Introduction

Neonates with very low birth weights (VLBWs) are born weighing 1500g or less; these cases suffer from high morbidity as well as growth and developmental deficiencies1. In adolescence, VLBW babies exhibit increased incidences of neurological, neuropsychological, cognitive, motor, and health deficiencies, including higher rates of asthma, high blood pressure, cerebral palsy, disturbances of sight and hearing, ASD, and ADHD, as well as a higher risk of developing eating disorders1.

The dentition of VLBW babies is predisposed to enamel hypoplasia, crown dilacerations, and palatal distortions in primary and permanent dentition2. Seow et al. reported high prevalence of generalized enamel hypoplasia in the primary dentition of 40–70% of preterm children, and they explained this result with low bone-mineral availability2. Also, the higher prevalence of enamel hypoplasia in VLBW babies over time can lead to increased susceptibility to dental caries3. Other oral findings associated with VLBW include higher prevalence of plaque, gingivitis, Streptococcus mutans, and lower saliva secretion compared to matched controls4, as well as alterations in bacterial counts5. In contrast, no difference was found in the levels of oral immunoglobulins among full-term and preterm infants6. Still, little is known about the immunological and protective mechanisms present in the oral cavities of VLBW children.

Seow et al. reported that the enamel in preterm primary dentition has an abnormally thinner surface quality probably due to reduced prenatal growth and small primary-dentition dimensions7. A meta-analysis and systematic review by Occhi‐Alexandre et al. found that the prevalence of dental caries among preterm children was 39% compared to 30% in full-term children8. Seow et al. also found that the prevalence of enamel defects in the permanent first molars of preterm babies was 21% (vs. 11% in healthy cases) and that the prevalence of enamel defects in lateral incisors was 12% (vs. 0% in healthy cases). These findings led to the hypothesis that persistent systemic imbalances during birth disrupted enamel formation2. Nelson et al. found a significant risk of increased enamel defects in the permanent incisors and first molars of VLBW children, but lower caries values compared to normal birth-weight children9. Low birth weight has been suggested as one of the causes of molar incisor hypomineralization10. Nevertheless, scarce evidence is available on the oral conditions of VLBW children in adolescence.

This study explored the oral and dental status, including bacteria and cytokines associated with dental decay and gingival inflammation, enamel defects, and salivary parameters, of VLBW children aged 10–13 years. We hypothesized that premature, VLBW infants will have different levels of cariogenic bacteria and salivary cytokines in their oral cavities due to differences in their immune systems.

Materials and methods

Study population

This study was approved by the Institutional Human Subjects Ethics Committee of the Rabin Medical Center in Petah Tikva, Israel (approval number 0315-13-RMC). The study cohort consisted of 32 cases of children born weighing 1500g or less in the period 2002–2004 (aged 10–12 years at the time of the study). All the children were born at the Schneider Children’s Medical Center of Israel and treated at the center’s neonatal unit. All the participants’ parents/guardians signed informed consent forms before the enrollment phase. The healthy controls were children born with normal weights and after full-term pregnancies, with age and sex matched to those in the VLBW group.

Clinical oral examination

Dental examinations were conducted by a pediatric dentist (ED) and were performed using a dental mirror, dental explorer, and dental probe. These examinations included the following parameters:

  1. 1.

    Oral hygiene, measured by the PI11.

  2. 2.

    Periodontal status, measured by the GI12.

  3. 3.

    Caries status, measured by the mean DMFT/dmft index—that is, the sum of the number of DMFT, which stands for decayed (present as an unmistakable cavity, undermined enamel, or a detectably softened cavity floor or wall), missing due to caries, and filled teeth13.

  4. 4.

    Enamel-defect prevalence and severity (number of teeth affected), measured by the DDE index14.

  5. 5.

    Dental-calculus formation, scored according to the World Health Organization (WHO) oral hygiene indices (WHO score)15.

pH measurements

Oral surface pH was measured using a flat glass-electrode pH meter (model CyberScan pH 11, Eutech Instruments) at seven sites: the hard and soft palates; the anterior, middle, and posterior tongue; and the right and left buccal mucosae. Two measurements were recorded for each site at 5-min. intervals, as described elsewhere16.

Saliva collection

Saliva was collected in a quiet room between eight in the morning and noon. The participants spat into wide test tubes for 3 min. All the children refrained from eating, brushing their teeth, and rinsing with mouthwash for at least 1 h. before saliva collection. The collected saliva was immediately stored at 4 °C and subsequently stored at − 80 °C until analysis. Unstimulated saliva flow was calculated by the amount of saliva collected for 3 min.

