# Insight into perinatal health by investigating the neonatal line: a systematic review.

Julia Estivals, Elsa Garot, Margaux Lamare, David John Manton, Adeline Le Cabec, Christine Couture

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Julia Estivals, Elsa Garot, Margaux Lamare, David John Manton, Adeline Le Cabec, et al. Insight into perinatal health by investigating the neonatal line: a systematic review. Archives of Oral Biology, 2025, pp.106335. 10.1016/j.archoralbio.2025.106335. hal-05112151

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# Insight into perinatal health by investigating the neonatal line: A systematic review

Julia Estivals<sup>a,b,c,*</sup> <sup>□</sup>, Elsa Garot<sup>a,b,d</sup> <sup>□</sup>, Margaux Lamare<sup>a</sup>, David John Manton<sup>e,f</sup> <sup>□</sup>, Adeline Le Cabec<sup>b</sup> <sup>□</sup>, Christine Couture<sup>b</sup>

## ARTICLE INFO

### Keywords:

Dental histology

Incremental marker

Primary teeth

Birth

## ABSTRACT

**Background:** The neonatal line (NNL) is an accentuated line visible in the enamel and dentine of primary teeth and in some of the cusps of the first permanent molars. It corresponds to the physiological stress associated with the event of birth. This study aimed to investigate the characteristics of the NNL, its relationship with prenatal and postnatal enamel, its correlation with health and life data of both contemporary and ancient individuals, and its potential to differentiate the prenatal from postnatal origin of enamel defects to better understand the life conditions of mother and child.

**Materials and methods:** Following PRISMA guidelines and using the PICO framework, a systematic review was conducted across MEDLINE, Scopus, DOSS, and Google Scholar covering literature from 1937 to 2023.

**Results:** A total of 1420 references were identified, from which 32 studies met the inclusion criteria. The selected studies examined the impact of maternal factors, perinatal conditions and gestation on the NNL. The width of the NNL varied according to maternal health and birth conditions. The NNL was also a marker of birth viability, with its presence indicating a live birth. Lastly, dietary shifts around birth were observed through variations in the perinatal enamel strontium/calcium ratio.

**Discussion and conclusion:** This study highlights the value of the NNL in reconstructing early life history by linking maternal health, prenatal and postnatal stress, and enamel development. Further research on the relationship between hypomineralized lesions in pathological enamel and the NNL could help identifying early (i.e., *in utero* or perinatal) risk factors.

## 1. Introduction

In modern humans, birth is a key event in an individual's life history, marking the transition from *in-utero* prenatal to postnatal biological development and socio-cultural skills learning during infancy and childhood to reach self-sufficiency later in ontogeny. This major life history event is also a specific point of interest for various scientific disciplines, each drawing valuable conclusions about health, identity, development, and human evolution. For biological anthropologists, birth can provide essential information about human evolution, particularly regarding the growth and lifespan of individuals, as well as the

temporal evolution of modalities related to gestation, weaning, and reproductive strategies (Schwartz, 2012; Smith, 2013). In forensics, accurately determining a child's age is crucial to help in their identification and to understand the circumstances associated with their death (Franklin et al., 2015). Determining whether a perinatal infant survived birth is of paramount importance for the same reasons (Witzel, 2014). Around the time of birth, several teeth are already in the process of mineralisation. By analyzing biological markers in these forming dental tissues, early risk factors for developmental defects can be identified. The impact of prenatal, perinatal, and postnatal conditions on dental development and oral health throughout life can be assessed, which is of

<footnote><sup>a</sup> Univ. Bordeaux, UFR des Sciences Odontologiques, Bordeaux 33076, France</footnote>

<footnote><sup>b</sup> Univ. Bordeaux, CNRS, Ministère de la Culture, PACEA, UMR 5199, Pessac F-33600, France</footnote>

<footnote><sup>c</sup> C.H.U. de Bordeaux, Bordeaux 33000, France</footnote>

<footnote><sup>d</sup> Centre de Compétence des Maladies Rares Orales et Dentaires, CCMR O-Rares, C.H.U. de Bordeaux, Bordeaux 33000, France</footnote>

<footnote><sup>e</sup> Department of Cariology, Center for Dentistry and Oral Hygiene, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands</footnote>

<footnote><sup>f</sup> Academic Center for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam, the Netherlands</footnote>

<footnote>* Corresponding author at: Univ. Bordeaux, UFR des Sciences Odontologiques, Bordeaux 33076, France.</footnote>

<footnote>E-mail addresses: julia.estivals@u-bordeaux.fr (J. Estivals), elsa.garot@u-bordeaux.fr (E. Garot), margaux.lamare@u-bordeaux.fr (M. Lamare), d.j.manton@umcg.nl (D.J. Manton), adeline.le-cabec@u-bordeaux.fr (A. Le Cabec), christine.veschambre-couture@u-bordeaux.fr (C. Couture).</footnote>
particular interest in dentistry (Mountain et al., 2021). The study of birth
in archaeology offers valuable clues about cultural practices, sanitary
conditions, and lifestyles of ancient societies, which are typically not
documented in written records for the time periods they exist. It also
allows for the establishment of links between the biological and social
events of an individual's life, thereby contributing to a better under-
standing of the history of health and well-being in past societies (Finlay,
2013).

Enamel is the most mineralized tissue in the human body (Ruschel et al., 2011), and therefore, by extension, teeth are the anatomical elements that best preserve after death. Dental tissues (i.e., enamel, dentin and cementum) grow incrementally and thus preserve developmental information, allowing for the tracing and study of significant life events such as particular stressful events that may translate into accentuated lines or “stress”. Birth is one of them in that it leads to significant physiological changes affecting the main vital functions of the infant, and translates in teeth into a specific accentuated line called the Neonatal Line (NNL). This birth line was first described by Schour (1936) and is visible in the enamel and the dentin that forms at the time of the physiological stress and intense metabolic changes associated with birth. The present study focuses on examining the NNL in enamel to better understand perinatal enamel defects affecting modern-day children, and assess the relevance of proposed etiologies for these pathologies by studying past populations from which teeth have been excavated.

In enamel, the NNL is considered to be an accentuated stria of Retzius that forms at the time of birth and thus delineates the boundary between enamel formed prenatally and postnatally (Eli et al., 1989). Therefore, the NNL is visible on teeth that initiate and begin mineralizing *in utero* – i.e., all primary teeth and sometimes the first permanent molars (the NNL is most frequently visible in the mesial cusps; Schour, 1936). The NNL in enamel is considered to result from a disturbance in amelogenesis (Eli et al., 1989). Some authors have associated its appearance with the stress caused by physiological changes experienced by the newborn at birth, the transition from the intra-uterine to the extra-uterine environment (Molnar & Ward, 1975), or changes in the quality and mode of nutrition (Gustafson & Gustafson, 1967). The activity of the easily ‘upset’ ameloblasts is thereby altered, causing a modification in enamel synthesis. According to Sabel et al. (2008), the neonatal line is an optical phenomenon due to alterations in the size and mineralization of enamel prisms. Sognnaes (1949) argues that it involves thickening of prism sheaths, whereas Weber and Eisenmann (1971) suggest localized changes in prism configuration and a possible reduction in crystalline concentration. The latter also explain that the number of ameloblasts appears to decrease during the neonatal period. The NNL is identified on histological sections examined under a microscope by its location in the enamel, its dark color, and by the difference in texture perceived between prenatal and postnatal enamel (Sabel et al., 2008). More specifically, the prenatally formed prisms are slightly more irregular than the more regular postnatal prisms (Sabel et al., 2008).

