHODS: In this model development and validation study, we studied all infants born in Finland between June 1, 1997, and May 31, 2020, and in Sweden between June 1, 2006, and May 31, 2020, along with the data for their parents and siblings. Infants were excluded if they died or were admitted to hospital for RSV within the first 7 days of life. The outcome was hospital admission due to RSV bronchiolitis during the first year of life. The Finnish study population was divided into a development dataset (born between June 1, 1997, and May 31, 2017) and a temporal hold-out validation dataset (born between June 1, 2017, and May 31, 2020). The development dataset was used for predictor discovery and selection in which we screened 1511 candidate predictors from the infants', parents', and siblings' data, and developed a logistic regression model with the 16 most important predictors. This model was then validated using the Finnish hold-out validation dataset and the Swedish dataset. FINDINGS: In total, there were 1 124 561 infants in the Finnish development dataset, 130 352 infants in the Finnish hold-out validation dataset, and 1 459 472 infants in the Swedish dataset. In addition to known predictors such as severe congenital heart defects (adjusted odds ratio 2·89, 95% CI 2·28-3·65), we confirmed some less established predictors for RSV hospital admission, most notably oesophageal malformations (3·11, 1·86-5·19) and lower complexity congenital heart defects (1·43, 1·25-1·63). The prediction model's C-statistic was 0·766 (95% CI 0·742-0·789) in Finnish data and 0·737 (0·710-0·762) in Swedish validation data. The infants in the highest decile of predicted RSV hospital admission probability had 4·5 times higher observed risk compared with others. Calibration varied according to epidemic intensity. The model's performance was similar to a machine learning (XGboost) model using all 1511 candidate predictors (C-statistic in Finland 0·771, 95% CI 0·754-0·788). The prediction model showed clinical utility in decision curve analysis and in hypothetical number needed to treat calculations for immunisation, and its C-statistic was similar across different strata of parental income. INTERPRETATION: The identified predictors and the prediction model can be used in guiding RSV immunoprophylaxis in infants, or as a basis for further immunoprophylaxis targeting tools. FUNDING: Sigrid Jusélius Foundation, European Research Council, Pediatric Research Foundation, and Academy of Finland.

Circulating extracellular vesicles in human cardiorenal syndrome promote renal injury in a kidney-on-chip system.

BACKGROUNDCardiorenal syndrome (CRS) - renal injury during heart failure (HF) - is linked to high morbidity. Whether circulating extracellular vesicles (EVs) and their RNA cargo directly impact its pathogenesis remains unclear.METHODSWe investigated the role of circulating EVs from patients with CRS on renal epithelial/endothelial cells using a microfluidic kidney-on-chip (KOC) model. The small RNA cargo of circulating EVs was regressed against serum creatinine to prioritize subsets of functionally relevant EV-miRNAs and their mRNA targets investigated using in silico pathway analysis, human genetics, and interrogation of expression in the KOC model and in renal tissue. The functional effects of EV-RNAs on kidney epithelial cells were experimentally validated.RESULTSRenal epithelial and endothelial cells in the KOC model exhibited uptake of EVs from patients with HF. HF-CRS EVs led to higher expression of renal injury markers (IL18, LCN2, HAVCR1) relative to non-CRS EVs. A total of 15 EV-miRNAs were associated with creatinine, targeting 1,143 gene targets specifying pathways relevant to renal injury, including TGF-ß and AMPK signaling. We observed directionally consistent changes in the expression of TGF-ß pathway members (BMP6, FST, TIMP3) in the KOC model exposed to CRS EVs, which were validated in epithelial cells treated with corresponding inhibitors and mimics of miRNAs. A similar trend was observed in renal tissue with kidney injury. Mendelian randomization suggested a role for FST in renal function.CONCLUSIONPlasma EVs in patients with CRS elicit adverse transcriptional and phenotypic responses in a KOC model by regulating biologically relevant pathways, suggesting a role for EVs in CRS.TRIAL REGISTRATIONClinicalTrials.gov NCT03345446.FUNDINGAmerican Heart Association (AHA) (SFRN16SFRN31280008); National Heart, Lung, and Blood Institute (1R35HL150807-01); National Center for Advancing Translational Sciences (UH3 TR002878); and AHA (23CDA1045944).

