[This retracts the article DOI: 10.1016/j.jvssci.2020.09.002.].

Background Spastic cerebral palsy, the most common pediatric-onset disabling condition with an estimated prevalence of 0.2% in children, is a complex condition characterized by stiff movement, muscle contractures, and abnormal gait that can diminish quality of life. Spastic CP accounts for approximately 83% of all CP cases and frequently co-occurs with other complex conditions, like epilepsy. An estimated 42% of spastic CP cases have co-occurring epilepsy. Unfortunately, CP is often difficult to diagnose. Although most children with CP are born with it or acquire it immediately after birth, many are not identified until after 19 months of age with CP diagnosis often not confirmed until 5 years of age. New bioinformatic approaches to identify CP earlier are needed. Recent studies indicate that altered DNA methylation patterns associated with CP may have diagnostic value. The potential confounding effects of co-occurrent epilepsy on these patterns are not known. We evaluated machine learning classification of CP patients with or without co-occurring epilepsy. Results Whole blood samples were collected from 30 study participants diagnosed with epilepsy (n=4), spastic CP (n=10), both (n=8), or neither (n=8). A novel Support-Vector-Machine learning algorithm was developed to identify methylation loci that have ability to classify CP from controls in the presence or absence of epilepsy. This algorithm was also employed to measure classification ability of identified methylation loci. After preprocessing of data, isolation of important methylation loci was performed in a binary comparison between CP and controls, as well as in a 4-way scheme, encapsulating epilepsy diagnoses. The classification ability was similarly assessed. CP Classification performance wasevaluated with and without inclusion of epilepsy as a feature. Median F1 scoreswere 0.67 in 4-class comparison, and 1.0 in the binary classification, outperforming Linear-Discriminant-Analysis (0.57 and 0.86, respectively). Conclusion This novel algorithm was able to classify study participants with spastic CPand/or epilepsy from controls with significant performance. The algorithm shows promise for rapid identification in methylation data of diagnostic methylation loci. In this model, Support Vector Machines outperformed Linear Discriminant Analysis in classification. In the evaluation of epigenetics-based diagnostics for CP, epilepsy may not be a significant confounding factor.

Cerebral palsy (CP) is a pediatric onset disorder with poorly understood molecular causes and progression, making early diagnosis difficult. Circular RNAs are regulatory RNAs that show promise as biomarkers in various diseases but the role of circular RNAs in CP is beginning to be understood. This study identified the role of circNFIX in regulating the expression of myocyte-specific enhancer factor 2C (MEF2C), an important transcription factor for sarcomere development. We found that circNFIX is downregulated in the muscle cells of individuals with CP, and its localization shifts toward the nucleus as visualized using single-molecule resolution imaging. The decreased expression of circNFIX, MEF2C, and MEF2C targets persisted throughout myoblasts to myotubes differentiation, and in the skeletal muscle tissue. Bioinformatic and experimental validation confirmed that circNFIX acts as a sponge for miR373-3p, a microRNA that represses MEF2C translation. In normal muscle, circNFIX derepresses MEF2C translation by sponging miR3733p, allowing for normal sarcomere generation. In CP, reduced circNFIX expression results in loss of miRNA sponging, leading to lower MEF2C expression and downregulation of sarcomere genes, potentially causing shortened and dysfunctional muscle fi bers. Knockdown (KD) of circNFIX reduced myogenic capacity of myoblasts to fuse and form myotubes similar to CP cells evident from the lower fusion index in CP and KD as compared to control myotubes. This is the fi rst study reporting reduction of MEF2C in CP and single-molecule resolution imaging of circNFIX's subcellular distribution and its role in CP, suggesting circNFIX as a potential therapeutic target and biomarker for early CP diagnosis.

