Preeclampsia (PE) is a complex, multi-organ disorder typically emerging after 20 weeks of gestation or postpartum, characterized by new-onset hypertension and proteinuria or significant end-organ dysfunction [1]. Although its etiology is not fully understood, a widely accepted two-stage model proposes that inadequate trophoblast invasion and disordered uterine spiral artery remodeling lead to placental ischemia/hypoxia, followed by systemic maternal endothelial dysfunction driven by an imbalance of circulating angiogenic and anti-angiogenic factors [2].

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases crucial for extracellular matrix (ECM) remodeling during reproductive processes, including gametogenesis, implantation, angiogenesis, placentation, and parturition. MMP activity is tightly regulated transcriptionally, via pro-enzyme activation, and through inhibition by tissue inhibitors of metalloproteinases (TIMPs). Dysregulation of the MMP/TIMP balance has been implicated in cardiovascular disorders, including PE [3].

This study focused on a key group of MMPs involved in trophoblastic invasion into the maternal uterine and vascular remodeling, specifically MMP-2 (Gelatinase A), MMP-9 (Gelatinase B), and MMP-3 (Stromelysin-1), along with their primary endogenous inhibitors, TIMP-2 (inhibits MMP-2) and TIMP-1 (inhibits MMP-9). MMP-2 and MMP-9 degrade various ECM components, including collagen types IV, V, VII, and X, as well as gelatin. MMP-3 targets collagen, proteoglycans, laminin, and fibronectin [3]. MMP-2 plays a vital role in implantation, as well as systemic and uterine vasodilation, MMP-9 in vascular basement membrane remodeling, and MMP-3 in trophoblast motility and activation of other MMPs, like MMP-1, MMP-7, and MMP-9, thereby amplifying ECM degradation [3, 4].

Altered levels of MMPs and TIMPs have been reported in the plasma, amniotic fluid, and endothelial cells of women with PE, suggesting a potential role in the disease’s pathogenesis through both uteroplacental and vascular dysfunction. However, prior studies have evaluated individual MMPs or TIMPs at specific time points during pregnancy, without accounting for their dynamic changes throughout gestation or their regulatory interactions [5, 6, 7]. This limitation reduces the translational value of such findings. In the present study, we measured plasma concentrations of MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2, as well as the MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios, in both normal and preeclamptic pregnant women during the second and third trimesters. We further assessed their associations with clinical indicators of PE and performed receiver operating characteristic (ROC) curve analysis to determine their trimester-specific predictive value.

MMP-2 (Gelatinase A), expressed by decidual cells and trophoblasts, degrades key ECM components. It is secreted in a latent form (pro-MMP-2), and its activation involves TIMP-2 and MT1-MMP (MMP-14) in a complex process in which TIMP-2 can both facilitate activation and inhibit active MMP-2. MMP-2 is crucial for early implantation, and systemic and uterine vasodilation to ensure adequate fetal perfusion, but its activity is tightly regulated by TIMP-2, maintaining a balance essential for normal placentation [3, 4, 8].

Our finding of significantly increased plasma MMP-2 and TIMP-2 levels in PE patients compared to controls in both trimesters and overall, aligns with previous studies [9, 10]. However, the MMP-2/TIMP-2 ratio declined significantly in the PE group in the third trimester and overall, which may reflect its role in PE pathogenesis. Placental ischemia/hypoxia may stimulate vascular endothelial growth factor (VEGF) production, subsequently upregulating MMP-2 expression [11]. Elevated MMP-2 may also be mediated by interleukin-8 (IL-8) from endothelial cells under a chronic inflammatory conditions [12]. However, the reduced MMP-2/TIMP-2 ratio might indicate decreased proteolytic potential of MMP-2, resulting in impaired trophoblast invasion and vascular remodeling [3]. Additionally, the MMP-2 dependent vasodilatory effect is even more pronounced in PE compared to normal pregnancies or non-pregnant women. This suggests that inhibition of MMP-2 by TIMP-2 may decrease its vasodilatory capacity and increase the risk of vasoconstriction, exacerbating hypertensive symptoms [8]. Thus, altered MMP-2/TIMP-2 balance appears linked to both early and later stages of PE.

Notably, MMP-2 showed strong predictive value for PE in the second trimester (AUC = 0.817), consistent with findings by Bahabayi et al. [13], although our optimal cut-off (260.8 ng/mL) differed slightly, potentially due to population variations. Combining MMP-2 with TIMP-2 further improved second-trimester prediction (AUC = 0.893). In the third trimester, TIMP-2 emerged as the best single predictor (AUC = 0.807), supported by studies linking TIMP-2 (often combined with insulin-like growth factor binding protein-7 [IGFBP7]) to PE-related acute kidney injury [14].

MMP-3 (Stromelysin-1) is secreted in a zymogen form (pro-MMP-3) by decidual stromal cells, macrophages, and extravillous trophoblasts. Pro-MMP-3 is activated by serine proteases like trypsin and plasmin via proteolytic cleavage of its propeptide domain. During pregnancy, MMP-3 facilitates the directional migration of trophoblasts into maternal tissues by cleaving IGFBP-1, a key regulator of trophoblast motility. Additionally, MMP-3 contributes to ECM remodeling both directly, by degrading ECM components, and indirectly, by activating other MMPs [4, 15].