Recontacting biobank participants to collect lifestyle, behavioural and cognitive information via online questionnaires: lessons from a pilot study within FinnGen.

OBJECTIVES: To recontact biobank participants and collect cognitive, behavioural and lifestyle information via a secure online platform. DESIGN: Biobank-based recontacting pilot study. SETTING: Three Finnish biobanks (Helsinki, Auria, Tampere) recruiting participants from February 2021 to July 2021. PARTICIPANTS: All eligible invitees were enrolled in FinnGen by their biobanks (Helsinki, Auria, Tampere), had available genetic data and were >18 years old. Individuals with severe neuropsychiatric disease or cognitive or physical disabilities were excluded. Lastly, 5995 participants were selected based on their polygenic score for cognitive abilities and invited to the study. Among invitees, 1115 had successfully participated and completed the study questionnaire(s). OUTCOME MEASURES: The primary outcome was the participation rate among study invitees. Secondary outcomes included questionnaire completion rate, quality of data collected and comparison of participation rate boosting strategies. RESULTS: The overall participation rate was 18.6% among all invitees and 23.1% among individuals aged 18-69. A second reminder letter yielded an additional 9.7% participation rate in those who did not respond to the first invitation. Recontacting participants via an online healthcare portal yielded lower participation than recontacting via physical letter. The completion rate of the questionnaire and cognitive tests was high (92% and 85%, respectively), and measurements were overall reliable among participants. For example, the correlation (r) between self-reported body mass index and that collected by the biobanks was 0.92. CONCLUSION: In summary, this pilot suggests that recontacting FinnGen participants with the goal to collect a wide range of cognitive, behavioural and lifestyle information without additional engagement results in a low participation rate, but with reliable data. We suggest that such information be collected at enrolment, if possible, rather than via post hoc recontacting.

Breast milk-derived extracellular vesicle miRNAs are associated with maternal asthma and atopy.

Background: Breast milk-derived extracellular vesicle (EV) miRNAs may program child health outcomes associated with maternal asthma and atopy. The authors investigated associations between maternal asthma/atopy and EV miRNAs in the Programming of Intergenerational Stress Mechanisms cohort. Methods: Breast milk-derived EV miRNAs collected 6.1 ± 5.9 weeks postnatally (n = 80 mothers) were profiled using the TaqMan OpenArray Human MicroRNA Panel. The authors assessed associations using adjusted robust regression. Results: Nine EV miRNAs were associated with asthma during pregnancy (a priori criteria: nominal p < 0.05; |Bregression| >0.2). miR-1290 was associated with asthma and atopy during pregnancy (p < 0.05; |Bregression| >0.2). Enriched Kyoto Encyclopedia of Genes and Genomes pathways included TGF-ß signaling and extracellular matrix-receptor interaction (false discovery rate <0.05). Conclusion: In this study, maternal asthma and atopy were associated with breast milk-derived EV miRNAs. Additional studies are needed to understand whether EV miRNAs have direct effects on infant and child health.

Maternal asthma is associated with child health outcomes, although the biological mechanisms involved are not fully understood. miRNAs are small molecules involved in regulating gene expression. miRNAs packaged into membrane-bound particles called extracellular vesicles (EVs) are present in human breast milk and may pass from mother to infant to signal which genes to translate into proteins. This study investigated the extent to which maternal asthma and atopy influenced levels of 130 EV miRNAs measured in breast milk. Nine EV miRNAs were associated with maternal asthma during pregnancy, and one EV miRNA was associated with maternal atopy. miRNAs associated with asthma target genes in pathways related to asthma; however, future research is needed to determine whether changes in breast milk-derived EV miRNAs impact child health.

Mir-30d Regulates Cardiac Remodeling by Intracellular and Paracrine Signaling.