Spastic cerebral palsy (CP) is a common pediatric-onset disability with an estimated prevalence of 0.2%. It is a complex condition characterized by muscle stiffness, contractures, and abnormal movement. Spastic CP is difficult to diagnose. Although nearly all affected children are born with it or acquire it immediately after birth, many are not identified until after 19 months of age with the diagnosis often not confirmed until 5 years of age. In addition, CP frequently co-occurs with other complex conditions that can complicate diagnosis and treatment. For example, an estimated 42% of spastic CP cases have co-occurring epilepsy. Recent studies indicate that altered DNA methylation patterns in peripheral blood cells are associated with CP and may have diagnostic value. Accordingly, the purpose of this study is to assess the diagnostic value of methylation in CP with more complex disease states. We evaluated machine learning classification for detecting CP based on DNA methylation pattern analysis in the context of co-occurrent epilepsy. Blood samples from 30 study participants diagnosed with epilepsy (n=4), spastic CP (n=10), both (n=8), or neither (n=8) were analyzed by Illumina MethylationEpic arrays. A novel machine learning algorithm using a Support Vector Machine (SVM) or Linear Discriminant Analysis (LDA) was developed to identify methylation loci that classified CP from controls and to measure the classification ability of identified methylation loci. The isolation of informative methylation loci was performed in a binary comparison between CP and controls, as well as in a 4-way comparison that included epilepsy. Median F1 scores for SVM-based analysis were 0.67 in 4- class comparison, and 1.0 in the binary classification. SVM outperformed LDA (median F1 0.57 and 0.86, respectively). Overall, the novel machine learning based algorithm was able to classify study participants with spastic CP and/or epilepsy from controls with significant performance.

BACKGROUND Higher neighborhood deprivation is associated with hypertension diagnosis in youth. In this study, we assess if there is an association between neighborhood deprivation and anti-hypertensive therapy prescription among insured youth with a primary hypertension diagnosis. METHODS Using a retrospective cross-sectional design, we assessed the proportion of youth with a diagnosis of primary hypertension prescribed anti-hypertensive therapy. We evaluated the proportion of youth prescribed anti-hypertensive therapy and compared prescribing patterns by area deprivation index, age, sex, obesity diagnosis, race, ethnicity, and duration of Medicaid coverage. RESULTS Of the 65452 non-pregnant Delaware Medicaid recipients, 8 to 18 years of age, 1145 (1.7%) had an ICD-9/ICD-10 diagnosis of primary hypertension; 165 of the 1145 (14%) were prescribed anti-hypertensive therapy. Factors associated with a greater odds of prescription by multivariable logistic regression were age, obesity diagnosis, and duration of Medicaid full benefit coverage. Odds of anti-hypertensive therapy prescription did not vary by race, ethnicity or by area deprivation index. CONCLUSIONS Anti-hypertensive therapy prescription rates are poor despite national guideline recommendations. Among youth receiving Delaware Medicaid between 2014 and 2019, prescription proportions were highest among youth of older age, with an obesity diagnosis, and among youth with longer duration of Medicaid benefit coverage. Although high area deprivation has been shown to be associated with the diagnosis of hypertension, high vs low area deprivation was not associated with greater anti-hypertensive therapy prescription among youth with primary hypertension. Our finding of a mismatch between hypertension diagnosis and anti-hypertensive therapy prescription highlights a potential disparity in anti-hypertensive therapy prescription in youth.

Introduction: AHA “Life’s Essential 8” scores cardiovascular health (CVH) as good, intermediate or poor based on diet, physical activity, sleep, tobacco exposure, cholesterol, blood pressure, blood glucose, and body mass index. CVH of the US population remains suboptimal, and the relationship between neighborhood deprivation and CVH in youth has not been fully explored. Hypothesis: We hypothesize that high neighborhood deprivation is associated with intermediate/poor CVH in adolescents. Methods: Using 2007-2018 National Health and Nutrition Examination Survey data, we examined the relationship between neighborhood deprivation index (NDI) and CVH status in adolescents. NDI was created from 2010 US Census data and reflects neighborhood level income, housing, education, and poverty status. Higher NDI indicates greater neighborhood deprivation. NDI values were divided into tertiles for analysis. We used proc survey logistic to assess the odds of poor/intermediate CVH status among youth living within high and intermediate compared to low deprivation communities. The model was adjusted for age, sex, ethnicity-race, household reference education and marital status, family poverty, rural vs urban residence and Citizenship status. All analyses were conducted using SAS 9.4, p<0.05 . Results: We evaluated data from 3894 individuals, representative of 57,195,556 US adolescents 13 to 18 years of age. Most adolescents were non-Hispanic white (67%), male (51%), US Citizens (96%), mean age of 15 years, with an even distribution of youth living within low, intermediate and high deprivation neighborhoods (Table 1). We identified 31% greater odds of intermediate/poor CVH status among youth living within the most deprived communities (Table 2). Conclusions: Greater NDI associates with worse CVH in US adolescents. Public health initiatives should consider using NDI to tailor efforts in designing intervention strategies to preserve CVH status in US adolescents.

