Neuron-specific enolase and angiogenic markers in preeclampsia

Preeclampsia (PE) is a dangerous complication that has been for many years a leading cause of maternal and perinatal mortality worldwide. Currently, the exact etiopathogenesis and mechanisms of PE are unclear [1].

It should be noted that PE occurs and can develop in the perinatal period (at 22 weeks of pregnancy), and coincides with the time of formation of the key structures of the central nervous system – neurocorticogenesis, maturation of the main cortical centers and associative pathways, neocortical regionalization, and formation of higher nervous activity and functions, active expression of  expression of foreign neuron-specific proteins (NSPs) at blood-brain barrier, when the structural and functional foundations for neurobiophysiological autonomy are formed in the fetus, as well as the neural correlates of consciousness and sensorimotor mapping, the ability to a wide range of cognitive functions, the foundation for integrated information processing on a global scale, response to stressors, that are  the basis for survival and viability [2].

Neuron-specific enolase (NSE), cerebrospinal fluid (CSF) S100B, Tau, Reelin, Neurofilament light chain (NfL), and other reflect the process of neurocorticogenesis, impairment of blood-brain barrier (BBB) integrity and permeability ​​in patients with PE [3–5]. Their elevated expression in the fetal brain and high levels in the maternal circulation in PE indicate abnormal neocortical neurogenesis and fetal neurodevelopment, fetal and maternal traumatic brain injury. These are the key etiopathogenetic factors of maternal-fetal neurogenic, neuroimmune conflict based on complement-mediated endothelial damage [2].

In the context of exploration of the neurogenic neuroimmune genesis of PE, it is relevant to investigate the relationship between the markers of fetal cortical neurogenesis and angiogenic imbalance as potentially valuable prognostic and diagnostic targets.

The objective of the study was to assess serum levels of neuron-specific enolase (NSE) and angiogenic factors – soluble FMS-like tyrosine kinase-1(sFLT-1) and placental growth factor (PlGF) in pregnant women with PE, in women at high risk of developing PE, and in women with normal pregnancy.

Materials and methods

This single-center study was conducted at V.F. Snegirev Clinic of Obstetrics and Gynecology, where 30 patients were selected and divided into three groups: the studied group comprised 10 patients with PE, the risk group comprised 10 patients with risk factors for developing PE, and the control group included 10 somatically healthy women. The study was conducted between November, 2025 and February, 2026. The method of continuous sampling was used. The patients who were admitted to hospital during conducting the study and who met the inclusion criteria were consequently enrolled in the study.

Inclusion criteria:

• For the main group of patients with PE: written informed consent from the patient to participate in the study; age over 18 years; singleton pregnancy; gestational age from 22 to 41⁺⁶; the diagnosis of PE according to the clinical guidelines of the Ministry of Health of Russia “Preeclampsia. Eclampsia. Edema, proteinuria and hypertensive disorders in pregnancy, childbirth and the postpartum period” of September 05, 2024.
• For the risk group patients: written informed consent from the patient to participate in the study; age over 18 years; singleton pregnancy; gestational age from 22 to 41⁺⁶, pregnancy complication; fetal growth restriction: gestational arterial hypertension, chronic arterial hypertension; isolated proteinuria, a history of early and/or severe PE, diabetes mellitus, obesity, antiphospholipid syndrome, pregnancy after assisted reproductive technology, a family history of PE, chronic kidney disease, connective tissue disease, factor V Leiden, high risk for PE according to the first screening results.
• For the control group: written informed consent from the patient to participate in the study; age over 18 years; singleton pregnancy; gestational age from 22 to 41⁺⁶weeks; normal pregnancy (without the signs of arterial hypertension, impaired utero-placental circulation, fetal growth restriction, kidney disease with proteinuria, isolated proteinuria, cardiovascular diseases, a history of liver and renal diseases, PE, HELLP syndrome).

Non-inclusion criteria were age below 18 years, multiple pregnancy; tobacco smoking (before and/or during pregnancy).

Exclusion criteria were patients’ refusal to participate in the study.

Venous blood samples from all patients were collected in Vacuette Premium blood collection tubes for subsequent determination of serum NSE, sFlt-1, PlGF levels and the sFlt-1/PlGF ratio. Blood samples were centrifuged for 10 minutes at 3000 rpm. Serum samples were then stored at -40°C until they were tested. The levels of marker spectrum were determined at the Interclinical Biochemical Laboratory of the Clinical and Diagnostic Center of Sechenov University using the enzyme-linked immunosorbent assay ELISA.