RATIONALE: Previous translational studies implicate plasma extracellular microRNA-30d (miR-30d) as a biomarker in left ventricular remodeling and clinical outcome in heart failure (HF) patients, although precise mechanisms remain obscure. OBJECTIVE: To investigate the mechanism of miR-30d-mediated cardioprotection in HF. METHODS AND RESULTS: In rat and mouse models of ischemic HF, we show that miR-30d gain of function (genetic, lentivirus, or agomiR-mediated) improves cardiac function, decreases myocardial fibrosis, and attenuates cardiomyocyte (CM) apoptosis. Genetic or locked nucleic acid-based knock-down of miR-30d expression potentiates pathological left ventricular remodeling, with increased dysfunction, fibrosis, and cardiomyocyte death. RNA sequencing of in vitro miR-30d gain and loss of function, together with bioinformatic prediction and experimental validation in cardiac myocytes and fibroblasts, were used to identify and validate direct targets of miR-30d. miR-30d expression is selectively enriched in cardiomyocytes, induced by hypoxic stress and is acutely protective, targeting MAP4K4 (mitogen-associate protein kinase 4) to ameliorate apoptosis. Moreover, miR-30d is secreted primarily in extracellular vesicles by cardiomyocytes and inhibits fibroblast proliferation and activation by directly targeting integrin α5 in the acute phase via paracrine signaling to cardiac fibroblasts. In the chronic phase of ischemic remodeling, lower expression of miR-30d in the heart and plasma extracellular vesicles is associated with adverse remodeling in rodent models and human subjects and is linked to whole-blood expression of genes implicated in fibrosis and inflammation, consistent with observations in model systems. CONCLUSIONS: These findings provide the mechanistic underpinning for the cardioprotective association of miR-30d in human HF. More broadly, our findings support an emerging paradigm involving intercellular communication of extracellular vesicle-contained miRNAs (microRNAs) to transregulate distinct signaling pathways across cell types. Functionally validated RNA biomarkers and their signaling networks may warrant further investigation as novel therapeutic targets in HF.

Profiling Extracellular Long RNA Transcriptome in Human Plasma and Extracellular Vesicles for Biomarker Discovery.

The recent discovery of extracellular RNAs in blood, including RNAs in extracellular vesicles (EVs), combined with low-input RNA-sequencing advances have enabled scientists to investigate their role in human disease. To date, most studies have been focusing on small RNAs, and methodologies to optimize long RNAs measurement are lacking. We used plasma RNA to assess the performance of six long RNA sequencing methods, at two different sites, and we report their differences in reads (%) mapped to the genome/transcriptome, number of genes detected, long RNA transcript diversity, and reproducibility. Using the best performing method, we further compare the profile of long RNAs in the EV- and no-EV-enriched RNA plasma compartments. These results provide insights on the performance and reproducibility of commercially available kits in assessing the landscape of long RNAs in human plasma and different extracellular RNA carriers that may be exploited for biomarker discovery.

MiR-574-5p: A Circulating Marker of Thoracic Aortic Aneurysm.

Thoracic aortic aneurysm (TAA) can lead to fatal complications such as aortic dissection. Since aneurysm dimension poorly predicts dissection risk, microRNAs (miRNAs) may be useful to diagnose or risk stratify TAA patients. We aim to identify miRNAs associated with TAA pathogenesis and that are possibly able to improve TAA diagnosis. MiRNA microarray experiments of aortic media tissue samples from 19 TAA patients and 19 controls allowed identifying 232 differentially expressed miRNAs. Using interaction networks between these miRNAs and 690 genes associated with TAA, we identified miR-574-5p as a potential contributor of TAA pathogenesis. Interestingly, miR-574-5p was significantly down-regulated in the TAA tissue compared to the controls, but was up-regulated in serum samples from a separate group of 28 TAA patients compared to 20 controls (p < 0.001). MiR-574-5p serum levels discriminated TAA patients from controls with an area under the receiver operating characteristic curve of 0.87. In the Fbn1C1041G/+ mouse model, miR-574-5p was down-regulated in aortic tissue compared to wild-type (p < 0.05), and up-regulated in plasma extracellular vesicles from Fbn1C1041G/+ mice compared to wild-type mice (p < 0.05). Furthermore, in vascular smooth muscle cells, angiotensin II appears to induce miR-574-5p secretion in extracellular vesicles. In conclusion, miR-574-5p is associated with TAA pathogenesis and may help in diagnosing this disease.

miRNA Profiles in Extracellular Vesicles From Serum Early in Pregnancies Complicated by Gestational Diabetes Mellitus.