Hypertension (HTN) is a primary cause of cardiovascular disease (CVD) and begins in youth. Among adults, low socioeconomic status (SES) is a demonstrated risk factor for HTN. Low SES has been associated with increased sodium intake and obesity. In this study, we assess the impact of family income, obesity and sodium intake during adolescence on average systolic blood pressure (SBP) to determine if these factors are associated with HTN and elevated BP during adolescence. Methods: Using 2007-2018 National Health and Nutrition Examination Survey data, representative of the non-institutionalized civilian population 13 to 18 years of age, we employed multivariable logistic regression (proc survey logistic) to assess the relationship between abnormal BP diagnosis (HTN or elevated BP), family income, obesity, and sodium intake. Poverty was defined as a family income less than 5-times the poverty level. Sodium intake was estimated based upon a combined 1-day total dietary and 1-day diet supplement recall and was adjusted for total caloric intake per day. The upper quartile of adjusted sodium intake was used to distinguish high from low sodium intake. Abnormal BP was defined as an average systolic blood pressure (SBP) at or above 120mmHg. Multivariable logistic regression was carried out. Results: 2.4% and 13% of the adolescent population had average SBP at or above 130mmHg (HTN) or at or above 120mmHg (elevated BP), respectively. According to multivariable logistic regression, predictors of the HTN/Elevated BP were overweight status (OR: 2.4, 95%CI: 1.8, 3.2), obesity (OR: 3.6 95%CI: 2.8, 4.7), male sex (OR:3.6, 95%CI: 2.7, 4.9), non-Hispanic black race (OR: 1.8, 95%CI: 1.2, 1.4) and age (OR: 1.3, 95%CI: 1.2, 1.4). Poverty status (OR: 1.2, 95%CI: 0.74, 1.9), Hispanic ethnicity (OR 1.2, 95%CI: 0.87, 1.5), and sodium intake (OR: 1.1, 95%CI: 0.86, 1.4) were not associated with HTN/elevated BP. Conclusion: While SES factors contribute to abnormal BP, sodium intake and family income were not associated with HTN/elevated BP in adolescents using a single day dietary intake recall. According to our results, additional SES factors contribute to HTN/elevated BP in adolescents and should be further explored.

AIM:To determine the dose-response relationship of collagenase Clostridium histolyticum (CCH) on collagen content and the change in muscle fiber bundle stiffness after ex vivo treatment of adductor longus biopsies with CCH in children with cerebral palsy (CP).METHOD:Biopsy samples of adductor longus from children with CP (classified in Gross Motor Function Classification System levels IV and V) were treated with 0 U/mL, 200 U/mL, 350 U/mL, or 500 U/mL CCH; percentage collagen reduction was measured to determine the dose-response. Peak and steady-state stresses were determined at 1%, 2.5%, 5%, and 7.5% strain increments; Young's modulus was calculated.RESULTS:Eleven patients were enrolled (nine males, two females, mean age at surgery 6 years 5 months; range: 2-16 years). A linear CCH dose-response relationship was determined. Peak and steady-state stress generation increased linearly at 5.9/2.3mN/mm2 , 12.4/5.3mN/mm2 , 22.2/9.7mN/mm2 , and 33.3/15.5mN/mm2 at each percentage strain increment respectively. After CCH treatment, peak and steady-state stress generation decreased to 3.2/1.2mN/mm2 , 6.5/2.9mN/mm2 , 12.2/5.7mN/mm2 , and 15.4/7.7mN/mm2 respectively (p < 0.004). Young's modulus decreased from 205 kPa to 100 kPa after CCH (p = 0.003).INTERPRETATION:This preclinical ex vivo study provides proof of concept for the use of collagenase to decrease muscle stiffness in individuals with CP.