Several parameters can be investigated to characterize the NNL, among which its presence in a tooth or a cusp, its position within the enamel (and dentine), its width, and its degree of mineralization. These variables can be correlated with biological data related to childbirth (such as the mode of delivery), the child (e.g., neonatal health status), the mother (including her health during pregnancy and any pathologies that may have influenced the course of delivery), as well as the environment, including the dietary habits of both the mother and the child, which can play a crucial role in perinatal development and long-term health. The NNL proves useful in various fields. In biological anthropology, several studies use it to trace individuals' early life history, such as their growth, development, and significant events such as birth, in order to investigate the evolution of these traits within human populations over time, particularly regarding physiological adaptations (Brickley et al., 2020; Canturk et al., 2014; Kurek et al., 2016; Sipovac

et al., 2023). In forensic medicine, the study of the NNL may contribute to help identify corpses through the estimation of age-at-death of a juvenile with primary or relatively unworn mixed dentition. In cases where the corpse cannot be identified by traditional means (for example, due to advanced decomposition), the presence or absence of the NNL can provide clues as to whether the infant was born alive or stillborn (Hurnanen et al., 2017; Srinivasan et al., 2017).

To guide our systematic exploration of the biological data provided by the study of the NNL, several research questions have been established: 1) What specific characteristics can be identified at and around the NNL? 2) How are these markers related to prenatal and perinatal health conditions? 3) How are the presence and characteristics of the NNL correlated with individual life history traits, such as growth patterns, health outcomes, and developmental milestones? 4) How can the study of the NNL be utilized to distinguish between prenatal and postnatal enamel, and what implications does this distinction have for understanding early life conditions? This systematic review aimed to enhance the current understanding of the biological data obtained from studying the NNL, especially in relation to an individual's life history traits.

## 2. Materials and methods

This systematic review was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) (Page et al., 2021) and was registered on the Open Science Framework (OSF). Its registration can be accessed at https://doi.org/10.17605/OSF.IO/QGNV3.

### 2.1. Eligibility criteria

This review included cross-sectional studies (studies comprising human teeth). Systematic reviews or meta-analyses, as well as studies using animal models were excluded. Articles in English were included, from the first definition of the NNL in 1937 until July 2023. The included articles had to investigate the NNL in enamel and/or use it to define the prenatal and postnatal enamel. The articles also needed to explore a measurable biological aspect and analyze how this aspect was associated with a specific parameter of the NNL, such as its width, its position within the enamel, or its degree of mineralization. This approach allows for connecting the observable characteristics of the NNL with measurable biological indicators.

### 2.2. Search strategy

In the present review, the SPIDER tool (Cooke et al., 2012) was used to effectively guide our qualitative research based on the following criteria: S (Sample: human teeth), P of I (Phenomenon of Interest: extracted teeth, physiologically shed or found in archeological sites, teeth prepared for sectioning and observation under a microscope), D (Design: contemporary case study, archeological observation or report), E (Evaluation: presence of the NNL in enamel, description and data on the NNL in enamel, prenatal enamel, and postnatal enamel), R (Research type: cross-sectional studies). The initial search was completed in July 2023, using four electronic databases - Medline via PubMed, DOSS via EBSCO, Scopus and Google Scholar. A search equation was constructed for each database (Table 1). The electronic search was complemented by browsing the reference lists of the included studies. If there were multiple studies based on the same sample, only the study which reported the most detailed data was included.

### 2.3. Study selection

A free and open-source reference management software was used to store and sort collected data (Zotero 5.0.96.2, GMU, VA, USA). Identified duplicates were removed. The selection process was conducted in
<caption>**Table 1**</caption>

Research equations developed for databases in order to compile a comprehensive list of articles measuring a biological aspect and analyzing how this aspect is associated with a specific parameter of the NNL.

<table><tr><td>Medline via Pubmed</td><td>"neonatal line" AND (teeth OR tooth OR dent*)</td></tr><tr><td>Scopus</td><td>TITLE-ABS-KEY "neonatal line" AND TITLE-ABS-KEY (teeth OR tooth OR dent*)</td></tr><tr><td>DOSS</td><td>"neonatal line" AND (teeth OR tooth OR dent*)</td></tr><tr><td>Google Scholar</td><td>"neonatal line" "teeth | tooth" "indicator | effect | change | marker | associated | information | result | factor"</td></tr></table>

two parts, based on inclusion and exclusion criteria: selection after reading the title and abstract, and selection after reading the full text. References were then exported into Rayyan software (Rayyan Systems Inc. "Rayyan: A Web-Based Systematic Review Tool." Rayyan, https://www.rayyan.ai/, Canada) to assess the eligibility of each paper based on titles and abstracts. Eligible references were then exported to an Excel spreadsheet (Version 16.82, Microsoft©, CA, USA) for evaluation upon reading the full text. Articles were selected by two evaluators (JE and ML), in a double-blind manner. Once the blinding was lifted, in the case of conflicts, consensus was obtained through discussion between the two authors.

## 2.4. Data extraction

A standardized data extraction form was used to record the following details: authors; title; publication year; number and type of teeth; parameters studied; biological data and results. In the various studies, there were not always, or not enough, figures clearly illustrating the analyses conducted, which in some cases complicated the full understanding of these studies.

## 2.5. Bibliometric analysis

The analysis of scientific domain mapping was conducted using the co-occurrence network of keywords in VOSviewer software (free software, version 1.6.18, Centre for Science and Technology Studies, Leiden University, Leiden, The Netherlands, 2022; (van Eck & Waltman, 2010)), based on the list of articles included in the review. From the matrix of selected articles, the keyword network analysis was created (threshold value set at 3). The term-article matrix allowed measuring the similarities of articles according to themes.

## 2.6. Assessment of study quality

The assessment of the risk of bias was conducted independently by two authors (JE and ML). The Critical Appraisal Checklist described by the Joanna Briggs Institute (Aromataris et al., 2024) was used for quality assessment of the selected studies. The risk of bias was assessed as per eight standardized and fixed items.

D1. Were the criteria for inclusion in the sample clearly defined? These should involve whether the types of analyzed teeth were identified, as well as their origins (clinical studies for modern teeth and/or archeological and/or forensic studies).

D2. Were the study subjects and the setting described in detail? This implied checking whether the subjects were well described (origin, characteristics, number) and whether the study setting was clearly detailed (sample provenience and analysis context).