CONTEXT: Underlying mechanisms leading to gestational diabetes mellitus (GDM) are still under investigation, and it is unclear whether the placenta plays a role in triggering glucose intolerance or if its functions are modified in response to the hyperglycemia. Circulating miRNAs are involved in placental development and function and are encapsulated in extracellular vesicles (EVs). OBJECTIVE: To compare differential expression of miRNAs in circulating EVs in pregnancies complicated by GDM vs controls. METHODS: This was a case-control study nested in a prospective pregnancy cohort including 23 women with GDM and 46 matched controls. The presence of serum EVs in early pregnancy was validated by transmission electron microscopy. Placental dimensions were assessed at 11 to 13 weeks of gestation. Differential expression of 17 miRNAs encapsulated in EVs (miR‒122-5p, miR‒132-3p, miR-1323, miR‒182-3p, miR‒210-3p, miR‒29a-3p, miR‒29b-3p, miR‒342-3p, miR‒517-5p, miR‒517a-3p, miR‒518b, miR-520h, miR‒525-5p, miR‒136-5p, miR‒342-3p, miR‒376c-5p, and miR‒494-3p) was assessed using quantitative reverse transcription PCR. RESULTS: EVs were present in the early phase of placentation (6 to 15 weeks of gestation) in both cases and controls. No differences were observed for placental dimensions and estimated placental volume between GDM and control groups. Ten miRNAs (miR‒122-5p; miR‒132-3p; miR‒1323; miR‒136-5p; miR‒182-3p; miR‒210-3p; miR‒29a-3p; miR‒29b-3p; miR‒342-3p, and miR-520h) showed significantly higher levels in GDM cases than in controls (P ≤ 0.05). Bioinformatics analysis showed that these miRNAs are involved in trophoblast proliferation/differentiation as well as in insulin secretion/regulation and glucose transport in pregnant women. CONCLUSION: The miRNA content of blood EVs may be a promising avenue for studying the early effect of impaired glucose metabolism on placental development.

Supraphysiological Concentrations of Bisphenol A Alter the Expression of Extracellular Vesicle-Enriched miRNAs From Human Primary Granulosa Cells.

Bisphenol A (BPA) is a widely used chemical that has been detected in follicular fluid and associated with adverse reproductive effects. Granulosa cells have an important role in follicular growth and oocyte maturation, however, little is known about the biological mechanisms of BPA toxicity on human granulosa cells. In this study, we exposed primary granulosa cells to different concentrations of BPA (0, 20, 200, 2000, and 20 000 ng/ml) and used quantitative polymerase chain reaction to measure the expression levels of miRNAs enriched in extracellular vesicles (EV-enriched miRNAs), and cellular levels of selected target genes of differentially expressed EV-enriched miRNAs. We found that exposure to 20 000 ng/ml BPA was associated with decreased levels of EV-miR-27b-3p (FC = 0.58, p = .04) and increased levels of its biologically relevant target genes FADD (FC = 1.22, p = .01), IGF1 (FC = 1.59, p = .06), and PPARG (FC = 1.73, p = .001) as compared with the control. In addition, we observed that under the same exposure conditions, the expression levels of miR-27b-3p in granulosa cells were also downregulated (FC = 0.65, p = .03) as compared with the control. Our findings suggest that both cellular and extracellular changes in gene expression may mediate BPA toxicity in granulosa cells.

Extracellular vesicle-enriched microRNAs interact in the association between long-term particulate matter and blood pressure in elderly men.

