Natal factors affecting developmental defects of enamel in preterm infants: a prospective cohort study

This study investigated natal factors influencing developmental defects of enamel (DDE) in premature infants using a newly refined preterm developmental defects of enamel (PDDE) index. Dental examinations were conducted on a cohort of 118 preterm infants (average age 3.5 ± 1.4 years) to record PDDE scores, while reviewing their medical records to examine natal factors. According to the logistic regression analysis, factors related to DDE prevalence were advanced maternal age, gestational age < 28 weeks, birth weight < 1000 g, 1 min APGAR score < 7, and hospitalization period > 2 months, which were significantly higher by 2.91, 5.53, 7.63, 10.02, and 4.0 times, respectively. According to regression analysis with generalized linear models, the PDDE scores were approximately 7.65, 4.96, and 15.0 points higher in premature children diagnosed with bronchopulmonary dysplasia, intraventricular hemorrhage, and necrotizing enterocolitis, respectively. When endotracheal intubation was performed, the PDDE score was 5.06 points higher. The prevalence of PDDE was primarily observed bilaterally in the maxillary anterior teeth. Extremely preterm infants showed significantly delayed tooth eruption, suggesting that the influence of gestational age on dental development rates. Identifying the factors related to DDE in premature children can inform early dental interventions to support the oral health of high-risk children.

Table 1.Demographics of the study population based on gestational age (GA).Values are presented as average (± SD) or number (%).For categorical data, the p-value was tested using the chi-squared test.For the numerical data, the p-value was tested using the Kruskal-Wallis test.Bonferroni post hoc tests were performed to conduct multiple pairwise comparisons after the initial analyses.*p < 0.05 indicates statistical significance.a indicates the data tested using the Kruskal-Wallis test.C-sec cesarean section; NSVD normal spontaneous vaginal delivery; NICU neonatal intensive care unit.www.nature.com/scientificreports/

Medical complications attributing to increased PDDE score in preterm infants
Preterm infants with BPD had significantly higher M (hypomineralization, p < 0.0001), P (hypoplasia, p = 0.0459), and T (M + P, p = 0.0001) scores than those without BPD.The T score of preterm infants with IVH was significantly higher than in those without IVH (p = 0.0391).Preterm infants with NEC had significantly higher M (p = 0.0047) and T (p = 0.0022) scores than those without NEC.Preterm infants with hypocalcemia had significantly higher P (p = 0.0340) than those without hypocalcemia (Fig. 1).

DDE distribution
Of the infants, 44 (43.1%) had DDE affecting only the primary incisors, and 55 (53.9%) had both primary incisors and molars, indicating a significantly higher proportion than preterm infants affecting only the primary molars (1.69%).DDE primarily occurred bilaterally in the maxillary anterior region (62.71% of cases).The prevalence of DDE and PDDE scores in both maxillary primary central and lateral incisors was significantly higher than that in primary canines (Fig. 2).

First primary tooth eruption in preterm infants
The primary tooth eruption in preterm groups was confirmed as follows: extremely preterm 8.07 ± 3.46, very preterm 8.58 ± 3.22, and late preterm 7.28 (± 2.73) months.No significant correlation was found between the gestational and eruption age (Table 1).However, as GA decreased, corrected age (CA) increased.Extremely Figure 1.Preterm developmental defects of enamel (PDDE) scores based on medical complications.Preterm infants with bronchopulmonary dysplasia had significantly higher M (p < 0.0001), P (p = 0.0459), and T (p < 0.0001) scores than those who were not diagnosed.The T-score was significantly higher in preterm infants with intraventricular hemorrhage than that in those who were not diagnosed (p = 0.0391).Preterm infants with necrotizing enterocolitis had significantly higher M (p = 0.0047) and T (p = 0.0022) scores than those without necrotizing enterocolitis.Finally, preterm infants with hypocalcemia had significantly higher P (p = 0.0340) score than those without hypocalcemia.M score assessment of hypomineralization in qualitative defects; P score assessment of hypoplasia in quantitative defects.