Salivary-cytokine quantification

The salivary levels of human IL-8, IL-6, IL-10, and TNFα were measured using ELISA kits according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, US).

Microbiological testing

Bacterial DNA was extracted from saliva using a DNA extraction kit (Qiagen). Afterward, the DNA was tested using specific primers for total bacteria17, S. mutans18, Lactobacillus19, and F. nucleatum17 with quantitative real-time polymerase chain reaction (RT-PCR). These bacteria were selected since they are the main pathogens in caries and gingivitis20. Bacteria levels are presented as the mean total amount per sample.

Statistical analysis

All the data were recorded on electronic sheaths (in an Excel file). Student’s t-test for independent samples was used to examine differences in continuous parameters between the two groups (VLBW participants and controls). Correlations among the parameters were tested using the Pearson correlation test. All the analyses were two-tailed, and significance was set at the 5% level. Statistical analysis was performed with SPSS Statistics (version 22, IBM Corporation, Armonk, NY, USA). Unless stated otherwise, the data are presented as means ± standard deviations. The study cohort included 32 very low birth weight (VLBW) cases along with age- and sex-matched healthy controls with normal birth weight (Table 1).

Table 1 Perinatal characteristics of the very low birth weight (VLBW) cohort.
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Results

The perinatal characteristics of the VLBW participants are detailed in Table 1. Controls were included in the study based on the parents’ general questionnaire, which confirmed that the child had a normal birth weight and gestational age. The mean birth weight for the cohort was 1086g, and the mean gestation age was 29.4 weeks. At the time of the examination, the average age of the VLBW group was 10.94 ± 0.17, while that of the controls was 11.75 ± 0.16. In both groups, the percentage of females was ≈50%.

A dental examination revealed robust decay and filling values in the VLBW group compared to the control group (P < 0.05 for the missing value, as shown in Table 2 and the supplementary table). The mean DMFT/dmft index was also greater in the VLBW group compared to the controls, albeit without statistical significance (Table 2). Moreover, both the plaque and gingival indices were greater in the VLBW group compared to the control group (P < 0.05).

Table 2 Dental parameters of children born with VLBW compared with healthy controls.
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The measurement of the enamel defects in the VLBW group revealed a mean of 2 and prevalence of 33% for all teeth (Fig. 4). The teeth that were most commonly affected by enamel defects were the first molars and incisors, both in terms of scoring and prevalence (Fig. 1). The salivary flow rate was found to be similar in the two groups. Oral pH was more acidic in the VLBW group than in the control, with statistical significance for all the sites, except for the right buccal mucosa (P < 0.05; Table 3).

Fig. 1
figure 1

Prevalence and distribution of enamel defects in VLBW children compared to healthy controls. The most affected teeth were the first permanent molars and incisors.

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Table 3 Oral mucosal pH measurements in VLBW and healthy groups.
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The salivary protein levels of the two groups were similar (Fig. 2). Salivary IL-10 and IL-8 were also at similar levels (Fig. 3), while TNFα was reduced in the VLBW group. IL-6 exhibited robust levels in the VLBW participants compared to the controls (P < 0.05; Fig. 3). Salivary bacteria (universal) were found to have reduced levels in the VLBW group compared to those of the control group (P < 0.05; Fig. 3). Moreover, F. nucleatum and Lactobacillus were reduced in the VLBW group compared to the control group (P < 0.05; Fig. 4). S. mutans was undetectable in both groups. It is important to note that total bacterial quantification was performed using universal 16S rRNA primers, whereas the other bacteria were quantified using species-specific qPCR assays. Due to potential differences in primer efficiency, amplification biases, and normalization methods, bacterial genera were not directly compared; rather, comparisons were made only between test and control groups within each bacterial quantification set.

Fig. 2
figure 2

Salivary protein levels in VLBW and control groups. No significant differences were observed between groups.

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Fig. 3
figure 3

Salivary cytokine and bacterial levels in VLBW versus controls. IL-10 and IL-8 were similar between groups, TNFα was reduced and IL-6 elevated in VLBW children. Salivary bacterial load (universal, F. nucleatum, and Lactobacillus) was reduced in the VLBW group.

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Fig. 4
figure 4

Prevalence and mean number of teeth affected by enamel defects in VLBW children. The graph highlights first permanent molars and incisors as the most commonly affected teeth.

