Prenatal opioid-exposed infant extracellular miRNA signature obtained at birth predicts severity of neonatal opioid withdrawal syndrome

Prenatal opioid exposure (POE) is commonly associated with neonatal opioid withdrawal syndrome (NOWS), which is characterized by a broad variability in symptoms and severity. Currently there are no diagnostic tools to reliably predict which infants will develop severe NOWS, while risk stratification would allow for proactive decisions about appropriate clinical monitoring and interventions. The aim of this prospective cohort study was to assess if extracellular microRNAs (miRNAs) in umbilical cord plasma of infants with POE could predict NOWS severity. Participants (n = 58) consisted of pregnant women receiving medications for opioid use disorder and their infants. NOWS severity was operationalized as the need for pharmacologic treatment and prolonged hospitalization (≥ 14 days). Cord blood miRNAs were assessed using semi-quantitative qRT-PCR arrays. Receiver operating characteristic curves and area under the curve (AUC) were estimated. The expression of three miRNAs (miR-128-3p, miR-30c-5p, miR-421) predicted need for pharmacologic treatment (AUC: 0.85) and prolonged hospitalization (AUC: 0.90). Predictive validity improved after two miRNAs (let-7d-5p, miR-584-5p) were added to the need for pharmacologic treatment model (AUC: 0.94) and another two miRNAs (let-7b-5p, miR-10-5p) to the prolonged hospitalization model (AUC: 0.99). Infant cord blood extracellular miRNAs can proactively identify opioid-exposed neonates at high-risk for developing severe NOWS.

www.nature.com/scientificreports/ a single alcohol biomarker at delivery and for each biomarker type were not different between groups. A substantially higher proportion of Pharmacologically-Treated infants (64.7%) were prenatally exposed to methadone compared to Not-Pharmacologically-Treated (24.4%; p < 0.01). There were no differences in maternal characteristics between the study groups except for a higher prevalence of Hepatitis C in Pharmacologically-Treated group (35.3%) compared to Not-Pharmacologically-Treated (22.0%) group (p = 0.03). Perinatal and infant characteristics are summarized in Table 3. In the study population of 58 infants, 17 received pharmacological treatment, and 15 had a hospital stay ≥ 14 days. There were differences in the mean gestational age at delivery (36.9 ± 1.0 vs. 38.8 ± 1.4 weeks; p < 0.0001) reflected in the higher incidence of preterm delivery in the Pharmacologically-Treated group compared to Not-Pharmacologically-Treated group (23.5% vs. 4.9%; p = 0.06). Length of hospital stay (LOS) was significantly longer in the Pharmacologically-Treated (19.5 ± 9.5 days) compared to Not-Pharmacologically-Treated group (6.0 ± 3.6 days; p < 0.01). Respiratory distress was also significantly more prevalent in the Pharmacologically-Treated compared to Not-Pharmacologically-Treated group (47.1% vs 9.8%, p < 0.01). Type of maternal MOUD exposure was buprenorphine for 37 (63.8%) infants and methadone for 21 (36.2%) infants. Higher percentages of infants with methadone exposure experienced the two main outcomes, pharmacological treatment and LOS ≥ 14 days. There was substantial overlap between these outcomes; of the 17 infants receiving pharmacological treatment, 13 (76.5%) had a LOS ≥ 14 days. Of the 6 infants with maternal buprenorphine exposure, 3 (50%) had a LOS ≥ 14 days, while among the 11 infants with maternal methadone exposure, 10 (90.9%) had a LOS ≥ 14 days. No other differences in infant characteristics were observed.
