Caitlin Grace Howe

MicroRNAs (miRNAs) are a class of small non-coding RNAs, 19 to 24 nucleotides in length, that regulate a range of developmental and physiological processes by modulating gene expression through pos...

Background: Circulating extracellular and vesicle particle (EVP) miRNAs have been associated with cardiovascular risk and adverse birth outcomes. Hypertensive disorders of pregnancy (HDP) increase risk for adverse birth outcomes and future cardiovascular outcomes in mothers and children and have been associated with altered maternal circulating EVP miRNA levels during pregnancy. Whether these relationships exist for elevated blood pressure (BP) in the subclinical range is unknown. We investigated associations between (1) hypertensive disorders of pregnancy and (2) maternal BP trajectories, including in the subclinical range, and circulating EVP miRNA levels during pregnancy in the MADRES (Maternal and Developmental Risks From Environmental and Social Stressors) Study (n=372). Methods: Latent class trajectory modeling was used to identify trajectories from BP measures abstracted from medical records. The NanoString nCounter platform was used to quantify 798 miRNAs extracted from maternal blood (median gestational age: 31.6 weeks). Covariate‐adjusted regression models assessed associations between each hypertensive disorders of pregnancy subtype or BP trajectory and levels of each miRNA. Results: Three BP trajectories were identified: Low, Moderate, and High. Chronic hypertension was associated with higher levels of miR‐1185‐2‐3p ( P _{false discovery rate} P <0.05). Target genes of BP‐associated EVP miRNAs were overrepresented in pathways involved in vascular inflammation, oxidative stress, endothelial dysfunction, and placental function. Conclusions: Circulating levels of placenta‐expressed EVP miRNAs previously implicated in adverse birth and cardiovascular outcomes are sensitive to elevated maternal BP during pregnancy, including in the subclinical range.

Introduction Quantification of placental histopathological structures is challenging due to a limited number of perinatal pathologists, constrained resources, and subjective assessments prone to variability. Objective standardization of placental structure is crucial for easing the burden on pathologists, gaining deeper insights into placental growth and adaptation, and ultimately improving maternal and fetal health outcomes. Methods Leveraging advancements in deep-learning segmentation, we developed an automated approach to detect over 9 million placenta chorionic villi from 1,531 term placental whole slide images from the New Hampshire Birth Cohort Study. Using unsupervised clustering, we successfully identified biologically relevant villi subtypes that align with previously reported classifications – terminal, mature intermediate, and immature intermediate – demonstrating consistent size distributions and comparable abundance. We additionally defined tertile-based combinations of villi area and circularity to characterize villous geometry. This study applies these cutting-edge AI methods to quantify villi features and examine their association with maternal and infant characteristics, including gestational age at delivery, maternal age, and infant sex. Results Increasing gestational age at delivery was statistically significantly associated (p=0.003) with an increase in the proportion of mature intermediate villi and a decrease in the proportion of the smallest, most circular villi (p < 0.001). Maternal age and infant sex were not statistically significantly associated with measures of villous geometry. Discussion This work presents a workflow that objectively standardizes chorionic villi subtypes and geometry to enhance understanding of placental structure and function, while providing insights into the efficiency, growth, and the architecture of term placentas which can be used to inform future clinical care.