D3. Was the exposure measured in a valid and reliable way? This involved the definition of the direct or indirect exposure for instance to environmental factors, the techniques employed, and the standardization of measurements (for example, the measurement of the NNL width).

D4. Were objective, standardized criteria used for measurement of the condition? This involved checking whether the variable measured was well-defined, whether the methods used were validated, and

whether the measurements were standardized and reproducible (for example, when measuring the NNL width, using a standardized and reproducible protocol).

D5. Were confounding factors identified? This implied verifying whether the study explicitly mentioned factors that could have influenced the variable of interest (e.g., the width of the NNL). These factors could have included, for example, the mode of delivery, maternal health conditions, prematurity, perinatal complications, maternal nutrition, medications or treatments administered during pregnancy or the environment.

D6. Were strategies to deal with confounding factors stated? It was essential to check whether the study mentioned statistical methods that adjusted for their results and confounding factors, whether it performed multivariate analyses, and whether it transparently discussed potential biases and their control.

D7. Were the outcomes measured in a valid and reliable way? This involved verifying whether the results had been obtained from validated methods, repeated multiple times, and across different samples.

D8. Was appropriate statistical analysis used? The statistical analyses were reviewed to ensure they were appropriate for the study's objectives. For example, researchers often use tests such as correlation analyses, or non-parametric tests, depending on the nature of the data and the research questions. These methods allow for evaluating relationships between variables, testing for significant differences between groups, and ensuring that the results are statistically reliable, with a well-defined significance threshold (e.g., p < 0.05). This approach ensures that the statistical methods are aligned with the study's objectives and data characteristics.

The authors answered 'Yes', 'No', 'Unclear' or 'Not Applicable' for each item and for each selected article. To categorize studies according to quality, an overall score for each study was calculated based on the number of 'Yes' answers, so that the resulting scores could range from 0 to 8. Finally, the studies were categorized according to their scores: low quality [score between 0 and 2]; moderate quality [3–5]; or high quality [6–8].

The methodological quality of these *in vitro* studies was illustrated by an accepted quality assessment tool for dental *in vitro* studies by means of the Robvis application (McGuinness & Higgins, 2021).

# 3. Results

## 3.1. Studies selection

The resulting article selection process (Fig. 1) based on the PRISMA guidelines first yielded a total of 1420 publications which were identified through searching of electronic databases. After applying the automatic filters (language and publication date) and removing duplicates, 1119 articles were retained for title and abstract screening. After reading the titles and abstracts, 1008 articles were excluded due to their non-compliance with the established inclusion criteria for this systematic review. Amongst the remaining 111 references, six were not accessible for reading. After full-text analysis and evaluation, 73 articles were excluded and for the remaining 32 articles, a risk of bias assessment was conducted (Figs. 2 and 3). After this analysis, all of the 32 articles were included in this review (Fig. 1), 22 of which were of high quality and 10 of moderate quality (Fig. 2).

## 3.2. Bibliometric analysis

The analysis of the keyword network created from the 32 included articles was developed and represented as a co-occurrence network (Fig. 4). Keywords are represented by points of different sizes and colors. The larger the point and the font size of the word, the more frequently the keyword appears in the data. Each color represents a group of words that are often associated with each other in the analyzed publications. Connecting lines between the points illustrate the associations among
Identification of studies via databases and registers

<figure><img src="image_3.png" /><figcaption>Fig. 1. Flowchart of study selection according to the PRISMA statement.</figcaption></figure>

the keywords.

In our case, the most frequently used keywords are: "humans", "enamel", "deciduous teeth", "neonatal line", "infant", "newborn", "child", and "pregnancy". These highlight the main focuses of the studies, such as the type of samples where the neonatal line is found (humans, enamel, primary teeth, infants or children) and one of the main a biological factor that may influence the NNL (pregnancy).

3.3. Data synthesis

Table 2 synthesizes the main data from the 32 studies included in this literature review. Their sample sizes ranged from two to 147 teeth, all involving primary or permanent teeth.

Several parameters were studied. On one hand, parameters directly related to the NNL: presence, width and position. On the other hand, parameters indirectly related to the NNL, such as prenatal and postnatal enamel thickness defined by the position of the NNL, the presence and/or number of accentuated lines relative to the NNL, the presence of accentuated lines around the NNL and of hypoplasia on the enamel outer surface, as well as the mineralization disturbance or artificially-induced demineralization rate near the NNL. The biological variables that may influence the aspect of the NNL were categorized into seven categories: maternal factors, factors related to the juvenile individual, factors related to gestation time and delivery mode, age at death, time period of archeological populations, mineralization/demineralization areas and dietary transition.

In these studies, several imaging techniques were used to analyze the
structural and chemical properties of enamel.

Optical microscopy, whilst providing less detail than electron mi-
croscopy, gives a clear overall view of dental sections and helps identify
areas of enamel where changes may have occurred. It is especially useful
for examining differences in enamel thickness between the prenatal and
postnatal periods.

Polarized light microscopy is a widely used technique to observe the

microstructure of dental enamel. It allows for the visualization of in-
cremental growth lines and the crystalline organization of enamel. This
makes it possible to locate the neonatal line and observe its relative
position with other enamel structures, providing insights into the tran-
sitions between the prenatal and postnatal periods.

Scanning electron microscopy offers the potential for imaging at a much higher resolution and enables the observation of enamel surface topography at a microscopic scale. It is particularly suited for studying the details of microstructures.

Finally, synchrotron X-ray fluorescence is a non-destructive tech-
nique that enables mapping the distribution of chemical elements in
enamel, including strontium, calcium, or zinc. This method allows for
the detection of variations in enamel’s chemical composition between
the prenatal and postnatal periods.

3.3.1. *Impact of maternal factors on the NNL*

Five studies examined the NNL to determine associations with maternal factors (Behie & Miszkiewicz, 2019; Hassett et al., 2020; Kurek et al., 2015, 2016; Mountain et al., 2021). Sample sizes ranged from 60 to 80 teeth studied (incisors, canines and molars). Maternal health factors were reported to influence the width of the NNL; including hypertension (Hassett et al., 2020), severe depression, psychiatric issues, severe anxiety, and depressive symptoms (Mountain et al., 2021), and occasional alcohol consumption during pregnancy (Behie & Miszkiewicz, 2019). Maternal factors that decreased NNL width were obesity (Hassett et al., 2020), psychosocial support at birth (Mountain et al., 2021) and antispasmodic medication by mothers during pregnancy (Kurek et al., 2015). Birth order within the sibling group influenced the width of prenatal enamel, with first-born children within a sibling group having thinner prenatal enamel compared to later-born children (Kurek et al., 2015). In the same study, no difference in enamel thickness was found when analyzing other maternal factors (age at childbirth, illness during pregnancy, Ządzińska et al., 2013).
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Risk of bias summary. Detail of the risk bias assessment (see color code) performed by two authors for each criterion on the 32 selected publications. D1: criteria for inclusion in the sample clearly defined; D2: study subjects and the setting described in detail; D3: exposure measured in a valid and reliable way D4: objective, standard criteria used for measurement of the condition; D5: confounding factors identified; D6: strategies to deal with confounding factors stated; D7: outcomes measured in a valid and reliable way; D8: appropriate statistical analysis.</figcaption></figure>