BACKGROUND: Several studies have shown that exposure to particulate matter (PM) may lead to increased systemic blood pressure, but the underlying biological mechanisms remain unknown. Emerging evidence shows that extracellular vesicle-enriched miRNAs (evmiRNAs) are associated with PM exposure and cardiovascular risk. In this study, we investigated the role of evmiRNAs in the association between PM and blood pressure, as well as their epigenetic regulation by DNA methylation. METHODS: Participants (n = 22, men) were randomly selected from the Veterans Affairs Normative Aging Study (NAS). Long-term (1-year and 6-month average) PM2.5 exposure was estimated at 1 × 1-km resolution using spatio-temporal prediction models and BC was estimated using validated time varying land use regression models. We analyzed 31 evmiRNAs detected in ≥ 90% of all individuals and for statistical analysis, we used mixed effects models with random intercept adjusted for age, body mass index, smoking, C-reactive protein, platelets, and white blood cells. RESULTS: We found that per each 2-standard deviations increase in 6-month PM2.5 ambient levels, there was an increase in 0.19 mm Hg (95% Confidence Interval [95%CI]: 0.11, 0.28 mmHg; p < 0.001) in systolic blood pressure (SBP). Per each 2-standard deviations increase in 1-year PM2.5 levels, there was an increase in 0.11 mm Hg (95% Confidence Interval [95% CI]: 0.03, 0.19 mmHg; p = 0.012) in SBP in older male individuals. We also found that both miR-199a/b (ß = 6.13 mmHg; 95% CI: 0.87, 11.39; pinteraction = 0.07) and miR-223-3p (ß = 30.17 mmHg; 95% CI: 11.96, 48.39 mmHg; pinteraction = 0.01) modified the association between 1-year PM2.5 and SBP. When exploring DNA methylation as a potential mechanism that could epigenetically regulate expression of evmiRNAs, we found that PM2.5 ambient levels were negatively associated with DNA methylation levels at CpG (cg23972892) near the enhancer region of miR-199a/b (ß = -13.11; 95% CI: -17.70, -8.52; pBonferroni< 0.01), but not miR-223-3p. CONCLUSIONS: Our findings suggest that expression of evmiRNAs may be regulated by DNA methylation in response to long-term PM2.5 ambient levels and modify the magnitude of association between PM2.5 and systolic blood pressure in older individuals.

Long noncoding RNA expression in the cervix mid-pregnancy is associated with the length of gestation at delivery.

Infants born preterm are at increased risk of multiple morbidities and mortality. Why some women deliver preterm remains poorly understood. Prior studies have shown that cervical microRNA expression and DNA methylation are associated with the length of gestation. However, no study has examined the role of long noncoding RNAs (lncRNAs) in the cervix during pregnancy. To determine whether expression of lncRNAs is associated with length of gestation at delivery, we analyzed RNA from cervical swabs obtained from 78 women during pregnancy (mean 15.5, SD 5.0, weeks of gestation) who were participating in the Spontaneous Prematurity and Epigenetics of the Cervix (SPEC) Study in Boston, MA, USA. We used a PCR-based platform and found that 9 lncRNAs were expressed in at least 50% of the participants. Of these, a doubling of the expression of TUG1, TINCR, and FALEC was associated with shorter lengths of gestation at delivery [2.8 (95% CI: 0.31, 5.2); 3.3 (0.22, 6.3); and 4.5 (7.3, 1.6) days shorter respectively]. Of the lncRNAs analyzed, none was statistically associated with preterm birth, but expression of FALEC was 2.6-fold higher in women who delivered preterm vs. term (P = 0.051). These findings demonstrate that lncRNAs can be measured in cervical samples obtained during pregnancy and are associated with subsequent length of gestation at delivery. Further, this study supports future work to replicate these findings in other cohorts and perform mechanistic studies to determine the role of lncRNAs in the cervix during pregnancy.

Advances, challenges, and opportunities in extracellular RNA biology: insights from the NIH exRNA Strategic Workshop.

Extracellular RNA (exRNA) has emerged as an important transducer of intercellular communication. Advancing exRNA research promises to revolutionize biology and transform clinical practice. Recent efforts have led to cutting-edge research and expanded knowledge of this new paradigm in cell-to-cell crosstalk; however, gaps in our understanding of EV heterogeneity and exRNA diversity pose significant challenges for continued development of exRNA diagnostics and therapeutics. To unravel this complexity, the NIH convened expert teams to discuss the current state of the science, define the significant bottlenecks, and brainstorm potential solutions across the entire exRNA research field. The NIH Strategic Workshop on Extracellular RNA Transport helped identify mechanistic and clinical research opportunities for exRNA biology and provided recommendations on high priority areas of research that will advance the exRNA field.

Extracellular RNAs: A New Awareness of Old Perspectives.