Plasma and RNA characteristics. Sample purity characteristics. There were no significant differences in hemolysis measures, including the presence of free hemoglobin and difference in cycle threshold (CT) for hemolytic markers (ΔCT hemolysis ), when comparing Pharmacologically-Treated to Not-Pharmacologically-Treated or LOS ≥ 14 days to LOS < 14 days (all p > 0. 10). There was also no significant correlation between sample absorbance at 414 nm and ΔCT hemolysis (r = 0.22, p = 0.094), therefore, samples were excluded from analysis only if it exceeded criterion on both measures 38 . There were no between-group differences in plasma total RNA concentration, qPCR amplification, or technical performance on focus panels ( Supplementary Fig. S2, all p > 0.10). Table 1. Maternal socio-demographic and medical characteristics. GED graduate equivalency degree, SD standard deviation. °Based on pooled variances t-test. 1 Based on Mann-Whitney test. 2 Based on Chi-square test. 3 Based on Fisher's exact test.  www.nature.com/scientificreports/  Table 4 and include 51 miRNAs that met eligibility criteria for at least one of the two outcomes, pharmacological treatment (n = 35) or LOS ≥ 14 days (n = 41). Among those, 25 miRNAs were predictive of both NOWS outcomes, with a greater mean effect size observed for LOS ≥ 14 days than for pharmacological treatment (0.77 ± 0.19 and 0.64 ± 0.14, respectively, p = 0.008). Ultimately, the final parsimonious models included a signature of three miRNAs which were predictive of both NOWS outcomes (miR-128-3p, miR-30c-5p, miR-421), plus two miRNAs which were predictive of one outcome but not another (i.e., let-7d-5p and miR-584-5p for the need of pharmacologic treatment; let-7b-5p and miR-10b-5p for the prolonged hospitalization).
Predictive validity of the common miRNA signature for identification of both NOWS severity outcomes. Receiver   www.nature.com/scientificreports/ www.nature.com/scientificreports/ (Model 3) also resulted in non-convergence and complete algorithm separation and two additional miRNAs were dropped (let-7b-5p and miR-128-3p). The AUC was slightly lower in Model 3 (0.967), but overall accuracy was 96.6%. As expected, AUC values for the bias-adjusted models were lower, and other model measures were also lower when using the same cutoff for predicted probability (≥ 0.55). Of the five miRNAs predictive of prolonged hospitalization, miR-128-3p and miR-421 were over-expressed and let-7b-5p, miR-10b-5p, and miR-30c-5p were under-expressed in LOS ≥ 14 days neonates compared to LOS < 14 days neonates.

Pathway enrichment analysis of miRNAs that predicted infant outcomes. Pathway enrichment
analysis (for miRNAs [PT,LOS] , the three core miRNAs that predict both outcomes; miRNAs [PT] , the five miRNAs that predict need for pharmacological treatment; and miRNAs [LOS] , the five miRNAs that predict prolonged hospitalization) identified 11 biological pathways, based on Kyoto Encyclopedia of Genes and Genomes (KEGG) classification, that were significantly targeted by miRNAs from all three miRNA prediction groups, 9 pathways were significantly targeted by miRNAs from two of the three miRNA prediction groups, and 7 pathways that were significantly targeted by miRNAs in only one of the miRNA prediction groups (Supplemental Fig. S5). Of the 11 KEGG pathways significantly targeted by all three miRNA prediction groups, 3 were classified as addiction pathways, including morphine addiction.

Discussion
The objective of this study was to identify circulating miRNAs obtained at birth from a readily available resource, infant cord blood, which were capable of risk stratification of opioid-exposed infants with respect to development of NOWS symptoms before those symptoms manifest. This study identified a signature of neonatal circulating miRNAs, detectable in umbilical cord blood plasma collected at birth, which predict severity of NOWS with more than 90% accuracy. Specifically, a panel of five miRNAs identified infants with severe NOWS requiring pharmacologic treatment with 88.2% sensitivity and 95.1% specificity. Similarly, five miRNAs (three overlapping with the predictive model for the need of pharmacologic treatment) accurately identified infants with prolonged hospitalization (93.3% sensitivity and 97.7% specificity). Moreover, even bias-adjusted models, used to reduce the influence of any one observation on model performance, resulted in overall accuracy of over 82%. Inclusion of gestational age at delivery and the type of maternal MOUD provided minimal improvement in the overall predictive performance of the miRNA panel. To our knowledge this is the first study reporting predictive utility of miRNA epigenetic markers for the risk stratification of infants born to women with OUD. The predictive models identified in our cohort consist of multiple miRNAs, consistent with prior reports which indicate that circulating miRNAs have predictive value, and possibly serve biological functions, as a group rather than individually. For instance, we previously reported that miRNAs are altered in infants prenatally exposed to alcohol 38 , and that groups of miRNAs partially mediated effects of prenatal alcohol on infant growth and neurodevelopmental deficits. In another study, we also identified a group of miRNAs elevated at mid-pregnancy in pregnant women with heavy alcohol exposure, whose infants were affected by that exposure, which collectively explained between 24 and 31% of the variation in infant growth parameters at birth 35 . Moreover, these elevated maternal miRNAs collectively impaired fetal and placental growth in pregnant mice, and collectively, but not individually, inhibited growth of human placental cells in vitro, suggesting molecular synergy 37 . We have also found that plasma miRNAs collected from pregnant women were differentially expressed among women with OUD, women with alcohol exposure during pregnancy, and unexposed controls 34 . These data collectively support the hypothesis that plasma miRNAs are affected by prenatal drug exposure and mediate effects of prenatal drug exposure on infant outcomes. Yet, care should be taken against the causal inference between prenatal opioid exposure and changes in miRNA expression, but instead the profile of miRNAs that are predictive of NOWS likely reflect changes across biological systems that contribute to both risk and resilience to the development of NOWS.