After getting approval of the Ethics Committee of Sechenov University (approval No. 23-25 от November 13, 2025, pregnant women who were admitted to the Obstetric hospital and met the inclusion criteria were invited to participate in the study. Written informed consent was obtained from all women who confirmed their willingness to participate in the study.

Statistical analysis

Statistical data analysis was performed using software program Jamovi Version 2.3.16.  The sample was analyzed using descriptive statistics. The quantitative variables (for example, blood pressure values, levels of serum markers) are represented as the median (Me) and interquartile range (Q1 and Q3). The categorical variables (for example, the presence of absence of arterial hypertension and proteinuria, age groups) were represented as absolute values (n) and relative frequencies (%). 

The clinical and anamnestic data were described to characterize the three groups. The intergroup comparison using statistical tests was not performed, since it was not the objective of the study.

The values ​​of the tested markers in the groups were compared using the nonparametric Kruskal–Wallis test. After finding statistical difference, pairwise comparisons of the groups were conducted using the Mann-Whitney U test with Bonferroni correction.  The level of statistical significance was set at p<0.05. For multiple comparisons with Bonferroni correction, the threshold value was set at p<0.017.

Results and discussion

The characteristics of patient sample based on the anamnestic and clinical data, as well as comparative analysis of neuron-specific and angiogenic markers in the groups are represented in Tables 1 and 2.

Distribution of marker levels (NSE, sFlt-1, PIGF) and the Flt-1/PIGF ratio in the three groups is represented in the box plots (Fig. 1–4).

The obtained results showed statistically significant differences in serum levels of neuron-specific and angiogenic markers between the groups of pregnant women with PE, high risk for developing PE, and women with normal pregnancy. This allows to consider neuronal factors as potential markers of this complication.

NSE as a marker of neuronal damage and impaired blood-brain barrier permeability

Comparison of NSE levels in the studied groups showed statistically significant differences. In the PE group, the median value of NSE was 13.5 (11.3; 14.0) μg/L, that was higher both in the high risk group – 8.0 (8.0; 8.8) μg/L (p<0.001), and in the control group – 4.0 (3.0; 5.0) μg/L (p<0.001). It is interesting to note that the gradient of serum NSE level in the control group increased towards the PE group. In women in the risk group, NSE level was significantly higher compared to the control group, that can indicate subclinical structural damage of neurons and impaired blood-brain barrier permeability before manifestation of the common symptoms of PE.

According to current data, NSE is a cytosolic enzyme found in neurons and neuroendocrine cells. When cell membranes are damaged, it is released into the blood [6, 7]. The studies by Bergman L. et al. demonstrated that in pregnant women with preeclampsia, the levels of NSE remain high throughout pregnancy, whereas in healthy women, these tended to decrease [8]. The authors reported that this phenomenon is associated with BBB integrity disruption and the leakage of brain-originated proteins into the peripheral circulation [9]. The data obtained in our study are fully consistent with these observations and to some extent support the concept that increased NSE in PE is not only an indicator of laboratory phenomenon, but also reflects pathogenetically significant neuronal damage.

Of particular interest is the fact that increased NSE level was found in the risk group. This suggests that impairment of structural and functional properties of BBB can precede the full clinical picture of PE. Due to this, exploration of NSE as an early prognostic marker is promising. In the study by Friis T. et al., human brain microvascular endothelial cell line (hCMEC/D3) was used. Their study found that plasma from women with PE directly increased BBB permeability, and this effect correlated with the levels of neurofilament light chain (NfL), Tau protein, NSE, and S100B [4]. Therefore, NSE can be considered not only as a marker of damage, but also as an indirect indicator of the functional status of BBB.

It is important to note that increased NSE level can have different pathogenetic trajectories and justifications depending on the phenotype of PE. It can be suggested that in early-onset PE (22–34 weeks of gestation), increased NSE is largely associated with impairment of fetal BBB against the backdrop of subcortical and cortical elements formation, immaturity of BBB, hypoxic-ischemic impairments associated with impaired placentation, hypoperfusion, and angiogenic imbalance. At the same time, in late-onset PE (after 34 weeks of gestation), high level of NSE can reflect the existing neuroimmune conflict with active production of maternal autoantibodies against the neuronal structures of the fetus, neocortex and subcortical-cortical connectivity. Unfortunately, the small sample size in our study did not lead us to perform stratified analysis of the time of manifestation of the disease, that determines the need for further exploration of this issue and increased number of patients in the subgroup with early-onset PE.