### 3.3.2. Associations between NNL parameters and perinate individual factors in primary teeth

Seven studies examined several parameters concerning the NNL to find an association with perinatal individual-dependent factors (Hershkovitz et al., 2019; Keinan et al., 2007; Norén, 1983; Ranggård et al., 1995; Seow et al., 2005; Teivens et al., 1996; Zilberman et al., 2010). Several studies examined the width of the NNL (Hershkovitz et al., 2019; Ranggård, 1994; Ranggård et al., 1995; Seow et al., 2005) and the relative thickness of prenatal and postnatal enamel in regards to

the position of the NNL (Keinan et al., 2007; Seow et al., 2005; Zilberman et al., 2010). Some analyzed the enamel structure in relation to the NNL (Norén, 1983; Ranggård et al., 1995), whilst others studied the incremental lines in the enamel in relation to the NNL (Teivens et al., 1996; Zilberman et al., 2010), with sample sizes ranging from 11 to 107 primary teeth including incisors, canines and molars.

Cerebral palsy had an influence on the width of the NNL (Hershkovitz et al., 2019) whereas neonatal hypocalcemia (Ranggård et al., 1995), blood ionized calcium (Ranggård, 1994) and birth weight
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Risk of bias graph. Overall proportions of each risk of bias as assessed by two authors, for all 32 selected publications.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Graphical representation of bibliometric mapping from the 32 included studies. Each node and word are proportional in size to their frequency in the 32 selected publications. The thickness of the connecting lines represents the tie strength between two nodes.</figcaption></figure>

(Seow et al., 2005) did not influence the width of the NNL. Down syndrome, cerebral palsy (Keinan et al., 2007), familial dysautonomia (Zilberman et al., 2010), as well as prematurity (Seow et al., 2005) could influence the thickness of prenatal enamel and postnatal enamel of primary teeth. In low birth weight infants affected by neonatal hypocalcemia, the enamel outer surface of their primary teeth was affected by pronounced and localized hypoplasia corresponding to the location of the NNL within the tissue (Norén, 1983; Ranggård et al., 1995). Furthermore, the cause of death (illness or sudden death) of infants influenced the number of accentuated lines observed in the postnatally formed tooth tissues. Infants who died from illness had one or more accentuated lines in the postnatal enamel, unlike infants who died of Sudden Infant Death Syndrome, who only occasionally exhibited such

lines (Teivens et al., 1996). Finally, the NNL could be a useful marker in sex determination of an individual. Jakobsen (1975) found that nearly half of the boys did not have any visible NNL on any of their first permanent molars, whilst all the girls had at least one. He further correlates this observation with the fact that, on average, boys are less dentally mature at birth than girls, and this difference could potentially be used for sex determination of juvenile skeletal remains in forensics (Jakobsen, 1975).

#### 3.3.3. Impact of gestation length and delivery mode on the NNL

Several parameters related to gestation duration and childbirth may influence the aspect of the NNL. Some studies investigated the width of the NNL (Behie & Miszkiewicz, 2019; Canturk et al., 2014; Eli et al.,
<caption>**Table 2**</caption>

Summary of the 32 selected publications investigating biological variables (pregnancy, nutrition, maternal and child health conditions, viability at birth, enamel mineralization and hypoplasia) and their direct or indirect association with the NNL in the children's teeth.