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The correlation analysis revealed a negative correlation of IL-10 with GI and pH and a positive correlation with enamel defects (Table 4). IL-8 had a positive correlation with total salivary protein and a negative correlation with total salivary bacteria. IL-6 was also found to correlate with total salivary protein. Interestingly, Lactobacillus was negatively correlated with PI. At the clinical level, GI was correlated with DMF and PI, while DMF was also significantly correlated with PI.

Table 4 Correlation between dental indices, salivary cytokines, microbial parameters, and enamel defects in the VLBW group.
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The correlation analysis also revealed distinct immune and microbial interactions between groups (Table 4); In VLBW children, GI, PI, and DMFT were strongly correlated, IL-10 negatively correlated with GI and pH but positively with enamel defects, and IL-8 was linked to salivary protein and bacteria. In contrast, controls showed stronger correlations between IL-10, TNFα, IL-6, total protein, and salivary bacteria, with F. nucleatum and universal bacteria closely linked.

A retrospective power analysis was conducted based on the observed differences in the Gingival Index (GI) between the VLBW and healthy groups. The analysis yielded a large effect size (Cohen’s d = 1.69), with a corresponding statistical power of 99.9% (α = 0.05), indicating that the sample size was adequate to detect significant group differences. Mean GI values were 1.88 for the VLBW group and 0.87 for the healthy group. While the Shapiro–Wilk test indicated non-normal distribution in the healthy group (p = 0.00002), the VLBW group followed a normal distribution (p = 0.67). Accordingly, a Welch’s t-test was used, revealing a statistically significant difference between groups (p < 0.00001). These parameters support the validity of the group comparisons made in the study.

Discussion

Recent advances in neonatal care have improved the survival rates of infants weighing between 500 and 1500g. Therefore, efforts have also been made to improve their long-term well-being21, albeit with little attention to their oral health. In the present study, we examined the oral-health status of 11-year-old children who were born with VLBWs. The results show that the oral conditions of VLBW babies differ from those of normal birth-weight babies even in adolescence, with robust decay and filling, plaque and gingival inflammation levels, enamel defects, and changes in salivary pH, cytokines, and bacterial load.

The survival rates of most preterm babies have improved dramatically, even at neonatal weights of ≈500 g21. Still, a preterm infant may suffer from several complications, such as birth asphyxia, apnea, hyaline membrane disease, patent ductus arteriosus, intracranial hemorrhage, renal immaturity, metabolic dysfunction, GI intolerance, and susceptibility to infections1.

Children born with VLBWs are at risk of oral-health problems due to a combination of factors, including prematurity, exposure to medications and medical procedures, and limited opportunities for oral hygiene. Indeed, the findings reveal that one of the primary concerns in VLBW children is the increased risk of dental caries. Several factors contribute to this elevated risk, including limited exposure to fluoride, difficulty with oral hygiene, and high sugar intake. The present study also found that VLBW cases showed higher plaque and gingivitis levels, both of which are key factors in cariogenic processes. This contradicts other studies that have reported that dental caries is not associated with gestational age or birth weight22,23. The authors of these studies explained the differences in their findings arguing that increased antibiotic use and delayed tooth eruption may explain the negative association between IUGR and dental caries. Another possibility, suggested by Peres et al.24 in their study of VLBW children aged six years, is that harmful social and biological risk factors accumulate in early life and contribute to the development of high levels of dental caries; however, low birth weight was not found to be one of these factors.

Developmental defects of enamel (DDE)—such as enamel hypoplasia, enamel opacity, and molar incisor hypomineralization (MIH)—have frequently been described in association with preterm birth. These defects might affect both the primary and permanent dentitions2. In a systematic review published in 2014, the relationship between DDE and preterm birth was analyzed25, and it was found that preterm children presented high rates of DDE. In our study, permanent teeth were examined due to the age of the participants. The teeth most commonly affected were the first permanent molars and the incisors9, which form and calcify at birth. This finding confirms previous studies. Interestingly, in our study, other teeth were also affected, which suggests that the consequences of being VLBW children continued to affect the participants’ teeth beyond the first year of life9.

Saliva plays a crucial role in maintaining oral health as it provides a range of protective functions, including lubrication, buffering, and antimicrobial action16. In the case of VLBW children, there is a greater variation in the composition of saliva compared to normal birth-weight children6,26. Interestingly, we were unable to find significant differences in flow rate. However, we did find an acidic oral environment in the VLBW group. This is extremely important since acidic saliva contributes to dental caries, which was also higher in this group. Furthermore, focusing on salivary composition, our study revealed a reduced prevalence of oral bacteria (total bacteria, F. nucleatum, and Lactobacillus) in VLBW children. This is in agreement with previous studies5,27 that indicated that VLBW infants may have altered oral microbiota, which are characterized by higher levels of potentially pathogenic bacteria and reduced diversity of beneficial bacteria.

