Pregnancy is a unique immunological state in which the maternal immune system adapts to tolerate fetal antigens while maintaining sufficient immune defense (1). This balance comes at the cost of increased susceptibility to infections and inflammatory conditions, including periodontal disease (2–4). It is well established that the severity of periodontal disease, particularly gingival inflammation, tends to increase during pregnancy, with rates of pregnancy gingivitis ranging from 30% to 100% (5–8). Clinical signs can escalate from mild inflammation to pronounced hyperplasia, profuse bleeding, an increase in pseudo-periodontal pockets, and further progression, particularly in women with pre-existing periodontitis (5, 7, 8). Hormonal fluctuations during pregnancy—most notably elevated estrogen and progesterone—modify the subgingival microbiome, promote angiogenesis, and enhance vascular permeability, collectively amplifying local inflammatory responses in periodontal tissues (7–9).

Clinically, periodontitis is increasingly recognized not just as a local oral health issue but as a potential contributor to systemic pregnancy complications (4, 9). Recent evidence suggests a modest but consistent association between periodontitis and preterm birth (PTB). A 2024 meta-analysis of over 2.5 million participants reported that periodontitis increases the risk of PTB by approximately odds ratio (OR) 1.87 (10). Large cohort studies also support a dose–response relationship, with greater periodontal severity linked to higher PTB risk (11). Translocation of periodontal bacterial components to the maternal fetal unit, along with the systemic release of inflammatory mediators such as IL-1β, IL-6, TNF-α, and C-reactive protein (CRP), has been implicated in pre-eclampsia, gestational diabetes mellitus, fetal growth restriction, and spontaneous preterm birth (sPTB) (9–15). Gingival crevicular fluid (GCF), which bathes the periodontal sulcus, contains a dynamic mix of host immune factors, microbial byproducts, enzymes, hormones, and placental molecules, making it a promising, non-invasive biofluid for detecting systemic and local molecular changes during pregnancy (16, 17).

At the molecular level, angiogenic imbalance has been identified as a critical factor driving placental dysfunction and sPTB (18). Placental growth factor (PlGF) and its soluble antagonist, sFlt-1, are central regulators of vascular development and endothelial placental health (18, 19). While these molecules are predominantly studied in the context of placental and maternal vascular health (20), their role in peripheral tissues such as the gingiva remains largely unexplored. Angiogenesis is a fundamental component of periodontal tissue maintenance and repair, yet dysregulated angiogenic activity can exacerbate chronic inflammation, impair tissue integrity, and promote periodontal disease progression (21, 22).

The possibility that PlGF and sFlt-1 levels in the GCF reflect both local periodontal status and broader systemic vascular shifts represents an intriguing, underinvestigated avenue of research with clear clinical implications, especially considering their usefulness in the early prediction of perinatal diseases (20, 23, 24). Therefore, this study aimed to bridge the gap between periodontal and obstetric research by (1) evaluating the association between first-trimester gingival crevicular fluid PlGF/sFlt-1 ratios and the risk of sPTB, and (2) exploring the relationship between angiogenic markers in the GCF and periodontal disease severity during pregnancy. By elucidating these connections, this study seeks to support the development of novel, non-invasive oral biomarkers for the early identification of women at risk for adverse pregnancy outcomes, offering opportunities for timely intervention and promoting maternal, oral, and fetal health.

To our knowledge, this is the first study to analyze an imbalance of angiogenic factors in gingival crevicular fluid as early predictors of spontaneous preterm birth risk and their association with periodontal diagnosis during early pregnancy. The main conclusions that may be drawn from this study are as follows: (1) there was an imbalance in gingival crevicular fluid angiogenic factors, particularly sFlt-1, in pregnant women with periodontitis compared to those with gingivitis and those who were periodontally healthy; (2) sFlt-1 was reduced in the pregnant women who developed spontaneous preterm birth since early pregnancy; (3) sFlt-1 and the PlGF/sFlt-1 ratio, when combined with the plaque index score and median arterial blood pressure in multiparametric regression models, have potential utility as oral biomarkers for predicting the risk of preterm birth, with a reasonably AUC-ROC curve (0.78), indicating a potential association between periodontal health status and alterations in angiogenic factor levels during pregnancy.

Pregnancy promotes the expression of a proinflammatory and immunosuppressing phenotype (1–3). It is believed that this immune-inflammatory shift, accompanied by high levels of pregnancy-related hormones and combined with an impaired neutrophil function and a proangiogenic environment, may enhance gingival inflammation and the severity of periodontal disease (22, 28, 29). Furthermore, pregnancy hormones could favor a proangiogenic state by augmenting the amount of angiogenic factors and protecting macrophages from apoptosis, thus prolonging the angiogenic effect in periodontal tissues (29). We propose that sFlt-1 and PlGF are potentially involved in the gingival inflammation observed during pregnancy. In this sense, in the present study, we observed an increased expression of sFlt-1 and PlGF in pregnant women with periodontitis compared to those with gingivitis or healthy pregnant women. Furthermore, a correlation between an increase in both angiogenic factors and periodontal inflammatory clinical parameters, such as probing depth, clinical attachment loss, and bleeding on probing, was also observed.