Importance:The association between degree of neighborhood deprivation and primary hypertension diagnosis in youth remains understudied. Objective:To assess the association between neighborhood measures of deprivation and primary hypertension diagnosis in youth. Design, Setting, and Participants:This cross-sectional study included 65 452 Delaware Medicaid-insured youths aged 8 to 18 years between January 1, 2014, and December 31, 2019. Residence was geocoded by national area deprivation index (ADI). Exposures:Higher area deprivation. Main Outcomes and Measures:The main outcome was primary hypertension diagnosis based on International Classification of Diseases, Ninth Revision and Tenth Revision codes. Data were analyzed between September 1, 2021, and December 31, 2022. Results:A total of 65 452 youths were included in the analysis, including 64 307 (98.3%) without a hypertension diagnosis (30 491 [47%] female and 33 813 [53%] male; mean [SD] age, 12.5 (3.1) years; 12 500 [19%] Hispanic, 25 473 [40%] non-Hispanic Black, 24 565 [38%] non-Hispanic White, and 1769 [3%] other race or ethnicity; 13 029 [20%] with obesity; and 31 548 [49%] with an ADI ≥50) and 1145 (1.7%) with a diagnosis of primary hypertension (mean [SD] age, 13.3 [2.8] years; 464 [41%] female and 681 [59%] male; 271 [24%] Hispanic, 460 [40%] non-Hispanic Black, 396 [35%] non-Hispanic White, and 18 [2%] of other race or ethnicity; 705 [62%] with obesity; and 614 [54%] with an ADI ≥50). The mean (SD) duration of full Medicaid benefit coverage was 61 (16) months for those with a diagnosis of primary hypertension and 46.0 (24.3) months for those without. By multivariable logistic regression, residence within communities with ADI greater than or equal to 50 was associated with 60% greater odds of a hypertension diagnosis (odds ratio [OR], 1.61; 95% CI 1.04-2.51). Older age (OR per year, 1.16; 95%, CI, 1.14-1.18), an obesity diagnosis (OR, 5.16; 95% CI, 4.54-5.85), and longer duration of full Medicaid benefit coverage (OR, 1.03; 95% CI, 1.03-1.04) were associated with greater odds of primary hypertension diagnosis, whereas female sex was associated with lower odds (OR, 0.68; 95%, 0.61-0.77). Model fit including a Medicaid-by-ADI interaction term was significant for the interaction and revealed slightly greater odds of hypertension diagnosis for youths with ADI less than 50 (OR, 1.03; 95% CI, 1.03-1.04) vs ADI ≥50 (OR, 1.02; 95% CI, 1.02-1.03). Race and ethnicity were not associated with primary hypertension diagnosis. Conclusions and Relevance:In this cross-sectional study, higher childhood neighborhood ADI, obesity, age, sex, and duration of Medicaid benefit coverage were associated with a primary hypertension diagnosis in youth. Screening algorithms and national guidelines may consider the importance of ADI when assessing for the presence and prevalence of primary hypertension in youth.

CircRNAs are a category of regulatory RNAs that have garnered significant attention in the field of regulatory RNA research due to their structural stability and tissue-specific expression. Their circular configuration, formed via back-splicing, results in a covalently closed structure that exhibits greater resistance to exonucleases compared to linear RNAs. The distinctive regulation of circRNAs is closely associated with several physiological processes, as well as the advancement of pathophysiological processes in several human diseases. Despite a good understanding of the biogenesis of circular RNA, details of their biological roles are still being explored. With the steady rise in the number of investigations being carried out regarding the involvement of circRNAs in various regulatory pathways, understanding the biological and clinical relevance of circRNA-mediated regulation has become challenging. Given the vast landscape of circRNA research in the development of the heart and vasculature, we evaluated cardiovascular system research as a model to critically review the state-of-the-art understanding of the biologically relevant functions of circRNAs. We conclude the review with a discussion of the limitations of current functional studies and provide potential solutions by which these limitations can be addressed to identify and validate the meaningful and impactful functions of circRNAs in different physiological processes and diseases.