Scholars that have evaluated oral bacterial colonization in VLBW and normal-born children have focused mainly on S. mutans, and they have detected it in 60% of six-month-old full-term infants and 50% of same-aged preterm infants28. They have also found an increasing presence of S. mutans with age; this bacterium was detected in 37% of one-year-old children, with higher prevalence in full-term children compared to preterm ones. Koberova et al.27 detected S. mutans in 13.19% of VLBW children and 48.84% of normal birth-weight children; however, preterm children had significantly lower prevalence of S. mutans and marginally significant low growth density. Thus, a higher risk of caries cannot be attributed to S. mutans. Our results agree with the literature regarding the alteration in salivary bacterial composition in VLBW children. However, in our study, in contrast to the abovementioned studies, the children were examined at the age of 11, which is when alterations in salivary bacterial composition are expected. Another important result is that the higher plaque and gingival indices that were found in our study were probably related to other pathogenic bacteria, which were not studied.

In our study, salivary cytokines did not reveal clear differences in total protein, IL-10, and IL-8 levels. However, salivary TNFα exhibited reduced levels, and IL-6 showed robust levels in the VLBW group compared to the control group. Previous studies6,26 also found that salivary cytokines in VLBW children may differ from those in full-term infants, including in terms of altered levels of IL-6, IL-10, and salivary IgA. The mean levels and frequencies of detection of the examined cytokines were higher in the VLBW group. The cytokine levels were different between the VLBW and control groups, and they appeared to be influenced by stressful situations and/or antigenic microbial challenges. These findings are in accordance with ours, and they confirm that cytokine levels appear to be influenced by birth weight and or gestational age, which might reflect the level of immunological maturity of the mucosal immune system26. Moreover, elevated levels of proinflammatory cytokines, such as TNFα, have been associated with adverse health outcomes, including increased risk for chronic conditions such as asthma as well as metabolic and neurodevelopmental disorders29. Therefore, the patterns and implications of salivary cytokines in VLBW children are valuable markers that may reflect the functioning of the immune system and health risks. The negative association that was found between IL-10 and the gingival index is interesting since IL-10 is an anti-inflammatory cytokine, and this correlation may show that saliva cytokine is aligned with gingival inflammation29. Another interesting correlation was the one between Lactobacillus and the plaque index, which may reflect the shifting of plaque to a dysbiotic phase as plaque levels grow.

Our study has several limitations. First, data on diet, weight, height, and oral healthcare habits—factors that could influence microbiological and immunological outcomes—were not available for the control group. Additionally, the extended time frame between birth and adolescence introduces the potential for numerous confounding variables, including socioeconomic status, environmental exposures, and access to dental care, which may have contributed to the oral health differences observed between groups. While we attempted to minimize confounding by matching participants on age and sex and recruiting from similar urban regions, comprehensive control of these factors was not feasible and should be acknowledged. Moreover, the relatively small sample size may limit the statistical power of our findings and their generalizability to broader populations. From a methodological standpoint, our qPCR-based approach aimed to quantify key bacterial species; however, S. mutans was undetectable in both groups, potentially due to limitations in primer–probe sensitivity, low bacterial load, or DNA extraction efficiency. The observed discrepancies between total bacterial counts and individual species (e.g., F. nucleatum and Lactobacillus) may also reflect differences in primer efficiency and normalization techniques. Future studies with larger, more diverse cohorts and standardized microbial quantification methods—such as metagenomic sequencing—are needed to validate these preliminary findings and further elucidate the long-term impact of very low birth weight on adolescent oral health.

In conclusion, our findings provide preliminary evidence that children born with very low birth weights (VLBWs) may be at increased risk for oral health challenges during adolescence, including higher rates of dental caries, enamel defects, and gingival inflammation. These results suggest a potential need for increased attention to oral health monitoring and preventive care in this population. Regular dental checkups and individualized preventive strategies, such as fluoride supplementation, may help support better oral health outcomes. However, due to the study’s limited sample size and potential for unmeasured confounding factors, these findings should be interpreted with caution. Further large-scale, longitudinal studies are needed to establish definitive conclusions and to clarify the long-term oral health implications of VLBW.