<table><thead><tr><th>Authors and year of publication</th><th>Number (N) and type of teeth</th><th>Studied parameters</th><th>Biological data tested</th><th>Results</th></tr></thead><tbody><tr><td>Behie and Miszkiewicz (2019)</td><td>N = 65 primary teeth<br>59 incisors<br>5 molars<br>1 canine</td><td>NNL width</td><td>Maternal factors<br>Factors related to gestation time and/or delivery mode</td><td>Significant association between the NNL width and the mother's alcohol consumption<br>No significant association between the width of the NNL and maternal weight, age, illness, stress, gestation time, and delivery mode</td></tr><tr><td>Canturk et al., (2014)</td><td>N = 48 primary teeth<br>incisors</td><td>NNL width.<br>Presence of NNL</td><td>Factors related to gestation time and/or delivery mode<br>Viability at birth</td><td>Significant association between NNL width and delivery type<br>Significant association between NNL presence and live birth</td></tr><tr><td>Dean et al., (2019)</td><td>N = 7 primary teeth<br>5 molars<br>2 canines</td><td>Mapping of Ca, Zn, and Sr</td><td>Enamel mineralization</td><td>Association between Zn in NNL and caries resistance</td></tr><tr><td>Eli et al., (1989)</td><td>N = 147 primary teeth<br>28 incisors<br>49 molars<br>70 canines</td><td>NNL width</td><td>Factors related to gestation time and/or delivery mode</td><td>Significant association between NNL width and delivery type</td></tr><tr><td>Hassett et al., (2020)</td><td>N = 80 primary teeth<br>canines</td><td>NNL width</td><td>Health maternal factors<br>Factors related to gestation time and/or delivery mode</td><td>Significant association between NNL width and maternal hypertension, obesity, gestation duration, season of child birth.<br>No significant association between the width of the NNL and delivery type and duration<br>Significant association between NNL width and cerebral palsy</td></tr><tr><td>Herskovitz et al., (2019)</td><td>N = 58 primary teeth<br>20 incisors<br>18 canines<br>20 molars</td><td>NNL width</td><td>Health factors related to the child</td><td></td></tr><tr><td>Humphrey, Dean, & Jeffries, 2007</td><td>N = 6 primary teeth<br>4 canines<br>2 molars</td><td>Sr/Ca ratio</td><td>Type of feeding at birth (breastfeeding vs formula)</td><td>The Sr/Ca ratio changes on either side of the NNL: decreases in breastfed infants and increases in infants fed with cow's milk-based formula</td></tr><tr><td>Hurnanen et al., (2017)</td><td>N = 129 primary teeth<br>12 incisors<br>62 canines<br>55 molars</td><td>NNL width</td><td>Factors related to gestation time and/or delivery mode</td><td>Significant association between NNL width and delivery duration.<br>No significant association between NNL width and delivery type</td></tr><tr><td>Hurnanen et al., (2019)</td><td>N = 88 primary teeth<br>3 incisors<br>58 canines<br>27 molars</td><td>Presence of staircase pattern enamel in NNL</td><td>Factors related to gestation time and/or delivery mode</td><td>No significant association between staircase pattern prevalence and duration or mode of delivery or pain-relieving medication</td></tr><tr><td>Jakobsen, (1975)</td><td>N = 108 First permanent molars</td><td>Presence of NNL</td><td>Factors related to the individual</td><td>Significant association between presence of NNL and sex (absence of NNL in males)</td></tr><tr><td>Janardhanan et al., (2011)</td><td>Primary tooth germs recovered from a deceased infant</td><td>Presence of NNL<br>Accentuated lines</td><td>Age at death<br>Viability at birth</td><td>Association between presence of NNL and live birth and the number of accentuated lines<br>Association between presence of NNL and survival duration</td></tr><tr><td>Keinan et al., (2007)</td><td>N = 39 primary teeth<br>canines</td><td>Thickness of prenatal and postnatal enamel</td><td>Health factors related to the child</td><td>Significant association between total enamel thickness and prenatal enamel thickness and Down syndrome and cerebral palsy</td></tr><tr><td>Kurek et al., (2015)</td><td>N = 60 primary teeth<br>incisors</td><td>NNL width</td><td>Maternal factors</td><td>Significant association between the NNL width and the season of birth of the child, and the maternal intake of spasmolytic medicine during pregnancy<br>No significant association between the NNL width and maternal age at delivery, birth order, number of children in family, diseases during pregnancy, preparations sustaining pregnancy, iron and vitamin preparation during pregnancy, birth by caesarean section</td></tr><tr><td>Kurek et al., (2016)</td><td>N = 57 primary teeth<br>incisors</td><td>NNL width</td><td>Time period of populations (archeological series)<br>Viability at birth</td><td>Significant negative association between the NNL width and the child's age at death<br>No significant association between the NNL width and time period</td></tr><tr><td>Kurek et al., (2022)</td><td>N = 55 primary teeth<br>incisors</td><td>Distance NNL - accentuated lines<br>Width of accentuated lines</td><td>Age at death<br>Viability at birth</td><td>Significant association between width of accentuated lines and age at death<br>No significant association between distance NNL and accentuated lines/age at death</td></tr><tr><td>Mishra et al., (2009)</td><td>N = 2 primary teeth<br>incisors</td><td>Rate of artificially-induced demineralization in the enamel</td><td>Progression of carious lesion</td><td>Decreased rate of demineralization in the NNL region</td></tr><tr><td>Mountain et al., (2021)</td><td>N = 70 primary teeth<br>canines</td><td>NNL width</td><td>Maternal factors</td><td>Significant association between the NNL width and psychosocial factors</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 2 (continued)**</caption><thead><tr><th>Authors and year of publication</th><th>Number (N) and type of teeth</th><th>Studied parameters</th><th>Biological data tested</th><th>Results</th></tr></thead><tbody><tr><td>Norén, (1983)</td><td>N = 107 primary teeth 102 incisors 5 cuspid</td><td>Hypoplasia</td><td>Factors related to the individual</td><td>Enamel hypoplasias only in the teeth of low-birth-weight infants, localized about the NNL and associated with neonatal hypocalcemia</td></tr><tr><td>Priya et al., (2015)</td><td>N = 10 primary teeth molars</td><td>NNL width</td><td>Factors related to gestation</td><td>Association between the NNL width and gestation duration</td></tr><tr><td>Ranggård, (1994)</td><td>N = 24 primary teeth canines</td><td>NNL width</td><td>Health factors related to the child</td><td>No significant association between the NNL width and blood calcium</td></tr><tr><td>Ranggård et al., (1995)</td><td>N = 11 primary teeth incisors</td><td>NNL width Hypoplasia Position of NNL</td><td>Factors related to the individual Factors related to gestation and/or delivery</td><td>Positive association between NNL position and gestation duration, unclear association between hypoplasia and hypocalcemia No association between the NNL width and hypocalcemia</td></tr><tr><td>Rythén et al., (2008)</td><td>N = 44 primary teeth 20 incisors 4 canines 20 molars</td><td>Mineralization defects</td><td>Factors related to gestation and/or delivery</td><td>Association between gestation duration and quality of postnatal enamel</td></tr><tr><td>Sebald et al., (2018)</td><td>N = 30 primary teeth No data about type of teeth</td><td>Presence of NNL Accentuated lines</td><td>Age at death Viability at birth</td><td>Comparison of histological and morphological age at death. Association between presence of NNL and viability at birth</td></tr><tr><td>Seow et al., (2005)</td><td>N = 90 primary teeth incisors</td><td>NNL width Position of NNL Thickness of prenatal enamel and postnatal enamel</td><td>Factors related to the mother and the infant: gestation duration, birth weight</td><td>Significant association between birth weight, gestation duration and enamel thickness Significance association between gestation duration and NNL position</td></tr><tr><td>Sipovac et al., (2023)</td><td>N = 59 primary teeth 33 incisors 26 canines</td><td>NNL width</td><td>Time period of archeological populations (Early Bronze and Modern Ages)</td><td>No association between birth weight and position of NNL Association (change in NNL width over archeological time period from the same geographical area)</td></tr><tr><td>Srinivasan et al., (2017)</td><td>undetermined incisors tooth germs</td><td>Presence of NNL</td><td>Age at death Viability at birth</td><td>Association between presence of NNL and viability at birth</td></tr><tr><td>Teivens et al., (1996)</td><td>N = 19 individuals</td><td>Accentuated lines</td><td>Factors related to the individual</td><td>Association between presence of accentuated lines and neonatal/postnatal disorder</td></tr><tr><td>Witzel, (2014)</td><td>N = 6 primary teeth 2 incisors 2 canines 2 molars</td><td>Accentuated lines</td><td>Age at death Viability at birth</td><td>Estimation of survival time of an individual with accentuated lines</td></tr><tr><td>Ządzińska et al., (2013)</td><td>N = 60 primary teeth incisors</td><td>Thickness of prenatal enamel and postnatal enamel</td><td>Health and genetic maternal factors Factors related to gestation and/or delivery</td><td>Significant association between prenatal enamel thickness and birth order, season of birth No significant association between prenatal enamel thickness and the mother's age at delivery, number of children, illness, medication use, iron or vitamin intake</td></tr><tr><td>Żądzińska et al., (2015)</td><td>N = 83 primary teeth incisors</td><td>NNL width Accentuated lines</td><td>Child's age at death</td><td>Significant association between NNL width and age of death Significant association between NNL width and number of accentuated lines</td></tr><tr><td>Zanolli et al., (2011)</td><td>N = 100 primary teeth incisors, canines and molars</td><td>NNL width</td><td>Factors related to gestation and/or delivery</td><td>Significant association between the NNL width and gestation duration No significant association between the NNL width and delivery type</td></tr><tr><td>Zilberman et al., (2010)</td><td>N = 17 primary teeth molars</td><td>Thickness of prenatal enamel and postnatal enamel Accentuated lines</td><td>Factors related to the individual</td><td>Significant association between thicker prenatal enamel and familial dysautonomia Significant association between more frequent presence of accentuated lines in postnatal enamel and familial dysautonomia</td></tr></tbody></table>