Extracellular RNA (exRNA) has recently expanded as a highly important area of study in biomarker discovery and cancer therapeutics. exRNA consists of diverse RNA subpopulations that are normally protected from degradation by incorporation into membranous vesicles or by lipid/protein association. They are found circulating in biofluids, and have proven highly promising for minimally invasive diagnostic and prognostic purposes, particularly in oncology. Recent work has made progress in our understanding of exRNAs-from their biogenesis, compartmentalization, and vesicle packaging to their various applications as biomarkers and therapeutics, as well as the new challenges that arise in isolation and purification for accurate and reproducible analysis. Here we review the most recent advancements in exRNA research.

Extracellular Vesicles in Cardiovascular Theranostics.

Extracellular vesicles (EVs) are small bilayer lipid membrane vesicles that can be released by most cell types and detected in most body fluids. EVs exert key functions for intercellular communication via transferring their bioactive cargos to recipient cells or activating signaling pathways in target cells. Increasing evidence has shown the important regulatory effects of EVs in cardiovascular diseases (CVDs). EVs secreted by cardiomyocytes, endothelial cells, fibroblasts, and stem cells play essential roles in pathophysiological processes such as cardiac hypertrophy, cardiomyocyte survival and apoptosis, cardiac fibrosis, and angiogenesis in relation to CVDs. In this review, we will first outline the current knowledge about the physical characteristics, biological contents, and isolation methods of EVs. We will then focus on the functional roles of cardiovascular EVs and their pathophysiological effects in CVDs, as well as summarize the potential of EVs as therapeutic agents and biomarkers for CVDs. Finally, we will discuss the specific application of EVs as a novel drug delivery system and the utility of EVs in the field of regenerative medicine.

Second trimester extracellular microRNAs in maternal blood and fetal growth: An exploratory study.

Healthy feto-maternal communication is critical during pregnancy and is orchestrated by the placenta. Dysfunction of the placenta leads to fetal growth complications; however, the underlying biological mechanisms have yet to be fully elucidated. Circulating extracellular microRNAs (exmiRNAs) in the blood have been implicated in cell-to-cell communication. Therefore, exmiRNAs may provide useful biological information about communication between the mother, the fetus, and the placenta during pregnancy. We used logistic regression to determine the association of exmiRNAs with abnormal fetal growth by comparing mothers of infants classified as small-for-gestational age (SGA) (n = 36) and large-for-gestational age (LGA) (n = 13) to appropriate-for-gestational age (AGA), matched by gestational age at delivery and infant sex. In addition, we used linear regression to determine associations between exmiRNAs and birth weight-for-gestational age (BWGA) z-score (n = 100), adjusting for maternal age, body mass index, and parity. We found that higher levels of miR-20b-5p, miR-942-5p, miR-324-3p, miR-223-5p, and miR-127-3p in maternal serum were associated with lower odds for having a SGA vs. AGA infant, and higher levels of miR-661, miR-212-3p, and miR-197-3p were associated with higher odds for having a LGA vs. AGA infant. We also found associations between miR-483-5p, miR-10a-5p, miR-204-5p, miR-202-3p, miR-345-5p, miR-885-5p, miR-127-3p, miR-148b-3p, miR-324-3p, miR-1290, miR-597-5p, miR-139-5p, miR-215-5p, and miR-99b-5p and BWGA z-score. We also found sex-specific associations with exmiRNAs and fetal growth. Our findings suggest that exmiRNAs circulating in maternal blood at second trimester are associated with fetal growth. Validation of our findings may lead to the development of minimally-invasive biomarkers of fetal growth during pregnancy.

Extracellular microRNAs in follicular fluid and their potential association with oocyte fertilization and embryo quality: an exploratory study.