Discussion
This study examined factors affecting amelogenesis disruption leading to DDE in preterm infants.The primary objective of this paper is to identify factors impacting the prevalence and severity of DDE, demanding early dental examination and intervention in high-risk preterm infants.Distinguishing itself from previous research, Results were similar to that of previous studies, indicating a significant increase in the prevalence and severity of DDE in preterm infants, with a GA < 28 weeks and a BW < 1000 g.This suggested the importance of calcium accumulation after 28 weeks of gestation for amelogenesis, significantly affecting both the maturation and secretion phases 25 .Fetal serum calcium increases exponentially between the GA of 24 and 37 weeks, contributing to 80% of the required mineral accumulation in the third trimester 26 .Consequently, the high prevalence and severity of DDE in preterm infants born before calcium accumulation is expected.This aligns with prior studies stating that DDE prevalence ranges from 46 to 96%, with shorter GA and lower BW associated with a higher prevalence 14,15,27,28 .Notably, this study confirms an increase in DDE severity compared to previous research.
Advanced maternal age increased the prevalence of DDE, however, it does not appear to affect severity.This implied that although advanced maternal age is associated with an increased risk of preterm delivery 29 , it is difficult to conclude that senescence directly disturbs amelogenesis by raising the severity of DDE.Only a correlation between young maternal age and the prevalence of DDE was confirmed; however, not with advanced maternal age 4,27,30 .Given the growing trend of advanced maternal age, further research into the relationship between the health status of older mothers and DDE is warranted.
APGAR scores, a straightforward assessment method for newborns, evaluate skin color, heart rate, muscle tone, respiration, and reflexes immediately after birth 31 .In this study, 1-min APGAR scores ≥ 7 and 5-min scores ≥ 8 were associated with reduced DDE prevalence and severity, consistent with normal APGAR scores ranging from 7 to 10 31 .This indicated that the APGAR score serves as an indicator of infant health and a predictive criterion for DDE occurrence and severity.
Common diseases in premature infants include BPD, rickets, IVH, NEC, hyperbilirubinemia, IUGR, hypocalcemia, and sepsis 1, 3,32 .Although these complications did not affect the prevalence of DDE, BPD, IVH, NEC, and hypocalcemia increased its severity.In other words, there was no significant difference in the prevalence of DDE between preterm infants with and without complications; however, there was a significant difference in severity.This suggested that the presence of complications does not necessarily lead to the development of DDE; however, it may exacerbate pre-existing DDE.Complications that disrupted enamel maturation included BPD and NEC, whereas those that affected enamel secretion included BPD, IVH, NEC, and hypocalcemia.
BPD, characterized by inadequate spontaneous breathing, often requires oxygen supplementation with endotracheal intubation.Intubation before tooth eruption can cause localized trauma to developing teeth 33 .Additionally, BPD directly affects mineral metabolism and reduces bone mineral content owing to drug treatment, potentially leading to DDE 19,34 .IVH is diagnosed when bleeding occurs in the germinal matrix beneath the ependyma of the brain ventricles, and can lead to neurological impairments 22,35 .Neurological abnormalities increase the incidence of DDE, and systemic disorders affecting neurodevelopment can impact amelogenesis [22][23][24] .NEC, a disease causing intestinal or colon necrosis in newborns often requiring surgical resection of the affected bowel, reduces the total absorptive surface area of the intestine, limiting mineral absorption 20,21 .Hypocalcemia, which results in a low concentration of crucial minerals during amelogenesis, leads to DDE.Postnatal factors such as the duration of endotracheal intubation and NICU admission significantly increase the PDDE score, aligning with previous research 27 .It is common for preterm infants to be admitted to the NICU for airway management competency with endotracheal intubation immediately after birth.
Localized trauma can result in defects in the affected area, which aligns with the findings of our study of a higher prevalence and severity of DDE in the anterior teeth, consistent with previous research 30,[36][37][38] .Notably, localized trauma from tracheal intubation induces this effect on primary incisors.In addition, premature birth can lead to incomplete calcification of the primary incisors as enamel formation of the teeth begins at approximately 4-5 months in the uterus, at the earliest.However, some studies have shown a high frequency of DDE Figure 2. Distribution of developmental defects of enamel (DDE).Forty-four infants (43.1%) had DDE affecting only the primary incisors, and 55 (53.9%) had defects in both primary incisors and molars, indicating a significantly higher proportion than that of preterm infants with defect affecting only the primary molars (1.69%).DDE primarily occurred bilaterally in the anterior maxillary region (62.71% of cases).The prevalence of DDE and PDDE scores in both maxillary primary central and lateral incisors was significantly higher than that in primary canine.
in the maxillary first primary molars 38,39 , emphasizing the need for further studies on the prevalence of DDE in each tooth.
The timing of tooth eruption is a key indicator of overall growth and development 40 .This study confirmed that as GA and BW decreased, the CA of the first primary tooth eruption was delayed, particularly in extremely preterm infants and those with BW < 1000 g infants.This implied that dental development rates in preterm infants are influenced by GA and BW, similar to their impact on other aspects of physical and cognitive development.Additionally, using CA instead of chronological age for dental development rate comparison is reasonable, especially in children aged three or younger 41,42 .These findings differ from those of prior research, which showed a correlation between tooth eruption age and GA when calculated chronologically; however, not when adjusted using CA 43 .The average GA of the preterm infants examined in previous studies was 36.5 weeks, and they were classified as moderate to late preterm infants.In contrast, this study investigated CA in extremely to late preterm infants, with a significant delay in CA among extremely preterm infants born before the exponential calcium accumulation 26 .
This study had several limitations.First, there were no comparisons between preterm and full-term infants, owing to difficulties in assembling a control group of full-term infants with common preterm birth complications.Second, the PDDE relies heavily on the visual inspection of the examiner, making it difficult to maintain objectivity.To mitigate this, we attempted to enhance the consistency of dental color assessment by capturing intraoral photographs of the infants during each visit and comparing them over time.Finally, gestational age as main variable was correlated with all variables, including birth weight, APGAR score, BPD, and NEC.Therefore, a univariate analysis was performed and a risk model through multivariate analysis could not be presented.
The strengths of the study included a well-documented cohort of preterm infants with medical and dental records, enabling simultaneous investigation of the relationship between various natal factors and DDE.Regular check-ups excluded post-eruption influences such as dental caries, enhancing DDE assessment reliability.Additionally, it proposed quantitative criteria for evaluating DDE, enabling the assessment of factor impact during the secretory and maturation phases.This study examined the factors affecting DDE severity and distribution in preterm infants, emphasizing esthetic concerns, tooth sensitivity, and caries risk, highlighting the importance of early dental intervention.