1989; Hassett et al., 2020; Hurnanen et al., 2017; Kurek et al., 2015; Priya et al., 2015; Zanolli et al., 2011), while others analyzed the position of the NNL (Ranggård et al., 1995), the quality of the enamel (Rythén et al., 2008) or the presence of staircase pattern enamel in NNL (Hurnanen et al., 2019). Żądzińska et al. (2013) used the NNL to differentiate and analyze the thickness of prenatal and postnatal enamel. All ten studies used primary teeth and the sample sizes ranged from 10 to 147 teeth (incisors, canines, molars). While some studies have suggested an effect of delivery type on the NNL width (Eli et al., 1989; Seow et al., 2005), others have found no association (Behie & Miszkiewicz, 2019; Hassett et al., 2020; Hurnanen et al., 2017; Zanolli et al., 2011). Similarly, although an impact of delivery duration has been suggested (Hurnanen et al., 2017), other studies have found no difference (Behie & Miszkiewicz, 2019; Hassett et al., 2020). Finally, birth season has been

identified as an influential factor in some studies (Hassett et al., 2020; Kurek et al., 2015). The duration of gestation affected the position of the NNL within the enamel (Ranggård et al., 1995) as well as the quality of the enamel in primary teeth (Rythén et al., 2008). On the other hand, a study highlighted that the average width of the neonatal line in children born with a shortened gestation period was thicker compared to children born with a normal gestation period (Priya et al., 2015). The season of birth influenced the thickness of prenatal and postnatal enamel: the prenatal enamel thickness of children born in spring or summer is significantly less than that of children born in autumn or winter (Żądzińska et al., 2013). Finally, the presence of a staircase pattern in the enamel at the NNL did not show any significant association with gestation time, delivery mode or duration, or the use of pain-relieving medication (Hurnanen et al., 2019).
### 3.3.4. The NNL: a marker of birth viability and early life events

In all publications concerned, the sample sizes ranged from six to 83 primary teeth (incisors, canines and molars). One study examined both primary and permanent teeth (Janardhanan et al., 2011). The NNL is often used to estimate the individuals' age at death and to assess neonatal viability (Janardhanan et al., 2011; Sebald et al., 2018; Witzel, 2014). The presence of accentuated lines in the postnatal enamel enables to discuss about early childhood mortality (Kurek et al., 2022; Żądzińska et al., 2015). The presence of the NNL has been linked to evidence of a live birth (Sebald et al., 2018), whilst its absence in tooth germs is associated with stillborn children (Canturk et al., 2014; Janardhanan et al., 2011; Srinivasan et al., 2017; Witzel, 2014). The duration of child survival after birth was assessed by quantifying the number of prism cross-striations from the NNL in the postnatal enamel (Janardhanan et al., 2011; Witzel, 2014). Retzius lines may also be used to estimate the time of child survival, they may however be poorly visible in the postnatal enamel, jeopardizing any reliable time estimation (Sebald et al., 2018). The age at death influenced the width of the NNL; one study concluding that the age at death was inversely proportional to the width of the NNL (Kurek et al., 2016). It has also been reported that children who died between two and three years of age had a wider NNL compared to those who died between 4 and 6.5 years of age (Żądzińska et al., 2015).

### 3.3.5. The NNL: a marker for refining chronological and anthropological interpretations

The width of the NNL was studied in primary teeth (sample sizes ranging from 57 to 59 teeth) to determine if it could be associated with the chronological period of individuals from archeological sites (Kurek et al., 2016; Sebald et al., 2018; Sipovac et al., 2023). Kurek et al. (2016) did not find an association between the width of the NNL and the chronological period of Neolithic, Medieval and Modern age samples. In contrast, Sipovac et al. (2023) found that the width of the NNL in children's skeletons dated to the Bronze Age was greater than that of children from the Modern age. Sebald et al. (2018) highlighted the fact that age at death estimation in perinates and infants could be performed using long bone length or ossification and fusion stages, which should however be complemented by assessment of dental development using histology. This is of great significance in biological anthropology, as overestimating the age at death could be misleading regarding the interpretation of parental investment, or the social perception of a newborn as a member of the society (Sebald et al., 2018).

### 3.3.6. Exploring the role of the NNL in enamel mineralization

Using synchrotron X-ray fluorescence, Dean et al. (2019) mapped elemental distributions in pre- and postnatal enamel and dentine, finding an increase in zinc in the NNL, putatively associated with high crystallinity and resistance to caries. Mishra et al. (2009) applied an etching procedure to the enamel in order to mimic the caries process, thus creating an artificial demineralized lesion. They used scanning microradiography and did not find any difference in demineralization between prenatal and postnatal enamel, while the rate of demineralization decreased near the NNL thus possibly limiting the progression of carious lesions. These two studies suggest that the NNL, due to its specific mineral composition, could act as a barrier against the propagation of carious lesions in the surrounding enamel in primary teeth (Dean et al., 2019; Mishra et al., 2009).

### 3.3.7. Strontium/calcium ratio across the NNL: insights into dietary shifts at birth

Humphrey, Dean, & Jeffries, 2007 studied the longitudinal thin sections of six primary teeth under polarized light microscopy to locate the NNL. Laser ablation inductively coupled plasma mass spectrometry was then performed to analyze the strontium/calcium (Sr/Ca) ratio on either side of the neonatal line to explore its association with dietary change occurring at birth (Humphrey, Dean, & Jeffries, 2007). The

Sr/Ca ratio was shown to vary depending on the type of diet received at birth, with a decreased ratio in breastfed children and increased ratio in children fed with infant formula derived from cow's milk (Humphrey, Dean, & Jeffries, 2007).

## 4. Discussion

This systematic literature review shows that multiple factors appear to influence the NNL and highlighted relevant biological variables that may affect the aspect of the NNL. These function as biomarkers, allowing for the tracking and revealing of certain of the living conditions of the individuals (mother and child). The characterization of the NNL and the surrounding dental tissues may provide valuable information for biological anthropology, forensic medicine, and pediatric dentistry. Teeth, particularly enamel due to its non-reparative nature, possess the capacity to record life events reliably. The mineralization of hard dental tissues follows a consistent incremental pattern. Among the techniques used in the studies included in this systematic review, dental histology enabled to correlate the chronology of dental development with an individual's life events (such as birth, death, and stress events, Dirks et al., 2002). However, some of these techniques are destructive, which may be a major constraint for the study of ancient specimens in biological anthropology. The study of the NNL provides valuable insights into various aspects, including the physical and mental health of the mother during the pregnancy, disorders or diseases affecting the children, sex, birth weight, gestational age, delivery parameters (i.e., type, duration, complications, season) and neonatal survival duration. The study of the NNL could provide broad hints regarding the historical period during which the individual lived, although this requires further research, as only one study with a small sample size addresses this question (Kurek et al., 2016). Several biological variables are frequently examined in the literature, some of them are shown to significantly impact the aspect of the NNL (e.g., maternal factors, pregnancy and delivery-related factors, and individual-specific factors, Behie & Miszkiewicz, 2019; Canturk et al., 2014). In contrast, some others (e.g., presence of neonatal/postnatal disorder, Teivens et al., 1996) remain underexplored and warrant further attention. This review underscores the multitude of factors influencing the aspect of the NNL and the extensive spectrum of NNL expression, the latter involving a substantial 'within population' variation (Behie & Miszkiewicz, 2019), putatively attributable to individual differences in ameloblast susceptibility to disturbances, which may be linked to genetic or immune factors (Townsend et al., 1998). Furthermore, conducting studies on modern populations with known medical histories is crucial to enhance our understanding of these biological processes. In fact, providing valuable reference data may help interpreting findings in archeological specimens. A better understanding of the influence of various factors on enamel formation and the features of the NNL will benefit from a more advanced correlation between the dental tissue microstructure and morphometry with detailed medical records. The combined study of modern and archeological populations enables improving our understanding of the physiological mechanisms driving tooth tissue formation around birth, especially since past populations are not exposed to the same kind of environmental conditions (e.g., diet, pollutants) as modern-day populations do.