Angiogenic factors sFlt-1 и PIGF: the classic markers and their association with the phenotypes of PE

Analysis of angiogenic factors confirmed the known patterns. The highest level of sFlt-1 was found in the group of patients with PE – 8962 (6401; 13417) ng/ml, and it was significantly higher compared to the risk group – 8131 (7313; 8711) pg/ml (p=1.000) and the control group – 2448 (2232; 2794) pg/ml (p<0.001). The absence of statistically significant differences between the PE group and the risk group in sFlt-1 levels can be due to the heterogeneity of the risk group, and possibly due to the fact that some women developed the pathology at a later period.

The level of PIGF demonstrated an unexpected, but clinically significant pattern. Significantly reduced level of PIGF was found in the risk group – 96.0 (94.3; 101.0) pg/ml compared to the control group – 111.0 (102.0; 150.0) pg/ml (p=0.033). However, PIGF level in the PE group was 125.0 (113.0; 137.0) pg/ml, and did not differ from that in the control group (p=1.000). This may be due to the fact that the women were rather late included in the study, as well as due to the features of pregnancy course in this cohort of patients. It is known that serum PlGF level is significantly low in early-onset PE, which is associated with placental insufficiency and fetal growth restriction, whereas in late-onset PE, the imbalance between pro-angiogenic and anti-angiogenic factors is possibly less important, reflecting their key role in placental angiogenesis as a pathogenetic link predominantly of the early phenotype [10, 11]. This observation highlights the heterogeneity of PE and the need for a differentiated approach to assessment of the biomarkers depending on the time of manifestation of the disease.

The sFlt-1/PIGF ratio that is widely used in clinical practice for the prediction and diagnosis of PE, showed an interesting result for discussion as a differential angiogenic indicator. The median ratio was 72.0 (52.2; 103,0) in the PE group, 80.0 (75.3; 91.8) in the risk group, and 21.0 (17.0; 23.8) in the control group. The differences were significant between the PE group and the control group, as well as between the risk group and the control group (p<0.001). It is noteworthy that the median sFlt-1/PIGF ratio in high-risk group for PE was higher compared to the group of patients with overt PE. This observation can be explained by the heterogeneity of the risk group, and some women in the groups probably had the preclinical stage of the disease. The pathological angiogenic cascade, including increased sFlt-1 and decreased PIGF levels, was activated, but the classic clinical symptoms of PE (hypertension and proteinuria) had not yet developed by the time of patient inclusion in the study. Thus, the risk group reflects a spectrum of subclinical angiogenic imbalances preceding manifestation of the disease, whereas the PE group represents patients with identified clinical phenotype, that explains the observed differences in biomarker levels. However, the absence of statistically significant differences between the PE group and the risk group (p=1.000) indicates common pathogenic mechanisms and allows to identify the risk group as a valid diagnostic category.

Possible interpretation of the obtained data within the framework of the concept of neuroimmune genesis of PE

Of particular interest is the interpretation of the obtained results in the context of the new concept of the pathogenesis of PE. According to this concept, this complication is considered as maternal-fetal neuroimmune conflict based on complement-mediated endothelial damage during neurocorticogenesis period. The key issue of this concept is recognition of the fundamental role of the perinatal period, when neocortical structures mediated by NSP expression are being formed [2].

The obtained data demonstrate that in this critical period (the median gestational age of patient inclusion in the study was 34.2±3.1 weeks in the PE group) statistically significant increase in NSE occurs in maternal blood. According to the neuroimmune concept, when the integrity of the fetal BBB is compromised due to its immaturity or damaging factors, NSPs penetrate into maternal circulation, where they are perceived by the immune system as foreign antigens due to the absence of immunologic tolerance to them (“barrier proteins”), leading to the formation of circulating immune complexes and hyperactivation of the complement system and the formation of the membrane attack complex (MAC) C5b-C9, that causes endothelial damage. This damage is characterized as destructive, rather than dysfunctional. Due to this PE is distinguished from other hypertensive disorders, that is confirmed by electron microscopy results showing edema, vacuolization and endothelial cell desquamation, and detachment of endothelial cells from the basement membrane [12].