We found significantly elevated MMP-3 levels in patients with PE throughout pregnancy, consistent with previous studies [6, 16]. A proposed pathogenesis involves stressed syncytiotrophoblasts secreting pro-inflammatory cytokines, including interleukin-1$\beta$ (IL-1$\beta$) and tumor necrosis factor alpha (TNF-$\alpha$), which upregulate MMP-3 gene expression by inhibiting histone deacetylation, and simultaneously enhance the enzymatic activity of serine proteases, thereby promoting the conversion of pro-MMP-3 to its active form. Elevated MMP-3 might contribute to or reflect both maternal and fetal complications in early-onset PE, such as fetal growth restriction, lower gestational age at birth, and lower birth weight [6, 16]. In this study, we also observed that MMP-3 correlated positively with plasma creatinine and proteinuria level, suggesting a link to renal involvement in PE. However, these mechanistic insights require further investigation and validation using functional assays in vitro or in vivo.

MMP-3 contributed significantly to PE prediction in the second trimester with high specificity. Although trimester-specific MMP-3 data are limited, Laskowska [6] also suggested its relevance for early-onset PE, a form of the disorder associated with placental dysfunction.

MMP-9, expressed at the maternal-fetal interface, degrades ECM components and activates VEGF, playing roles in angiogenesis, particularly in microvascular development and tissue remodeling during labor [7, 17]. Its activity is inhibited by TIMP-1, and the MMP-9/TIMP-1 balance regulates trophoblast invasion [8].

We observed significantly lower plasma MMP-9 levels and a lower MMP-9/TIMP-1 ratio in the PE group, while TIMP-1 levels were not significantly different. This finding contrasts with studies reporting increased TIMP-1, but aligns with others demonstrating decreased placental MMP-9 expression and lower MMP-9/TIMP-1 ratios in hypertensive pregnancies [7, 18]. The strong positive correlation between MMP-9 and TIMP-1 (r = 0.773) in our PE group suggests a potential coordinated regulation or compensatory mechanism. Reduced MMP-9 activity could impair vascular remodeling and excess collagen deposition, leading to abnormal spiral artery development and contributing to placental ischemia. Syncytiotrophoblast stress resulting from ischemia might further suppress MMP-9 expression via inflammatory mediators, potentially creating a detrimental feedback loop [3]. Consistent with studies in hypertension, we found positive correlations between MMP-9, TIMP-1, and SBP in PE patients [18, 19].

We evaluated the trimester-specific predictive value of MMP-9, TIMP-1, and the MMP-9/TIMP-1 ratio; however, their performances were limited (AUC 0.5). Interestingly, the MMP-2/MMP-9 ratio demonstrated stronger predictive potential for PE during the second trimester, which is consistent with previous findings [20]. This ratio may serve as a more promising biomarker for future validation.

This study has some limitations that should be considered. First, it was conducted at a single center, which may limit the generalizability of the findings. Second, the use of a single blood sample provides only a snapshot of biomarker levels and does not reflect the dynamic changes that occur throughout pregnancy; therefore, longitudinal follow-up is necessary. Third, the study did not include functional assays such as zymography to evaluate the enzymatic activity of MMPs, which would have provided deeper mechanistic insight into their biological roles. Despite these limitations, our findings highlight trimester-specific alterations in MMPs and TIMPs in PE. The strong predictive performance of the combined MMP-2 and TIMP-2 in the second trimester, and of TIMP-2 alone in the third trimester, supports their potential utility as components of a multi-marker panel for PE risk assessment or diagnosis. While plasma analysis may not fully reflect placental events, its feasibility supports potential integration into prenatal screening protocols. Further longitudinal studies in larger and more diverse populations are needed to validate these findings and explore their clinical applicability.

Plasma levels of MMP-2, and TIMP-2, along with the MMP/TIMP ratio, are significantly altered in women with PE compared to controls, showing distinct trimester-specific patterns. Elevated MMP-2 and TIMP-2 levels, particularly their combination in the second trimester, and elevated TIMP-2 levels in the third trimester, show promise as potential predictive biomarkers for PE. These findings warrant further validation before clinical application.

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

TTN and NAL designed the research study. NAL, TDD, TNDN, TNTN, and SHD performed the research. TNTN, TDD and NAL analyzed the data. TNTN and TTN drafted the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The study was carried out in accordance with the guidelines of the Declaration of Helsinki. This study was reviewed and approved by the Ethics Committee of the Hanoi Obstetrics & Gynecology Hospital (Approval No. 734 CN/BVPS – TT ĐT CĐT, Date: June 24, 2022). Informed consent was obtained from all individual participants included in the study.

This research was funded by the Hanoi Department of Science and Technology (01C-08/14-2021-3).

The authors declare no conflict of interest.

During the preparation of this work, the authors used ChatGPT-4o in order to check spelling and grammar. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.31083/CEOG42755.