Background: MicroRNAs (miRNAs) are increasingly being investigated as potential biomarkers for child development and disease. Although a growing number of studies are utilizing infant and toddler stool for transcriptomic analyses, no studies have compared protocols for preserving and extracting miRNAs from this specimen type, despite unique challenges, including abundant levels of RNAses and microbial RNA. Methods: To address this, we first compared three commercially available kits and four preservation methods for their ability to yield high quality RNA from infant and toddler stool (Phase 1). RNA quality was determined by fragment analyzer. Results: Of the three RNA extraction kits compared, Zymo BIOMICs yielded the highest overall RNA Quality Number (RQN) (median (range) RQN 9.4 (5.7-10.0)). Of the four preservation methods tested, stool collected in RNAlater and Zymo DNA/RNA Shield Fecal Collection Tubes yielded the highest two RQNs (median (range) RQN 9.8 (5.7-10.0) and 9.4 (5.4-10.0), respectively), which did not differ significantly from each other ( p = 0.47). Second, using miRNA-seq we directly compared miRNA profiles for RNA extracted using the Zymo BIOMICs kit from paired aliquots of the same stool sample from four infants collected into RNAlater and Zymo DNA/RNA Shield Fecal Collection Tubes (Phase 2). Given that microbial sequences greatly outnumber human miRNAs in stool, reads were first classified as human versus microbial prior to aligning human-classified reads to miRBase v22.1. The percentage of reads classified as human and the percentage of human reads aligning to miRBase did not differ for samples collected in RNAlater versus Zymo Shield ( p = 0.12 and p = 0.86, respectively). Furthermore, after multiple testing correction, normalized miRNA counts did not differ significantly between the two preservatives for any of the 42 human miRNAs detected across the eight samples. Conclusions: Collecting infant and toddler stool in either RNAlater or Zymo DNA/RNA Shield Fecal Collection Tubes, when paired with RNA extraction using the Zymo BIOMICs extraction kit, yielded high-quality RNA with similar human miRNA profiles. Moreover, of the 42 miRNAs that were detected, several (i.e., miR-194a-3p, miR-200c-3p, miR-26a-5p) are thought to contribute to overall gut homeostasis. These findings may inform protocols for future studies that aim to profile miRNAs in infant and toddler stool to evaluate their potential utility as biomarkers for children’s health.

In this study, the effect of cumulative ACEs experienced on human maternal DNA methylation (DNAm) was estimated while accounting for interaction with domains of ACEs in prenatal peripheral blood mononuclear cell samples from the Maternal and Developmental Risks from Environmental Stressors (MADRES) pregnancy cohort. The intergenerational transmission of ACE-associated DNAm was also explored used paired maternal (N = 120) and neonatal cord blood (N = 69) samples. Replication in buccal samples was explored in the Children’s Health Study (CHS) among adult parental (N = 31) and pediatric (N = 114) samples. We used a four-level categorical indicator variable for ACEs exposure: none (0 ACEs), low (1–3 ACEs), moderate (4–6 ACEs), and high (>6 ACEs). Effects of ACEs on maternal DNAm (N = 240) were estimated using linear models. To evaluate evidence for intergenerational transmission, mediation analysis (N = 60 mother-child pairs) was used. Analysis of maternal samples displayed some shared but mostly distinct effects of ACEs on DNAm across low, moderate, and high ACEs categories. CLCN7 and PTPRN2 was associated with maternal DNAm in the low ACE group and this association replicated in the CHS. CLCN7 was also nominally significant in the gene expression correlation analysis among maternal profiles (N = 35), along with 11 other genes. ACE-associated methylation was observed in maternal and neonatal profiles in the COMT promoter region, with some evidence of mediation by maternal COMT methylation. Specific genomic loci exhibited mutually exclusive maternal ACE effects on DNAm in either maternal or neonatal population. There is some evidence for an intergenerational effect of ACEs, supported by shared DNAm signatures in the COMT gene across maternal-neonatal paired samples.

MicroRNAs (miRNA) in extracellular vesicles and particles (EVPs) in maternal circulation during pregnancy and in human milk postpartum are hypothesized to facilitate maternal–offspring communication via epigenetic regulation. However, factors influencing maternal EVP miRNA profiles during these two critical developmental windows remain largely unknown. In a pilot study of 54 mother–child dyads in the New Hampshire Birth Cohort Study, we profiled 798 EVP miRNAs, using the NanoString nCounter platform, in paired maternal second-trimester plasma and mature (6-week) milk samples. In adjusted models, total EVP miRNA counts were lower for plasma samples collected in the afternoon compared with the morning (p = 0.024). Infant age at sample collection was inversely associated with total miRNA counts in human milk EVPs (p = 0.040). Milk EVP miRNA counts were also lower among participants who were multiparous after delivery (p = 0.047), had a pre-pregnancy BMI > 25 kg/m² (p = 0.037), or delivered their baby via cesarean section (p = 0.021). In post hoc analyses, we also identified 22 specific EVP miRNA that were lower among participants who delivered their baby via cesarean section (Q < 0.05). Target genes of delivery mode-associated miRNAs were over-represented in pathways related to satiety signaling in infants (e.g., CCKR signaling) and mammary gland development and lactation (e.g., FGF signaling, EGF receptor signaling). In conclusion, we identified several key factors that may influence maternal EVP miRNA composition during two critical developmental windows, which should be considered in future studies investigating EVP miRNA roles in maternal and child health.