Introduction: Hypertension (HTN) begins in youth and accounts for ~80% of the cardiovascular disease burden in the US. Socio/environmental factors are critical, and greater knowledge of the relationship between neighborhood level deprivation and blood pressure (BP) in youth is needed. Hypothesis: We hypothesized that greater neighborhood deprivation is associated with intermediate/poor BP status in adolescents even after adjusting for other factors. Methods: Data from 2009 to 2019 were extracted from PEDSnet (a PCORI-funded aggregate of pediatric health data). We modeled the relationship between BP and neighborhood deprivation based on national Area Deprivation index (ADI; scaled 1-100) tertiles (ADI<25=best/low, 25-52, > 52=worst/high). The BP status of each youth was coded as good (BP score: 80-100) or intermediate/poor (BP score: 0-80) where BP score=100 if median BP <120/80mmHg and score=75 if median BP 120-129/<80mmHg. Analyses were carried out using SAS 9.4 with p<0.05 considered significant. The outcome of interest was poor/intermediate BP score. Results: Data from 122,177 youth, 13-17 years of age were analyzed. 51% were female, 58% were non-Hispanic white, and 22% non-Hispanic Black. 24% lived in neighborhoods with ADI <25; 35% lived in neighborhoods with ADI > 52. In a multivariate logistic model, ADI > 52 was associated with a 49% greater odds of intermediate/poor BP score. Due to significant interactions between ethnicity-race and ADI, compared to non-Hispanic Black youth, non-Hispanic White and Other youth had lower odds of intermediate/poor BP score by univariate analysis, but greater odds in the full model (Table). Female sex was associated with lower odds of HTN. The model’s goodness of fit was strong with a ROC, AUC of 0.73 (Figure). Conclusions: Higher neighborhood deprivation is associated with greater odds of hypertension in adolescents. Greater emphasis on measures to mitigate this risk are needed.

Abstract Institutional Development Award (IDeA) programs build research infrastructure in regions with historically low access to NIH funds. The Mentored Research Development Award (MRDA), a professional development program embedded in our IDeA-funded center, provides junior investigators with mentorship and effort offset to write a grant. We evaluated outcomes from the first eight years (2013–2021; N = 55) using administrative records, publicly available data, and a self-report survey (n = 46, 84% response rate). Fifteen MRDA recipients (27%) went on to receive NIH funding. Providing just-in-time grant-writing support may launch early career clinician-scientists in an IDeA state context.

Introduction: Preservation of cardiovascular health (CVH) across the lifespan is essential to reducing cardiovascular disease burden. Greater knowledge of the relationship between neighborhood level deprivation and CVH in youth is needed. Hypothesis: We hypothesized that worse socio/environmental deprivation is associated with poor/intermediate CVH status in adolescents. Methods: Data from 2009-2019 were extracted from PEDSnet (a PCORI-funded network of pediatric health data). We modeled the relationship between CVH and neighborhood deprivation. National Area Deprivation Index values (ADI; scaled 1-100) were coded into PEDSnet and divided into tertiles (ADI<25=best, 25-52, > 52=worst) for analysis. CVH status was scored from a subset of available AHA Life’s Essential 8 (LE8) scores including blood pressure, blood glucose, blood cholesterol, body mass index, smoking/tobacco exposure, and sleep. Overall CVH scores were derived as the average of the sum of the individual scores. Univariate and multivariate analyses were performed. SAS 9.4 was used ( p<0.05) . Results: Data from 122,177 youth, 13-17 years old were analyzed. 51% were female, 24% lived in neighborhoods with ADI <25; 35% lived in neighborhoods with ADI > 52. 58% were non-Hispanic white and 22% non-Hispanic Black. According to a multivariate logistic model, ADI > 52 was associated with a 48% greater odds of poor/intermediate CVH status compared to ADI <25 (Figure). Female sex, ethnic-race categories of Non-Hispanic White and Other, as well private or other forms of non-public insurance coverage were associated with a lower odds of intermediate/poor CVH (Table). Interaction terms for age, ADI, race and ethnicity were not significant. Conclusions: Higher neighborhood deprivation is associated with poor/intermediate CVH status. We recommend greater attention to preserving CVH status within high deprivation communities to preserve and promote CVH status across the lifespan.