### 4.1. Process of NNL formation

All studies included in the present review that analyzed the presence of the NNL and viability at birth concur that the presence of the NNL in the enamel is a proof of live birth (Canturk et al., 2014; Witzel, 2014). Nevertheless, the NNL is only visible if the individual survived a few days after birth; therefore, the presence of the NNL indicates a live birth, but its absence does not necessarily indicate a stillbirth. The NNL is however consistently absent in the tooth germs of stillborn children, as these individuals did not undergo the birth process and the associated environmental change (Srinivasan et al., 2017). There is actually a delay
between birth and the appearance of the NNL in the enamel which is
visible as a band separating prenatal enamel from postnatal enamel,
only if the infant survived at least seven to ten days after birth (Sabel
et al., 2008; Zanolli et al., 2011). The structural disturbance of the
enamel caused by birth could be due to a drop in calcium levels in the
first two to three days after birth (Seow et al., 2005). Whether the NNL is
hypomineralized is still subject to debate (see Dean et al., 2019 for a
review and quantitative data).

## 4.2. Width of the NNL

Hassett et al. (2020) and Zanolli et al. (2011) agree that prematurely
born children have a wider NNL than those born post-term. Yet their
findings differ regarding full-term children: their NNL width is similar to
that of premature children for Hassett et al. (2020), while, in contrast,
for Zanolli et al. (2011), it is comparable to that of post-term children.
Eli et al. (1989) estimate that the width of the NNL is determined at 63 %
by environmental change and at 37 % by the birth process. The NNL
width increases in children born through assisted delivery (such as
breech, vacuum, forceps) and decreases in those born by cesarean sec-
tion (Eli et al., 1989). This suggests that the transition from the intra-
uterine environment to the extrauterine environment is only partially
responsible for the disruption of amelogenesis and that birth trauma
impacts cellular function (Eli et al., 1989). Although two studies re-
ported a significant link between NNL width and the type of delivery
(Canturk et al., 2014; Eli et al., 1989), others did not find any association
(Behie & Miszkiewicz, 2019; Hassett et al., 2020; Hurnanen et al., 2017;
Zanolli et al., 2011). Moreover, the examination of the NNL width in
teeth from different archeological times (Early Bronze age and Modern
age) did not reveal any association with the type of delivery in the
modern age cohort (Sipovac et al., 2023). Hurnanen et al. (2017) suggest
that the duration of delivery is inversely proportional to the width of the
NNL, which could result from a temporary disruption of amelogenesis
due to the stress of prolonged labor. There is however no consensus,
since this association is not systematically found (Behie & Miszkiewicz,
2019; Hassett et al., 2020). Compared to a cesarean section, vaginal
delivery is generally longer and induces more intense stress; neverthe-
less, a cesarean section can be preceded by a long period of physical and
psychological stress, which may explain these conflicting results.

The width of the NNL may also be useful for studying past pop-
ulations. Although there is no reported significant correlation between
NNL width and chronological age, a study with relatively small sample
sizes suggests a tendency towards a wider NNL in a Neolithic population
compared to medieval and modern populations (Kurek et al., 2016). The
median NNL width of children from modern-day populations (6.96 µm)
(Sipovac et al., 2023) is different that of others studies (Eli et al., 1989;
Hurnanen et al., 2017; Kurek et al., 2016); however the median NNL
width of children from the Bronze Age (12.57 µm) (Sipovac et al., 2023)
is comparable to the results from a medieval Polish population (Kurek
et al., 2016). Children from Neolithic or medieval period likely experi-
enced more stressful events than those from the modern period, which
could explain a wider NNL due to more difficult living conditions (e.g.,
higher stress levels, famine, disease) (Lorkiewicz, 2011). These results
could reflect similar maternal and individual factors amongst in-
dividuals of the same archeological sample; therefore, additional studies
on the NNL width could provide more information about the health
conditions of past populations.

The discrepancies in the results obtained may be due to selection
errors, sample preparation (e.g., section obliquity), limited sample size,
observation, and/or measurement errors. The width of the NNL depends
on the rate of enamel secretion, which has been shown to vary in speed,
accelerating and decelerating in different parts of the tooth (Mahoney,
2008). It is also important to note that there is significant variation in the
expression of the NNL (Behie & Miszkiewicz, 2019), particularly
depending on the type of tooth studied (Hassett et al., 2020). Janard-
hanan et al. (2011) studied the developing dentition of a single baby and

observed that, in this individual, the NNL was best visible in the primary
incisors in comparison to canines and molars, which led them to further
recommend this tooth type to estimate postnatal survival in neonates
(Janardhanan et al., 2011).

## 4.3. Prenatal and postnatal enamel thickness

The NNL is a useful landmark to compare prenatal and postnatal
enamel thickness. Several factors may affect enamel thickness such as
gestational duration, timing of amelogenesis initiation, and enamel
secretion rate, the latter being influenced by genetics factors and envi-
ronmental factors like stress or vitamin D deficiency (Seow et al., 2005;
Wright, 2023; Żadzińska et al., 2013). Birth season serves as an indicator
of maternal sunlight exposure during pregnancy and is linked to
maternal vitamin D synthesis (Sayers et al., 2009). In Kurek et al. (2015),
lower vitamin D exposure during winter births was associated with
delayed amelogenesis onset. Firstborn children in a sibling group typi-
cally exhibit thinner prenatal enamel compared to second or later-born
siblings, potentially due to maternal experience with nutrition and
reduced stress (Żadzińska et al., 2013). Preterm children often who a
delayed dental development and eruption in early childhood, which is
then compensated by later catch-up mechanisms (Seow et al., 2005).
Their thinner prenatal enamel thus reflects a shorter intrauterine
development period, while their postnatal enamel may only partly
exceed that of full-term children (Seow et al., 2005). The overall enamel
thickness of preterm children remains smaller than that of fullterm
children (Seow et al., 2005). Consequently, crown size decreases due to
halted ameloblast activity and mineralization defects related to calcium
and phosphate deficiencies (Seow et al., 2005). In children with Down
syndrome, accelerated growth during early intrauterine stages may
explain thicker prenatal enamel compared to healthy children (Keinan
et al., 2007). This primarily concerns primary incisors, canines, and first
molars, which begin forming and mineralizing during the first trimester
of pregnancy (Barden, 1983).