PURPOSE: The purpose of this study is to determine the profile of extracellular microRNAs (exmiRNAs) in follicular fluid (FF) and explore their association with fertilization potential and embryo quality. METHODS: We collected FF from single follicles containing mature oocytes from 40 women undergoing IVF and we screened for the expression of 754 exmiRNAs in FF using the TaqMan OpenArray® qPCR platform. To determine the association of exmiRNAs and IVF outcomes, we compared their expression levels in FF samples that differ by fertilization status (normally, abnormally, and failed to fertilize) and embryo quality (top vs. non-top). RESULTS: We detected 207 exmiRNAs, of which miR-30d-5p, miR-320b, miR-10b-3p, miR-1291, and miR-720 were most prevalent. We identified four exmiRNAs with significant fold change (FC) when FF that contained normally fertilized was compared to failed to fertilize oocytes [miR-202-5p (FC = 1.82, p = 0.01), miR-206 (FC = 2.09, p = 0.04), miR-16-1-3p (FC = 1.88, p = 0.05), and miR-1244 (FC = 2.72, p = 0.05)]. We also found four exmiRNAs to be significantly differentially expressed in FF that yielded top quality versus non-top quality embryos [(miR-766-3p (FC = 1.95, p = 0.01), miR-663b (FC = 0.18, p = 0.02), miR-132-3p (FC = 2.45, p = 0.05), and miR-16-5p (FC = 3.80, p = 0.05)]. In-silico analysis revealed that several of these exmiRNAs are involved in pathways implicated in reproductive system diseases, organismal abnormalities, and organ development. CONCLUSIONS: Our findings suggest that exmiRNAs in the follicular fluid can lead to downstream events that will affect fertilization and day 3 embryo morphology. We encourage further observational and experimental studies to confirm our findings and to determine the role of exmiRNAs in human reproduction.

Prenatal lead exposure and fetal growth: Smaller infants have heightened susceptibility.

BACKGROUND: As population lead levels decrease, the toxic effects of lead may be distributed to more sensitive populations, such as infants with poor fetal growth. OBJECTIVES: To determine the association of prenatal lead exposure and fetal growth; and to evaluate whether infants with poor fetal growth are more susceptible to lead toxicity than those with normal fetal growth. METHODS: We examined the association of second trimester maternal blood lead levels (BLL) with birthweight-for-gestational age (BWGA) z-score in 944 mother-infant participants of the PROGRESS cohort. We determined the association between maternal BLL and BWGA z-score by using both linear and quantile regression. We estimated odds ratios for small-for-gestational age (SGA) infants between maternal BLL quartiles using logistic regression. Maternal age, body mass index, socioeconomic status, parity, household smoking exposure, hemoglobin levels, and infant sex were included as confounders. RESULTS: While linear regression showed a negative association between maternal BLL and BWGA z-score (ß=-0.06 z-score units per log2 BLL increase; 95% CI: -0.13, 0.003; P=0.06), quantile regression revealed larger magnitudes of this association in the <30th percentiles of BWGA z-score (ß range [-0.08, -0.13] z-score units per log2 BLL increase; all P values<0.05). Mothers in the highest BLL quartile had an odds ratio of 1.62 (95% CI: 0.99-2.65) for having a SGA infant compared to the lowest BLL quartile. CONCLUSIONS: While both linear and quantile regression showed a negative association between prenatal lead exposure and birthweight, quantile regression revealed that smaller infants may represent a more susceptible subpopulation.

Detection of long non-coding RNAs in human breastmilk extracellular vesicles: Implications for early child development.

Breastmilk has many documented beneficial effects on the developing human infant, but the components of breastmilk that influence these developmental pathways have not been fully elucidated. Increasing evidence suggests that non-coding RNAs encapsulated in extracellular vesicles (EVs) represent an important mechanism of communication between the mother and child. Long non-coding RNAs (lncRNAs) are of particular interest given their key role in gene expression and development. However, it is not known whether breastmilk EVs contain lncRNAs. We used qRT-PCR to determine whether EVs isolated from human breastmilk contain lncRNAs previously reported to be important for developmental processes. We detected 55 of the 87 screened lncRNAs in EVs from the 30 analyzed breastmilk samples, and CRNDE, DANCR, GAS5, SRA1 and ZFAS1 were detected in >90% of the samples. GAS5, SNHG8 and ZFAS1 levels were highly correlated (Spearman's rho > 0.9; P < 0.0001), which may indicate that the loading of these lncRNAs into breastmilk EVs is regulated by the same pathways. The detected lncRNAs are important epigenetic regulators involved in processes such as immune cell regulation and metabolism. They may target a repertoire of recipient cells in offspring and could be essential for child development and health. Further experimental and epidemiological studies are warranted to determine the impact of breastmilk EV-encapsulated lnRNAs in mother to child signaling.