Interestingly, while the three miRNAs in the final signature were predictive of both NOWS outcomes, need for pharmacologic treatment and length of stay, two additional miRNAs were identified that were specific for each outcome, suggesting that biological mediators of each outcome are likely to be similar, though not completely overlapping. When miRNA models were restricted to the three miRNAs predictive of both NOWS outcomes, the accuracy, AUC, and PPV were good, but all values improved with the addition of the two outcome-specific miRNAs and sensitivity was greatly improved. In this context, within the larger set of 51 miRNAs which met initial eligibility criteria, the majority (n = 26) were predictive of both outcomes, though outcome-specific miRNAs were also identified. It should be noted that among infants with prolonged hospitalization (n = 15) the majority required treatment (86.7%) and among the 26 miRNAs predictive of both outcomes, effect sizes were larger for prolonged hospitalization. Both the need for pharmacologic treatment and prolonged hospitalizations are the commonly used measures of NOWS severity 39,40 ; however, there is currently, no 'gold standard' definition of NOWS, and substantial variability in clinical scoring tools and treatment protocols 41,42 . NOWS symptoms of maladaptation are highly variable and typically manifest as physiologic and/or behavioral dysregulation in four key domains-state control/attention, motor and tone control, sensory processing, and autonomic control 27 . While we hypothesize that miRNAs predictive of both outcomes might constitute the most robust signature, variability in miRNA expression may be influenced by the dominance of specific NOWS phenotypes, a possibility that requires further investigation.
Previous studies primarily focused on the role of maternal MOUD, polysubstance use, infant sex, gestational age at birth, and use of psychotropic medication on NOWS severity 29,[43][44][45] . A more limited number of studies have evaluated the role of epigenetic factors, such as DNA methylation and miRNA expression. Epigenetic analysis indicates that CpG-rich regions in the promotor for the µ opioid receptor (OPRM1) are hypermethylated following acute exposure to prescription opioids 46  www.nature.com/scientificreports/ therapy 49,50 . Likewise, neonates prenatally exposed to maternal methadone have higher methylation across assessed promotor and exon-1 regions of the OPRM1 gene than non-opioid exposed neonates 51 and increased OPRM1 promoter methylation has been associated with the need for two or more medications for pharmacological intervention for NOWS 52 . miRNAs can also impact OPRM1 expression and, reciprocally, exhibit altered expression due to opioid exposure. Of the candidate miRNAs identified in our study, 13 (25.5%) have been previously implicated as targeting OPRM1 53,54 and 22 (43.1%) have been previously shown to have altered expression following opioid exposure 33,[53][54][55][56][57] . Interestingly, both groups in our study were opioid-exposed, and therefore, any differences in miRNA expression between neonates pharmacologically treated for NOWS and non-treated infants cannot be attributable solely to the activation of opioid signaling pathways by MOUD. Yet, the candidate miRNAs included three members of the let-7 miRNA family, which target OPRM1 53,54,58 and increase morphine tolerance through reduced MOR protein expression 58 . Therefore, these altered miRNAs may alter biological responses to MOUD exposure, including tolerance, thereby exacerbating or prolonging maladaptive responses to MOUD exposure. A recent study examined changes to placental DNA methylation that occur in response to maternal OUD and may be predictive of NOWS development 59 . None of the miRNAs identified in our study (Table 4) had differential DNA methylation at their genomic loci when comparing placenta from POE neonates who would develop NOWS to neonates who did not develop NOWS. One CpG site (cg19782652) in promoter/enhancer regions upstream of let-7e and miR-125b was hypomethylated in POE neonates who develop NOWS when compared to POE-naïve neonates. Interestingly, both let-7e and miR-125b met criteria for inclusion in the model to predict prolonged hospitalization, and both also exhibited decreased expression in neonates with LOS ≥ 14 days. These data indicate that the placenta may play a role in modulating the expression of candidate plasma miRNAs present in umbilical cord blood.
