Dysregulation of synaptogenesis in fetal growth restriction

Fetal growth restriction (FGR) remains one of the most clinically significant challenges in contemporary obstetrics, associated not only with a high burden of adverse perinatal outcomes [1–4] but also with the development of long-term neurological sequelae. [5–8]. Chronic placental insufficiency and sustained fetal hypoxia disrupt normal development of the fetal CNS, with the severity and character of these disturbances dependent on the gestational age at FGR onset [9–11].

In recent years, considerable attention has been directed toward extracellular vesicles of neuronal origin [12, 13], as they enable the non-invasive assessment of fetal brain status in a liquid biopsy format [14, 15]. Our previous work demonstrated that various forms of FGR are associated with altered expression of neurogenesis-related proteins within FNVs [16–18], and that these alterations correlate with early neonatal complications, potentially reflecting the emergence of fetal neurological injury.

The present study extends this line of investigation to the subsequent stage of neuroontogenesis, synaptogenesis [19, 20]. Synaptic formation and maturation are essential for the integration of neurons into functional networks, and disruptions at this stage may underlie subsequent cognitive, motor, and behavioral disorders [19, 21].

This study aimed to evaluate the expression of synaptic proteins within FNVs in early- and late-onset FGR and determine their associations with neonatal complications.

Materials and methods

This study was conducted at the V.I. Kulakov NMRC for OG&P of the Ministry of Health of the Russian Federation and was approved by the Institutional Research Ethics Committee. Written informed consent was obtained from all the participants. A total of 120 pregnant women were enrolled in the study. The study group comprised 60 patients with a postnatally confirmed diagnosis of FGR: 30 with early-onset FGR and 30 with late-onset FGR. The control group consisted of 60 pregnant women matched by gestational age at delivery. Obstetric management and delivery were conducted in accordance with the clinical guidelines on «Insufficient fetal growth requiring maternal care (fetal growth restriction)» (2025), approved by the Ministry of Health of the Russian Federation.

The inclusion criteria were as follows: singleton pregnancy at 22–41 weeks of gestation complicated by FGR (study group) or uncomplicated by FGR (control group); maternal age 18–45 years; and provision of written informed consent. The exclusion criteria were as follows: pre-eclampsia, multiple gestation, severe maternal comorbidities (including autoimmune and oncological conditions), sexually transmitted infections, and fetal chromosomal abnormalities or congenital malformations.

The assessment of synaptic protein expression involved the isolation of the FNV subpopulation by immunoprecipitation, followed by electrophoretic protein separation and detection using western blotting. Densitometric analysis was performed using the Bio-Rad ImageLab 6.0 software.

Neonatal anthropometric parameters, including birth weight and length, were assessed using established criteria, the INTERGROWTH-21st standards, and Fenton growth charts. Neonatal growth restriction was diagnosed according to the consensus-based definition of growth restriction in newborns. All neonates underwent cranial ultrasonography (neurosonography).

Statistical analysis

Statistical analysis was performed using StatTech v. 4.7.0 software (Stattech LLC, Russia). The distribution of continuous variables was assessed using the Shapiro–Wilk test. Normally distributed data are presented as mean (M) and standard deviation (SD), and non-normally distributed data are presented as median (Me) with interquartile range (Q1; Q3). Comparisons of three independent groups on non-normally distributed continuous variables were performed using the Kruskal–Wallis one-way analysis of variance. Categorical variables across the three groups were compared using Pearson's chi-squared test for contingency tables. Where statistically significant differences were identified (p<0.05), post-hoc analysis was conducted. Pairwise comparisons of proportions were performed using Bonferroni correction, applying an adjusted significance threshold of p<0.017. Correlation analysis was performed using Spearman's rank correlation coefficient. Correlation strength was classified as follows: 0–0.3, weak; 0.3–0.7, moderate; >0.7, strong. Statistical significance was set at p<0.05.

Results

The clinical and anamnestic characteristics of the pregnant women are shown in Table 1.

Patients with early-onset FGR had a significantly higher prevalence of chronic arterial hypertension (p=0.012) and higher pre-pregnancy body weight (p=0.006) than those in the control group. Ultrasound and Doppler assessments demonstrated statistically significant between-group differences in fetal abdominal circumference (p<0.001), estimated fetal weight (p=0.003), and uterine artery pulsatility index (p<0.001) measurements. Similar findings were observed for late-onset FGR (Table 2).

During the early neonatal period, newborns with early FGR demonstrated a significantly higher incidence of disseminated intravascular coagulation (DIC) syndrome (p<0.001), central nervous system (CNS) depression syndrome (p<0.001), and intraventricular hemorrhage (IVH) detected by neurosonography (p<0.001). Notably, 50% of these cases (15/30) had grade 2 IVH. In late-onset FGR, grade 1 IVH was diagnosed in 10% (3/30) of the cases and grade 2 IVH in 3% (1/30).