Patient selection
The cohort was composed of 178 preterm infants who visited the Department of Pediatric Dentistry of Yonsei University Dental Hospital between 2019 and 2022.Most of them had been admitted to the NICU at Severance Hospital, Yonsei University College of Medicine.After discharge, dental examinations were performed through the consultation system from the Neonatology Department of the same hospital.The inclusion and exclusion criteria were as follows: Inclusion criteria: 1. Premature infants who visited the Department of Pediatric Dentistry before the eruption of primary teeth was completed.2. Premature infants who had been admitted to NICU. 3. Patients who underwent regular dental check-ups at intervals of 1-6 months.

Exclusion criteria:
Patients with a history of trauma that could hinder accurate PDDE scoring.Patients who developed dental caries before the evaluation of their enamel defects, making it difficult to calculate an accurate PDDE score.Patients with < 20 evaluable primary teeth due to early extraction or congenital missing teeth.
Sixty infants were excluded based on the exclusion criteria, and 118 preterm infants were included in the study.Written informed consent for participation in the study was obtained from parents of preterm infants.This study was approved by the Institutional Review Board of Yonsei University Dental Hospital (IRB no.2-2019-0045).

Preterm developmental defects of enamel index
This study quantitatively assessed the DDE of preterm The timing of tooth eruption is a key indicator of overall growth and development 40 .This study confirmed that as GA and BW decreased, the CA of the first primary tooth eruption was delayed teeth based on the PDDE index (Table 4), which was devised by revising the DDE index proposed by Clarkson 10 and the Modified DDE Index 44 .The PDDE index categorizes DDE as enamel hypoplasia or hypomineralization, assigning P and M scores to severity.Enamel hypoplasia was assessed based on the extent of hard tissue defects as follows: normal tooth morphology (0 points), structural defects confined to the enamel (1 point), and defects affecting both enamel and dentin (2 points).The sum of the scores for the 20 primary teeth was defined as the P score.Enamel hypomineralization was graded according to tooth color and opacity as follows: normal (0 points), white opacity (1 point), yellowish (2 points), and yellowish to brownish (3 points).The sum of the scores of all 20 primary teeth was defined as the M score.The final T score was the sum of the P and M scores.

Variables
The independent variables were classified into three categories: prenatal factors (maternal age, paternal age, and maternal abortion history), neonatal factors (sex, GA, BW, APGAR score, delivery mode, and single or multiple pregnancy status), postnatal variables (duration of NICU hospitalization, parenteral feeding, and endotracheal intubation), and medical complications (BPD, rickets, IVH, NEC, hyperbilirubinemia, sepsis, IUGR, and hypocalcemia).
Advanced maternal age was defined as ≥ 35 years at delivery 45 .APGAR scores were measured 1 min and 5 min after birth, referred to as 1-min and 5-min APGAR scores, respectively.The groups were categorized based on a 7-point and 8-point cutoff for the 1-min and 5-min APGAR scores, respectively, with each group demonstrating statistically significant differences.These variables were investigated by reviewing the NICU admission, discharge, and birth records of preterm infants.The chronological age and CA were investigated in this study.Chronological

Table 4 .
Classification of developmental defects of enamel using the preterm developmental defects of enamel (PDDE) index.confined to enamel 1Dental structural defects that affect both enamel and dentin 2

Table 3 .
Regression analysis of potential risk factors for increasing the preterm developmental defects of enamel score.For descriptive analysis, the p-value was tested using the Wilcoxon rank-sum test.For logistic regression, the p-value was tested using a generalized linear model.C-sec cesarean section, NSVD normal spontaneous vaginal delivery, NICU neonatal intensive care unit.*p < 0.05 indicates statistical significance.a Indicates that the cutoff value was determined using the receiver operating characteristic (ROC) curve.
this study not only explores the prevalence of DDE based on natal factors but also introduces a PDDE index to assess the severity of DDE.The PDDE index was developed by modifying the previously used DDE Index proposed in 1989 and applying it to preterm deciduous teeth.According to the PDDE index proposed in this study, quantitative defects of enamel were scored as P scores, while qualitative defects were scored as M scores.The quantitative and qualitative defects of enamel are induced by different processes during amelogenesis.By evaluating them separately with their respective scores, it becomes possible to investigate which amelogenesis stages are more influenced by natal factors.