Interestingly, our study found no similar trends in intergroup gradients of NSE level and angiogenic factors, that can indicate the relative autonomy of the neurogenic and placental-angiogenic links in the etiopathogenesis of PE. At the same time, neuroimmune conflict may act as a trigger factor, and placental hypoperfusion as an enhancing or modifying factor. Moreover, it can be suggested that contribution of each of these links differs in early-onset and late-onset PE. The placental-angiogenic mechanism dominates in early-onset PE, while neuroimmune interactions come to the fore in late-onset PE.

Base on the results of our study we can consider NSE and angiogenic factors as potential markers reflecting key constructs embedded in the concept of neuroimmune genesis of PE. In the light of this concept, increased NSE level can be considered as a marker of compromised integrity of the fetal BBB and damage to neuronal structures, while pathological changes in the levels of sFlt-1 and PlGF can be considered as a marker of placenta-mediated angiogenic imbalance. This opens up prospects for further research aimed at assessing the prognostic value of NSE and angiogenic factors with regard to the development of different phenotypes of PE, as well as exploring these markers as potential diagnostic and therapeutic targets.

Clinical significance and prospects for further research

The obtained results suggest the potential for using neuron-specific markers (in particular, NSE) in complete medical examination of pregnant women at high risk for PE. A combination of classic angiogenic factors and neuromarkers can improve the accuracy of predicting both PE and its severity. At the same time, comparative assessment of the severity of maladaptation in neuronal-glial interactions and placental mechanisms can enable prediction of its phenotype.

Promising areas for further research include exploration of the differences in biomarker profiles between early-onset and late-onset PE and their comparative effectiveness in determining the risk for developing a specific PE phenotype; assessment of the prognostic value of neuromarkers with regard to long-term neurological outcomes in mothers and their offspring, as well as the search for therapeutic agents that can modulate the neuroimmune response and restore BBB integrity. Expanding the range of explored markers including the indicators of neurocorticogenesis (S100B, NfL, Tau, GFAP, and other), BBB integrity (claudin-5, ZO-1, and other) and complement activation (C5, C1, C3, FB, FD, and other) will make it possible not only a comprehensive approach to the diagnosis of PE as a neurogenic neuroimmune process, which is associated with maladaptation in neuronal-glial interactions and placental mechanisms, but also subsequent deeper understanding of etiopathogenesis of different phenotypes of PE.

Limitations of the study

Interpreting the results obtained, the limitations of this study should be taken into account. The small sample size (n=30) does not allow to perform stratified analysis based on phenotypes of PE (early-onset or late-onset PE, with or without fetal growth restriction). The lack of dynamic monitoring of marker levels throughout pregnancy limits the possibility to assess their prognostic value. Also, within the framework of this study, assessment of the markers of damage to the glial structures of the central nervous system, BBB, and dysregulation of complement system was not performed, that could significantly expand the understanding of their role as valuable prognostic markers reflecting the etiopathogenetic constructs of PE. Moreover, In the near future, it is worth to  consider the possibility of assessing the levels of neuroimmune markers in umbilical cord blood, that could confirm the concept of penetration of NSPs in fetal brain through the damaged BBB and placental barrier and their role in initiation of maternal immune response. This will make it possible to determine the involvement of the fetus and the fetal central nervous system in the pathological process and to establish correlations between maternal and fetal parameters, that is an important direction for further research. In addition, it should be noted that our study did not include clinical and morthological comparisons, reflecting the relationship between the changes in the levels on neuron-specific markers and impairments of structural and functional properties of the placenta.  In this context, it seems quite promising to perform morphological and immunohistochemical examination of placentas, including assessment of NSP levels, tight junction proteins, complement components, and angiogenesis factors in placental tissue, that will make it possible to verify the damage to the placental barrier and to identify morphological correlations with the level of the explored biomarkers.

Conclusion

Thus, the obtained data allow us to suggest the following:

• Statistically significant increase in serum NSE level was observed in pregnant women with PE, that can indicate impairment of neuronal structures and impairment of BBB integrity.
• Increased NSE level was found in the high-risk group, that potentially indicates the subclinical involvement of the brain structures in the pathological process even before manifestation of classic symptoms of PE.
• Combined assessment of NSE and angiogenic markers (sFlt-1, PIGF) will help to characterize more fully the pathogenetic mechanisms of PE, integrating placental-vascular and neuroimmune disturbances.

The obtained results indicate the feasibility of further study of NSPs as potential diagnostic and therapeutic targets, taking into account a differentiated approach to different phenotypes of the disease and possible introduction of neurogenic markers into widespread clinical practice.