The miRNAs in circulation we identified as predictive of both need for pharmacologic treatment and prolonged hospitalization, miR-421, miR-128-3p, and miR-30c-5p have been previously implicated in modulating inflammatory tone. For instance, miR-421 was increased in human leukocytes following environmental particulate-exposure and its elevation correlated with altered expression inflammatory signaling pathway genes 60 . In human bone mesenchymal cells, miR-128-3p overexpression exacerbated tumor necrosis factor-α-induced pro-inflammatory cytokine release 61 , and overexpression of miR-30c-5p in a human macrophage cell line was associated with decreased release of pro-inflammatory cytokine, IL-1β 62 . Moreover, in our previous work examining infant circulating miRNA expression following prenatal alcohol exposure, miR-30c-5p clustered with a group of miRNAs that targeted the immune system and inflammation and, as a group, partially mediated the effects of prenatal alcohol exposure on infant length and neurodevelopment 38 . POE itself is documented to increase inflammatory cytokines and potentiate response to immune challenges in a rat model 7 . Here, both miR-421 and miR-128-3p were significantly increased, while miR-30c-5p was significantly decreased in cord blood plasma of neonates with worse outcomes which is consistent with a hypothesis that these miRNAs are associated with a pro-inflammatory state that may predict the onset of NOWS.
While miRNAs in the plasma can come from multiple tissues, thereby reflecting the biological state of many different cells, we have previously found that miRNAs within the plasma do act in concert to control biological outcomes 37,38 . Using pathway enrichment analysis, we found that miRNAs in all three prediction groups, miRNA [PT,LOS] , miRNAs [PT] , and miRNAs [LOS], target gene transcripts associated with generalized addiction. Additionally, miRNAs [LOS] target transcripts associated with intestinal barrier integrity and function (e.g. mucin type o-glycan synthesis 63 , extracellular matrix-receptor signaling 64 , amoebiasis 65 , focal adhesion and PI3K-Akt signaling 66 ), and miRNAs [PT] target transcripts associated with immunomodulation (e.g. TGF-β signaling 67 , N-glycan biosynthesis 68 , retrograde endocannabinoid signaling 69 ). These associations require further testing, particularly the pathways targeted could be organ/tissue dependent or may not be the primary site of action for the altered miRNAs. Furthermore, we do not as yet have enough information to understand the biology of these miRNAs. However, previous studies found that some miRNAs that are elevated in disease pathologies are protective while others are maladaptive 37 and we expect the same outcome is likely to be true with NOWSpredictive miRNAs.