## 4.4. Composition and structure of the NNL

Hypoplasia correspond to a quantitative developmental defect
involving the outer enamel surface, associated with a substantial enamel
thickness reduction. They result from physiological disruptions during
the secretory phase of amelogenesis through alterations of ameloblasts
(Goodman & Rose, 1990). In some modern-day neonates, prolonged
neonatal hypocalcemia and subsequent blood exchange transfusion
occurring during the first days of life may be responsible for enamel
hypoplasia very close to the neonatal line (Ranggård et al., 1995). Other
undocumented factors may as well induce enamel hypoplasia in these
infants (Ranggård et al., 1995). However, caution is advised regarding
the conclusions of this study due to its small sample size. Additionally,
calcium levels are rarely measured in newborns unless doctors suspect
hypocalcemia, making it challenging to establish a link between
neonatal hypocalcemia and enamel hypoplasia.

The microstructure and composition of enamel, specifically at the
NNL, helps for a better understanding of the demineralization processes
in primary teeth such as carious lesions. Zinc interacts with hydroxy-
apatite formation, influences crystal growth, and participates in a
dissolution/precipitation reaction, thereby reducing acid dissolution of
enamel and limiting demineralization (Mohammed et al., 2014). The
structural characteristics of the NNL have been shown to inhibit acid
diffusion, thus slowing demineralization progression - the NNL acts as a
barrier or obstacle against the progression of carious lesions extending
through postnatal to prenatal enamel (Mishra et al., 2009). It cannot be
excluded that an impurity, possibly zinc, inhibits enamel demineral-
ization when exposed to acid challenge (Mishra et al., 2009). The zinc
level in maternal blood decreases towards the end of pregnancy but
remains higher in the fetus than in the mother (Terrin et al., 2015).
Therefore, higher levels of zinc are found in prenatal enamel and dentin
compared to postnatal enamel and dentin. In the early days following birth, elevated zinc levels are released in breast milk, therefore a breastfed infant at birth could incorporate more zinc into the NNL and postnatal enamel than a non-breastfed infant (Dean et al., 2019). Strontium in prenatal enamel is enriched up to the NNL, where its concentration abruptly decreases, reflecting the reduced physiological availability of Sr in breastfed infants (Humphrey, 2010). Strontium follows a physiological concentration gradient and is not actively transported through the mammary glands, which limits its passage into breast milk (Rossipal et al., 2000). In contrast to calcium, of which 10-14 % of the intake is transferred into milk, only 1 % of ingested strontium reaches it due to metabolic processes that discriminate against its incorporation (Humphrey, 2010, 2014). These observations explain why the strontium/calcium ratio in the enamel and dentine of a breastfed infant is likely reduced at birth until an external dietary source of strontium is introduced, either through infant formula supplementation or complete replacement of breast milk during weaning (Humphrey, 2010). A significant reduction in the Sr/Ca ratio has been measured on either side of the NNL in the teeth of a small sample of three breastfed infants, suggesting a breastfeeding signal (Humphrey, Dean, & Jeffries, 2007). However, the required duration of breastfeeding to observe this reduction is not specified; and in two out of three infants there was an increase in Sr/Ca ratio, while the third child, who was not breastfed due to premature birth despite attempted breastfeeding, did not show similar results (Humphrey, Dean, & Jeffries, 2007). These findings provide a basis for interpreting dietary transition occurring around the time of birth through the elemental composition of perinatal enamel.

## 5. Conclusion

The results of the present review first presented several variables enabling to describe the NNL and the perinatally formed enamel: width and thickness of the NNL, the presence of the NNL, the mapping of Ca, Zn, and Sr and the Sr/Ca ratio in perinatal enamel, the presence of a staircase pattern in enamel at the NNL, the prenatal and postnatal enamel thickness, the distance between the NNL and accentuated lines, the number of accentuated lines, the resistance to enamel demineralization (i.e. carious lesions) at the NNL, the occurrence of perinatal hypoplasia, and the position of the NNL. Each of these variables was investigated using a variety of analytical methods, sometimes a combination of several of them, which complicates not only the interpretation of the resulting data but also any comparison across studies.

In forensic medicine and in biological anthropology, the study of the NNL offers various applications, particularly in enabling to determine the age at the time of death of a liveborn child. The identification of the NNL allows to distinguish stillbirths from postnatal deaths, a crucial criterion in certain medico-legal contexts. The presence of accentuated lines in the enamel beyond the NNL could be correlated with stress events that occurred during the neonatal or postnatal period. These accentuated lines indicate significant alterations occurring during enamel mineralization and may reveal signs of nutritional deficiencies, infections, or other stress factors, such as hormonal or metabolic disturbances. These stress events could contribute to identify specific disorders that took place before or after birth.

Overall, this review provides insights into the relationship between the NNL and the child's development, the physical and psychological health of the mother during pregnancy, and factors related to pregnancy and childbirth. Further studies are needed to explore the long-term risks and effects of these various types of environmental exposure. This has concrete applications in dentistry to enable intervening early as needed in the child's development, before signs or symptoms arise and limit or avoid irreversible damages. Such research could be beneficial for investigating the causes of enamel hypomineralization in children teeth, such as MIH (Molar Incisor Hypomineralisation) and HSPM (Hypomineralized Second Primary Molars), whose causes remain unknown.

Understanding whether these hypomineralized lesions initiate before, after, or during the formation of the neonatal line could help determine when and why the factors responsible for this enamel anomaly occur.

The access to medical records facilitates the analysis of various environmental or medicinal exposure and enables exploring their correlation with the observed dental characteristics. Case studies in modern day patients with known medical histories could further validate some of the results presented in this review and enrich the understanding and interpretation of data from anthropological and archeological samples. However, interpreting results in archeology is complicated by the absence of medical context, living conditions and the lack of knowledge of the factors influencing the NNL. When maternal and/or child medical data are unavailable, dental samples alone can provide insights into an individual's biology, sometimes supplemented by available skeletal data.

## Funding

The research was supported by the EVODIBIO team (PACEA, UMR 5199, CNRS, University of Bordeaux, French Ministry of Culture). The study received financial support from the French Government in the framework of the University of Bordeaux's IdEx "Investments for the Future" programme/GPR Human Past.

## Credit authorship contribution statement

**Julia Estivals:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Christine Couture:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **David John Manton:** Writing – review & editing, Visualization, Validation, Supervision. **Adeline Le Cabec:** Writing – review & editing, Visualization, Validation, Supervision, Methodology, Conceptualization. **Elsa Garot:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Margaux Lamare:** Writing – original draft, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

The authors would like to express gratitude to all the researchers who published studies that made this literature review possible.

## Data availability statement

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

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