Analysis of the expression of synaptogenesis-related proteins within fetal neuronal vesicles (FNVs) in early-onset FGR demonstrated a statistically significant decrease in SYN1 (-0.89; 0.55; p≤0.001) and SYT1 (-0.74; 0.49; p≤0.04) levels compared with the control group (Fig. 1). SYP levels were also significantly elevated (0.59; -0.72; p≤0.001) in the early-onset FGR group. No significant differences were observed in SYNPO or PSD95 levels. In late-onset FGR, a distinct pattern of protein expression changes was identified (Fig. 2). This group demonstrated a significant reduction in SYN1 expression (-0.74; 0.91; p≤0.001), whereas SYNPO (0.62; -0.77; p≤0.03) and SYP (0.82; -0.70; p≤0.02) levels were significantly increased compared with those in the control group. Changes in SYT1 and PSD95 expression in the late-onset FGR group were not statistically significant.

A comparison of the proteomic profiles of early- and late-onset FGR (Fig. 3) revealed the most pronounced differences in SYP expression and distinct involvement patterns of individual synaptic proteins. SYP levels were significantly higher in late-onset FGR, whereas reduced SYN1 expression was common to both forms. While decreased SYT1 expression was predominantly associated with early-onset FGR and increased SYNPO expression was more characteristic of late-onset FGR, these differences did not reach statistical significance.

Particular interest was directed toward the relationship between the levels of the investigated proteins and the hemodynamic parameters. Correlation analysis in early-onset FGR demonstrated a strong positive correlation between SYN1 and the pulsatility index in the left uterine artery (r=0.95), and a strong negative correlation between PSD95 and the same parameter (r=-0.88). In addition, SYNPO and SYP levels were negatively correlated with the cerebroplacental ratio (CPR) (r=-0.76) (Fig. 4, Table 3).

In late-onset FGR, PSD95 levels were negatively correlated with the pulsatility index in the right uterine artery (r=0.79), whereas SYT1 levels were positively correlated with the pulsatility index percentile in the umbilical arteries (r=0.76) (Fig. 5, Table 4).

Among the neonatal complications identified in newborns with growth restriction, alterations in synaptic protein expression were associated with CNS depression syndrome and IVH. In early-onset FGR, newborns with CNS depression syndrome demonstrated reduced expression levels of SYN1 (-0.67; 0.52; p≤0.01) and PSD95 (-0.8; 0.37; p≤0.04), along with increased SYP expression (0.5; -0.7; p≤0.005) (Fig. 6A). In cases of IVH, a statistically significant reduction in SYN1 levels was observed (-0.89; 0.55; p≤0.01) (Fig. 6B). In late-onset FGR, statistically significant changes were identified only in association with IVH. This group demonstrated significantly reduced levels of SYN1 (-1.18; 0.54; p≤0.004) and SYT1 (-1.48; 0.66; p≤0.007), accompanied by increased SYNPO (0.98; -0.64; p≤0.02) and SYP (1.09; -0.32; p≤0.004) expressions (Fig. 7B).

Logistic regression models were developed to assess the probability of IVH development in newborns with growth restriction (Fig. 8), demonstrating high sensitivity and specificity (Table 5).

Discussion

The findings of this study demonstrate that fetal growth restriction (FGR) is associated not only with impaired neurogenesis but also with a protein profile reflecting the dysregulation of synaptogenesis. Synaptogenesis is not completed during the intrauterine period; rather, it is a dynamic process that actively continues after birth, supporting the maturation of neuronal networks [19–21]. Accordingly, the observed changes in the composition of FNVs may reflect not only the acute detrimental effects of chronic placental insufficiency and hypoxia but also the long-term risk of impaired postnatal synaptic network formation [21–23].

Early-onset FGR was associated with decreased SYN1 expression. Given the role of SYN1 in regulating synaptic vesicle transport and neurotransmitter release, altered expression of this protein may indicate reduced presynaptic transmission [24, 25]. The observed increase in SYP expression may represent a compensatory response involving alterations in the composition of secreted FNVs under chronic hypoxia [19, 26, 27].

Notably, there was an association between synaptic protein levels in both early- and late-onset FGR and IVH. In late-onset FGR, alterations affected all assessed synaptic proteins, including SYN1, SYT1, SYNPO, and SYP. These findings may reflect differences in the pathogenesis of FGR phenotypes and the impact of these alterations on the developing fetal brain.

The observed correlations between synaptic protein expression, Doppler parameters, and neonatal complications support their potential clinical significance. Logistic regression and receiver operating characteristic (ROC) curve analyses identified SYN1 and SYT1 as potential biomarkers for predicting IVH in growth-restricted neonates.

Conclusion

These findings highlight the significance of synaptic proteins in FNVs for the noninvasive monitoring of neuroontogenetic processes in fetuses with growth restriction. The use of these biomarkers during pregnancy may complement existing methods of longitudinal fetal surveillance and contribute to risk stratification for neurological complications in newborns.