From a clinical perspective, risk stratification in infants with prenatal opioid exposure before manifestation of NOWS symptoms is of great importance. Per national guidelines, infants born to women who used opioids during pregnancy are monitored for at least 96 h in the hospital for signs of NOWS 15 . Current clinical tools guiding treatment decisions rely on the presence of withdrawal signs, which typically manifest by day 4 and are common on days 1 and 2 after birth, and are thus reactive in nature. Proactive identification of low-risk infants with high degree of accuracy will allow clinicians to shorten observation periods. Shorter hospitalization will not only result in healthcare cost savings but will improve maternal-infant bonding during the first days after birth-a crucial protective factor for long-term neuro-behavioral outcome in substance-exposed infants 70,71 . Identification of newborns at lower risk for needing pharmacological treatment might allow rural and community hospital to keep more of these infants in their postpartum units avoiding costly and socially disruptive transfer of these infants to tertiary care hospitals. A 2017 national survey demonstrated that while observation for NOWS is common in Level 1 nurseries, 87% of the Level 1 nurseries transferred to NICUs when pharmacological treatment was indicated 72 . On the other hand, identification of high-risk infants by the rapid laboratory assessment tool within the first hours of life will inform decisions about most appropriate clinical monitoring and access to care (e.g., timely transfer of high-risk infants from rural/underserved areas to tertiary care facilities) as well as supporting proactive intervention approaches. Increased attention to non-pharmacological interventions for high-risk infants including additional breastfeeding support when appropriate, and consideration of a lower threshold for pharmacological treatment are potential responses for infants identified as high risk if the miRNA results may be obtained within the first few days of life. www.nature.com/scientificreports/ Several strengths of the study also should be mentioned. First, a prospective cohort design allowed for collection of detailed information on substance use (MOUD, illicit drugs, alcohol, tobacco) by repeated prospective interviews and urine drug panels. Secondly, miRNAs were assayed non-invasively and using tissues that would otherwise be waste tissues. Thirdly, miRNA assessment by qPCR is a transferrable technology that is already employed in clinical settings. Moreover, this screen identified a number of abundant plasma miRNAs that can be further investigated for their role as mediators of adaptive biology, as we have reported for other adverse pregnancy outcomes 37 . Fourth, we examined the effect of the strongest confounders-maternal type of MOUD and gestational age at delivery, as well as applied a bias reduction approach in statistical modelling when estimating validity indices of signature miRNAs. Fifth, our cohort included a high percentage of Latinx population, reflecting the general population of New Mexico, addressing an unmet need to better understand the impact of prenatal MOUD exposure and NOWS in the Latinx community which has historically been underrepresented in studies of this nature.
Our findings should be weighed within the context of several study limitations. First, the sample size is relatively modest, and study findings will require validation in other population. Study findings can only be generalized to infants prenatally exposed to MOUD (with or without other opioids), while prenatal exposure to other opioids without MOUD may result in a different circulating miRNA signature. Moreover, further validation studies are needed to characterize the impact of maternal MOUD type, i.e., buprenorphine or methadone, on the accuracy of miRNAs in NOWS risk stratification. Second, polysubstance use is a common phenomenon in women with MOUD, and while every effort has been made to carefully control for the effect of co-exposures (e.g., exclusion of stimulant co-exposures, careful quantification of other substances), residual confounders cannot be ruled out. It should be noted though, that the prevalence of co-exposures was similar among the study groups, though types of co-exposures and methods of opioid use may impact the miRNA profile and biomarker sensitivity and specificity. Future larger studies are warranted in populations with polysubstance use to assess the stability of the miRNAs identified here in those populations especially in the context of prevalent co-exposures, such as tobacco 73,74 , and the rising use of methamphetamines among pregnant women with opioid use disorder 75 . Third, similar to other studies in the field 76,77 , we have excluded infants born before 34 weeks of gestation, but included 6 preterm infants (two at 34 weeks, four at 36 weeks). The length of hospitalization is particularly sensitive to gestational age at delivery, but here we found that for these 6 infants, outcomes were still variable, as two did not receive pharmacological treatment and one had a length of stay < 14 days. Additionally, there is increasing recognition that gestational age, while being the most commonly adjusted for covariate in obstetrics and perinatal epidemiology, might be causally linked to outcomes [78][79][80] . Therefore, analyses were conducted before and after adjustment for gestational age. However, models with adjustment for gestational age, and additional adjustment for type of maternal MOUD exposure, performed only slightly better than models without. Accuracy of the prolonged hospitalization model using miRNA alone was 96.6% and inclusion of gestational age and MOUD resulted in a surplus of predictive information as indicated by the lack of model convergence. We also recognize that some signs of prematurity and NOWS (e.g. respiration difficulties, poor feeding) might be hard to distinguish clinically, and common epigenetic mechanisms might play in role in both conditions. Future studies should examine the predictive ability of miRNAs in larger samples of full-term, late preterm, and early preterm infants. Fourth, while miRNAs were assayed using clinically-relevant qPCR technology, there are advantages to performing genome-wide RNA sequencing, including the quantification of the entire miRNA transcriptome and the identification of splice variants. Therefore, additional work using sequencing to identify miRNA biomarkers could provide additional insight into miRNAs that are predictive of NOWS.