PlGF is a member of the VEGF family, and it binds to Flt-1, VEGF-R1, or its soluble form (30). PlGF was originally identified in the placenta, contributing to trophoblastic growth and differentiation, but is now known to be expressed by multiple organs, such as the heart, lungs, thyroid, and muscles (31, 32). The PlGF functions described to date include acting as a powerful chemoattractant for monocytes and macrophages and contributing to the growth of vessels and the migration and survival of endothelial and bone marrow progenitor cells (33, 34). The plasma expression of PlGF is low in most healthy tissues but can be significantly increased in inflammatory conditions (33–35). It has also been linked to increased toll-like receptor signaling activity, producing a prolonged proinflammatory state, with the release of various proinflammatory cytokines and acute phase reactants (35). Flt-1 acts as a receptor for PlGF (36), and it has two forms: the transmembrane and its soluble form, which lacks cytoplasmic and transmembrane domains (37). Its main sources are endothelial cells, monocytes, and the placenta, and it prevents angiogenesis (38). Different studies suggest that altered levels of PlGF and sFlt-1 may contribute to impaired placental function, oxidative stress, and endothelial dysfunction, potentially leading to the onset of preterm labor (20, 23). An imbalance between the two angiogenic factors has been associated with various pregnancy complications, including sPTB (39, 40). Previously, we demonstrated that increased PlGF concentration in GCF was associated with the subsequent risk of gestational diabetes mellitus (GDM), where PlGF levels in GCF combined with first-trimester fasting glycemia were a predictor of GDM risk (AUC-ROC: 89%) (41). Moreover, sFlt-1 has also been associated with an increased risk of pre-eclampsia, with increased concentrations of sFlt-1 in the saliva and GCF samples of pre-eclamptic women compared to those who had a term delivery and healthy pregnancies (42).

We speculate that an imbalance in the PlGF/sFlt-1 ratio leads to increased vascular permeability and periodontal inflammation since early pregnancy. As an example, the binding of PlGF to Flt-1 in patients with rheumatoid arthritis stimulated the production of proinflammatory cytokines, including TNF-α and IL-6 in monocytes, enhancing inflammation (43). There is limited evidence regarding the direct role of the two angiogenic factors in pregnant women with periodontal disease. However, it has been described that the Flt-1 gene is highly upregulated in periodontitis-associated fibroblasts (21). In addition, Horton et al. investigated the sFlt-1 levels in maternal plasma in pregnant women before 26 weeks' gestation, but found no differences regarding periodontitis severity (44). Nevertheless, the specific mechanisms by which sFlt-1 and PlGF impact the severity of periodontal disease physiopathology are still being investigated, and the precise implications of the two angiogenic factors in the context of maternal periodontitis during pregnancy and the risk of adverse pregnancy outcomes remain to be fully elucidated.

The relationship between the severity of maternal periodontitis and PTB has been evaluated in previous studies, with the relative risk of PTB ranging from 1.6 to 3.4 (45–53). In this sense, a study conducted in 1,020 pregnant women before the 26th week of gestation found an RR of 1.6 (95% CI: 1.1–3.3) for moderate to severe periodontitis and PTB (45). Another study in over 1,000 pregnant women showed that those with periodontitis and preeclampsia had a higher risk of PTB with a hazard ratio of 4.11 and 11.00, respectively, when they were affected by moderate to severe periodontitis (46). In contrast, other prospective studies have not shown associations between periodontitis and PTB (47, 48). In this study, we did not find a direct association between maternal periodontitis and sPTB. However, we observed an increase in the severity of periodontal clinical parameters, including probing depth, percentage of periodontal pockets, and PISA, with more inflammation since the beginning of pregnancy in the women who subsequently had a sPTB compared to those who had a healthy term delivery.

Regarding their usefulness as early biomarkers for the prediction of PTB, the evidence indicates that plasmatic PlGF and sFlt-1 can be used separately, or the PlGF/sFlt-1 and sFlt-1/PlGF ratios can be used (18–20). Both ratios reflect an imbalance of angiogenic factors and vascular pathology in the placental tissues (39, 40). In the present study, we observed a similar trend, with an increased PlGF/sFlt-1 ratio found in the first-trimester GCF samples from pregnant women who had a sPTB. We propose that assessing the angiogenic vascular status in GCF through liquid biopsies in the first trimester of pregnancy represents a novel and non-invasive approach, furthering our understanding of the association between periodontitis and PTB and of early prediction of sPTB risk. Furthermore, the understanding of the intricate balance between angiogenic factors in periodontal tissues will be crucial for elucidating the relationship between periodontal disease severity and adverse pregnancy outcomes.

A critical limitation of our study is the small number of sPTB cases observed (n = 12, 3.4% of the cohort), which may affect the statistical power, precision of the estimates, and generalizability of the results. Although our multiparametric regression models yielded promising AUC values, the small event number raises the risk of model overfitting and wide confidence intervals. Future studies with larger and more diverse populations are essential to validate the diagnostic performance of GCF angiogenic markers in predicting sPTB. Ideally, a priori or post hoc power calculations should be incorporated in subsequent studies to formally assess whether the sample size provides sufficient power to detect clinically meaningful associations. Importantly, while we report significant associations between GCF angiogenic markers, maternal periodontal status, and sPTB risk, our findings should not be interpreted as evidence of causality. The observed relationships may be influenced by unmeasured confounding factors, and the cross-sectional nature of the early pregnancy sampling limits the ability to infer directionality. Future mechanistic and longitudinal studies are warranted to unravel the causal pathways linking local periodontal angiogenic changes to systemic pregnancy outcomes. Specifically, investigating the biological mechanisms, such as the potential translocation of inflammatory mediators or bacterial products from periodontal tissues to the systemic circulation, or the systemic vascular adaptations that may concurrently affect placental and periodontal microenvironments, would enrich our understanding of how oral health influences pregnancy outcomes.

Angiogenic factors could play a significant role in identifying women at risk of spontaneous preterm birth and may exacerbate periodontal inflammation during pregnancy. These findings should be further validated in subsequent prospective cohort studies that may yield valuable insights into oral biomarkers for the prediction of preterm birth and the pathophysiology of pregnancy periodontal disease and potentially pave the way for novel diagnostic and therapeutic strategies aimed at modulating angiogenic responses and inflammation within the periodontal microenvironment.