Introduction: Identification of factors associated with preservation of cardiovascular health (CVH) during adolescence may inform strategies to reduce CV disease burden in adulthood. Hypothesis: CVH status is worse in later (16-18 years) vs early (13-15 years) adolescence and is influenced by socioeconomic factors. Methods: National Health and Nutrition Examination Survey (NHANES) 2007 to 2018 data (6 most recent complete survey cycles) analyzed. CVH status was characterized by AHA Life Simple 7 score (AHAS7). A representative sample (N=5874) of the US adolescent population was analyzed and weights used to estimate trends for the entire US adolescent population. The proportion of US adolescents with ideal (AHAS7 score 12-14), intermediate (8-11), and poor (0-7) CVH status calculated. Consecutive 2-year NHANES cycles were rescaled to consecutive integers to assess change in CVH score over time. Regression models were used to assess trend in CVH score over time and separately by age. Nonlinearity of secular trend was tested by assessing the significance of adding a quadratic term to the regression model (significance, p<0.01). CVH status by early vs late adolescence was assessed via categorical analysis as was the association between CVH status in early vs late adolescence and social determinants such as insurance status, poverty status (above and below 5x’s the national poverty level) and household reference (e.g., parent) education level. Results: CVH scores improved from 2007 to 2018 ( p<0.0001 ), however, the proportion of youth with ideal CVH status declined with age and was highest in early and lowest in later adolescence (early 59% vs later 41%) ( p<0.0001 ). The proportion of adolescents with ideal physical activity, smoking status, blood pressure, and cholesterol was lowest among older adolescents. Older adolescents are more likely to have no insurance (early 44% vs later 56%, p<0.0001 ). Other social determinants were not significantly different. Conclusions: Efforts to improve CVH status should begin no later than adolescence. Greater focus on preserved physical activity, BP and cholesterol management, reduced smoking and maintenance of health insurance coverage may lead to sustained CVH status throughout adolescence.

Spastic type cerebral palsy (CP) is a complex neuromuscular disorder that involves altered skeletal muscle microanatomy and growth, but little is known about the mechanisms contributing to muscle pathophysiology and dysfunction. Traditional genomic approaches have provided limited insight regarding disease onset and severity, but recent epigenomic studies indicate that DNA methylation patterns can be altered in CP. Here, we examined whether a diagnosis of spastic CP is associated with intrinsic DNA methylation differences in myoblasts and myotubes derived from muscle resident stem cell populations (satellite cells; SCs). Twelve subjects were enrolled (6 CP; 6 control) with informed consent/assent. Skeletal muscle biopsies were obtained during orthopedic surgeries, and SCs were isolated and cultured to establish patient–specific myoblast cell lines capable of proliferation and differentiation in culture. DNA methylation analyses indicated significant differences at 525 individual CpG sites in proliferating SC–derived myoblasts (MB) and 1774 CpG sites in differentiating SC–derived myotubes (MT). Of these, 79 CpG sites were common in both culture types. The distribution of differentially methylated 1 Mbp chromosomal segments indicated distinct regional hypo– and hyper–methylation patterns, and significant enrichment of differentially methylated sites on chromosomes 12, 13, 14, 15, 18, and 20. Average methylation load across 2000 bp regions flanking transcriptional start sites was significantly different in 3 genes in MBs, and 10 genes in MTs. SC derived MBs isolated from study participants with spastic CP exhibited fundamental differences in DNA methylation compared to controls at multiple levels of organization that may reveal new targets for studies of mechanisms contributing to muscle dysregulation in spastic CP.

Individuals with spastic cerebral palsy (CP) often exhibit altered sensitivities to neuromuscular blocking agents (NMBAs) used for surgical intubation. We assessed usage of the NMBA rocuronium in patients with spastic CP and evaluated potential modifiers of dosing including gross motor function classification system (GMFCS) level, birthweight, gestational age, and the use of anticonvulsant therapy. In a case-control study, surgical patients with spastic CP (n = 64) or with idiopathic or non-neuromuscular conditions (n = 73) were enrolled after informed consent/assent. Patient data, GMFCS level, anticonvulsant use, and rocuronium dosing for intubation and post-intubation neuromuscular blockade were obtained from medical records. Findings reveal participants with CP required more rocuronium per body weight for intubation than controls (1.00 ± 0.08 versus 0.64 ± 0.03 mg/kg; p < 0.0001). Dosing increased with GMFCS level (Spearman’s rho = 0.323; p = 0.005), and participants with moderate to severe disability (GMFCS III-V) had elevated rocuronium with (1.21 ± 0.13 mg/kg) or without (0.86 ± 0.09 mg/kg) concurrent anticonvulsant therapy. Children born full-term or with birthweight >2.5 kg in the CP cohort required more rocuronium than preterm and low birthweight counterparts. Individuals with CP exhibited highly varied and significant resistance to neuromuscular blockade with rocuronium that was related to GMFCS and gestational age and weight at birth.