In summary, this is the first proof-of-principle report laying out the foundation for the utilization of miRNAs derived from umbilical cord at birth for risk stratification of opioid-exposed infants. While validity indices of the identified miRNA panel are very encouraging, results should be interpreted with caution before validation in different study populations. Potentially, miRNA panels might need to be developed for different patient subpopulations with scoring algorithms taking into considerations results of miRNA analysis and key clinical indicators (e.g., gestational age at delivery, maternal exposures, known comorbidities).The ability to identify infants with MOUD exposure that are at lower and high risk for prolonged LOS and pharmacological treatment can result in improved utilization of health care resources and minimize maternal-infant separation that often accompanies transfer to tertiary care hospitals.

Methods
Study design and population. This analysis used a study population derived from the 'Ethanol, Neurodevelopment, Infant and Child Health' (ENRICH) prospective cohort study, with recruitment and sample collection from March 2015 to November 2019. The primary objective of the parent ENRICH study was to identify early indices of functional brain damage associated with prenatal alcohol exposures 5,6,81 . In addition to unexposed controls, patients receiving MOUD were recruited as another comparison group due to similarities in pre-and post-natal environment between substance-using populations. The cohort was formed by recruiting pregnant women during one of the first prenatal care visits. Children born to cohort participants were followed for up to 20 months of age. The ENRICH study included 4 prospective visits, and data obtained from Visit 1 (prenatal) and Visit 2 (delivery/birth) were used in this analysis. The general eligibility criteria for the ENRICH study were: (1) ≥ 18 years old; (2) singleton pregnancy confirmed by ultrasound; (3) no fetal diagnosis of a major structural anomaly; (4) no more than minimal use of cocaine, crack-cocaine, MDMA (ecstasy), or methamphetamine use (per repeated prospective self-reports and repeated urine drug screen (UDS) tests). Minimal use is defined as no more than 1 urine drug test or incidence of self-report during the first trimester and abstention from use of these substances during the second and third trimesters 5 . For this analysis, the following additional eligibility criteria were employed: (1) participants recruited into MOUD group; (2) no self-reported alcohol use in the www.nature.com/scientificreports/ third trimester and no more than one positive ethanol biomarker in a comprehensive panel (described below); (3) gestational age at delivery ≥ 34 weeks; (4) available umbilical cord blood sample. Human subjects' approval was obtained from the University of New Mexico Human Research Review Committee and Texas A&M University Institutional Review Board. All participants provided written informed consent. All procedures were followed according to the relevant guidelines.

Characterization of maternal MOUD and other prenatal factors. The MOUD group was recruited
from the University of New Mexico Milagro Clinic, an interdisciplinary clinic specializing in treating pregnant women with substance use disorders. The objective was to identify miRNAs capable of risk stratification of opioid-exposed infants with respect to development of NOWS symptoms rather than to identify miRNAs associated with prenatal opioid exposure. Therefore, the current study did not include unexposed infants. The type of MOUD (methadone vs. buprenorphine) and dose before delivery were abstracted from electronic medical records. Co-exposure with illicit drugs was assessed throughout pregnancy (timing and frequency) by prospective repeated interviews, based on the 2011 National Survey on Drug use and Health 82 . Self-reported information was verified by study-specific Urine Testing Drug Panel-7 (UDS-7; US Drug Testing Lab, Des Plaines, IL) conducted at Visit 1 and Visit 2. The UDS-7 panel measured amphetamines, barbiturates, benzodiazepines, cocaine, opiates, PCP, and cannabinoids/THC metabolites. Alcohol use was captured by three timeline followback (TLFB) interviews which captured use a month around conception, 30 days before enrollment, and 30 days before delivery. Quantity and frequency of reported alcohol at each day were converted into the ounces of absolute alcohol per day (AA/day 83

NOWS severity measures, infant and postnatal environment measures. The primary outcomes
of interest were to identify miRNAs that predict: (1) which opioid-exposed neonates will be Pharmacologically-Treated, compared to neonates Not-Pharmacologically-Treated, and (2) which opioid-exposed neonates will have an extended length of hospital stay, defined as a LOS ≥ 14 days compared to neonates with LOS < 14 days. These measures are widely used in clinical studies for assessment of NOWS severity 42,87,88 . According to the University of New Mexico Hospital (UNMH) guidelines, pharmacological treatment of NOWS was initiated if an infant scored ≥ 8 on the Finnegan Neonatal Abstinence Scoring Tool 89 on three consecutive assessments, if the mean of three scores is ≥ 8, or if two consecutive scores are ≥ 12 90 , or was initiated based on the Eat, Sleep, and Console evaluation model. Newborns are scored at admission (~ 2 h of age). All infants with confirmed or suspected prenatal opioid exposure remain inpatient at UNMH for a minimum of 96 h.
Circulating miRNA analyses. Umbilical cord blood collection and RNA isolation. Immediately following delivery, blood samples were collected from umbilical cord in EDTA-coated tubes. Plasma was separated by centrifugation at 2000g for 10 min, collected in 200 µL aliquots, placed at − 80 °C, shipped to Texas A&M University on dry ice, and stored at − 80 °C until processing. Total RNA was isolated from 125 to 200 µL of plasma using the miRNeasy mini kit (Qiagen Sciences, Germantown, MD) with the addition of 1.2 µg carrier MS2 phage RNA (Roche Diagnostics, Mannheim, Germany), according to manufacturer's protocols. RNA was eluted using 20 µL nuclease-free water and stored at − 80 °C until cDNA synthesis. cDNA synthesis and qPCR. cDNA was synthesized from 6.25 ng RNA, diluted to 5 ng/µL using nuclease free water, in a 10 µL cDNA reaction mixture using the miRCURY LNA RT Kit (Qiagen Sciences, Germantown, MD). At the time of cDNA synthesis, spike-in miRNAs cel-miR-39-3p and UniSp6 (RNA Spike-In Kit, for RT, Qiagen Sciences, Germantown, MD) were added to the reaction mixture per the manufacturer's directions to allow for the detection of potential RT and PCR inhibitors. cDNA was diluted 110× and combined with an equal amount of 2× miRCURY SYBR Green Master Mix (Qiagen Sciences, Germantown, MD). Two independent samples were loaded onto the 384-well miRCURY LNA miRNA Human Serum/Plasma Focus miRNA PCR Panels (YAHS-106Y, Qiagen Sciences, Germantown, MD) and qPCR was performed on the 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). Two additional proof-of-principle samples had been previously assessed using miRCURY LNA Human miRNome panels v3. miRNA expression values were extracted (1) for primers which were unchanged across panel versions and (2) primers that were altered across versions but had comparable amplification to the Focus panel Supplementary information. Cycle threshold (CT) and target amplification were assessed using SDS 2.4 software (Applied Biosystems/Thermo Fisher Scientific Waltham, MA). We used qPCR to assay plasma miRNA expression as it has high specificity and sensitivity compared to other methods of miRNA analysis and is considered the 'gold standard' technique to validate sequencing and microarray studies [91][92][93] . We have previously found excellent correlation of expression of miRNAs with assay replication in the same biological samples, indicating the reliability of this qPCR protocol 94 . Assessment of erythrocyte contamination of plasma RNA. Plasma samples were assayed to assess potential erythrocyte contamination as previously described 35,36,38 , since lysed erythrocytes are a contaminating source of miRNAs. Spectrophotometric analysis of plasma was used to detect the presence of free hemoglobin, with absorbance at 414 nm > 0.3 as an indicator of possible hemolysis. Following RNA isolation, expression of erythrocyte-enriched transporter SLC4A1/BAND3 was assessed using the qScript cDNA Synthesis Kit (Quantabio, Beverly, MA) and PerfeCTa SYBR Green FastMix (Quantabio, Beverly, MA), and enrichment of erythrocyte www.nature.com/scientificreports/ miRNA was determined by comparing the expression of erythrocyte-enriched miR-451 to the expression of miR-23a which is stably-expressed in plasma (ΔCT hemolysis = CT miR-23a − CT miR-451 ) 95 . A ΔCT hemolysis of > 7 has been found to be a positive indicator of hemolysis 95 . In samples where SLC4A1/BAND3 was below the limit of detection for qPCR, we used absorbance at 414 nm and ΔCT hemolysis in tandem as an additional indicator of hemolysis due to previous findings that in infants absorbance alone is not a reliable hemolysis marker 38 , likely due to the conversion of fetal to adult hemoglobin.
Data analysis. Exclusion of cord blood samples. 66 umbilical cord samples were collected from infants with prenatal maternal MOUD exposure. One sample was excluded due to insufficient volume available for analysis. Six were excluded due to potential hemolysis, with three samples positive for the erythrocyte transporter SLC4A1/BAND3 and three samples that were SLC4A1/BAND3 negative but positive for both the additional hemolysis markers absorbance at 414 nm (> 0.3) and a ΔCT hemolysis (> 7; see Supplementary Fig. S2). miRNA expression in the two proof-of-principle samples was compared miRNA expression for all Focus panel samples. One proof-of-principle sample was determined to be an outlier based on principal component analysis of miRNA expression and was excluded. Resultant sample size of 58 subjects was used in the analysis.
Data processing and modifications. CTs were assessed for each miRNA. CT values were normalized to the sample global mean of expression for all detected miRNAs (ΔCT) for each sample. We further examined miR-NAs which were expressed in > 80% of samples within a group. miRNAs with undetected CTs were considered expressed below the detectable limit and, therefore, were assigned at CT value 1 unit above the highest CT for that miRNA, as assigning these values has been shown to reduce bias 96 . Comparisons of normalized miRNA expression (ΔCT) between outcome groups is denoted as ΔΔCT and 1 ΔΔCT approximates a two-fold change in expression between groups. Out of 179 assessed miRNAs, 171 miRNAs were expressed in > 80% of the samples, had consistent amplification across panels, and were further analyzed.
Pathway enrichment analysis. We performed pathway enrichment analysis to determine possible biological pathways that are targeted by three clusters of miRNAs: miRNAs [PT,LOS] , the three core miRNAs that predict both outcomes; miRNAs [PT], the five miRNAs that predict need for pharmacological treatment; and miRNAs [LOS] , the five miRNAs that predict prolonged hospitalization. Using DIANA-mirPath v3.0 97 , mRNA targets of the selected groups of miRNAs were determined using the in silico target prediction tool DIANA-microT-CDS v5.0 and pathways were identified that had significant overrepresentation for targeted mRNA transcripts. We used the KEGG analysis pipeline with the following parameters: microT threshold of 0.8, p-value > 0.01; and FDR correction. Briefly, this tool uses Fisher's exact test and unbiased empirical distributions to determine if a miRNA has enriched pathway targeting compared to other annotated miRNAs and, subsequently, uses meta-analysis statistics to determine the pathways targeted by a set of miRNAs 97  Predictive models for each of the outcomes were constructed by including miRNAs meeting the following criteria: (1) d > 0.40 for mean difference in miRNA expression in unadjusted analyses; (2) p < 0.10 for the differences in miRNA expression. The final 'miRNA signature' parsimonious models were developed for each outcome using step-wise logistic regression (Model 1) 101 . Firth's penalized likelihood approach was used to determine model predicted probabilities as it controls for potential regression model lack of convergence, or separation, related to the small sample size 102,103 . In the event that complete separation of the model still occurred (indicating an overspecified model or not enough data given the predictors), the least significant miRNA was dropped from the model 104 .
Since the sample size precluded having separate algorithm development and validation models, a second model applied a bias reduction method that estimated the predicted probability for a given observation by approximating the exclusion of that given observation 105 . This adjustment to model fit is provided as 'biasadjusted' estimates. ROC curves were constructed and AUC was estimated. Final predictive models were adjusted for (1) gestational age (Model 2) and (2) gestational age and type of maternal MOUD (Model 3). A predicted probability cut-off point was selected optimizing sensitivity and specificity 106 . Sensitivity, specificity, PPV, and NPV and associated 95% confidence intervals (95% CI) are reported. All statistical analyses were conducted using R 107 and SAS 9.4 (Cary, NC).

Data availability
Mean expression difference, effect size, and significance for all miRNAs analyzed for this study are included in the supplementary information files (see Supplementary Table S3). Raw miRNA qPCR amplification data generated and/or analyzed during the current study are not publicly available due the lack of data sharing acknowledgement of de-identified data in the consent and IRB protocol. The request for data sharing can be considered on the case-by-case basis with a formal data sharing agreement between institutions.