Introduction
Recurrent spontaneous abortion (RSA), defined as two or more consecutive spontaneous abortions with the same sexual partner, is a prevalent complication of pregnancy, with its incidence escalating with increased frequency. Recurrent abortion is characterized by an intricate and diverse etiology, encompassing established factors such as chromosomal abnormalities, autoimmunity, anatomical anomalies, endocrine elements, and infections [1,2]. According to previous studies, the expression of KISS-1 in patients with spontaneous abortion decreases significantly; notably, KISS-1 plays an important role in pregnancy implantation and continuation [3]. However, a subset of patients experiencing recurrent miscarriage exhibit an unknown etiology. Several studies reveal a significant association between recurrent miscarriage and impaired blood vessel formation in the placental tissue [4,5]. Notably, vascular endothelial growth factor (VEGF) plays a pivotal role in promoting angiogenesis, exerting a positive influence on vascular remodeling during early pregnancy stages [6,7].
VEGF is involved in oocyte maturation, trophoblast cell proliferation, embryo implantation and development, placental angiogenesis, and maternal and fetal vessel growth in utero. Its absence or downregulation can cause vascular network aberrations in chorionic villi or anomalies in cell infiltration and differentiation, culminating in miscarriage [8]. Conversely, hypoxia-inducible factor-1α (HIF-1α) regulates trophoblast differentiation in response to oxygen tension, thereby influencing embryonic growth and development during early pregnancy [9,10]. Its expression primarily impacts energy metabolism, functional activity of trophectoderm cells, and embryonic vasculature formation in a hypoxic environment.
Matrix metalloproteinases (MMPs) are peptide chain endonucleases containing Zn2+, which are involved in extracellular matrix (ECM) degradation. Increased MMP expression in the recurrent miscarriage metaphase suggests that they may contribute to recurrent miscarriage via intercellular matrix degradation [11].
The significance of VEGF, HIF-1α, and MMPs in recurrent miscarriage has been reported in past studies; however, the correlation between their expression in the placental villous tissue remains unclear. To enhance the prevention and treatment of recurrent miscarriages, fundamental indicators should be measured and their fluctuations elucidated. Therefore, in this study, the correlations between VEGF, HIF-1α, and MMP2 expression in the placental villous tissue of patients who have experienced RSAs were analyzed, providing a clinical reference for the management and treatment of such patients.
Materials and methods
1. General information
Fifty-one patients with recurrent miscarriage who attended the Fourth Hospital of Shijiazhuang between January 2022 and December 2023 were selected for this study (RSA group). Their age ranged from 23-43 years, with a mean age of 29.78±4.02 years. The gestation period varied from 56-73 days, with a mean of 61.78±3.05 days. The inclusion criteria were adherent to the diagnostic standards outlined in the expert consensus on the diagnosis and management of recurrent miscarriage: 1) history of three or more spontaneous miscarriages; 2) gestation period of 6-12 weeks; 3) no history of cardiovascular, endocrine, or immune system diseases; 4) clinical symptoms indicating a history of amenorrhea with little or no vaginal bleeding; and 5) ultrasound suggesting intrauterine pregnancy without germ or cardiac tube pulsation. Fifty cases of voluntary early pregnancy termination within the same timeframe were selected as the control group. These participants ranged in age from 20-38 years, with a mean age of 28.68±3.13 years. The gestation period ranged from 55-74 days, with an average of 60.88±2.41 days. The inclusion criteria were: 1) gestation period of ≤12 weeks; 2) ultrasound showing normal embryonic development with visible embryo and fetal heartbeat; 3) no previous history of abortion; and 4) no cardiovascular, endocrine, or immune system disorders, or other pregnancy complications.
Sample size assessment is the estimation of the minimum number of observed cases required for a clinical trial to ensure that the conclusion of a particular clinical trial is scientific, accurate, and reliable. In this study, the minimum sample size required was calculated to satisfy statistical precision and reliability (controlling class I errors and ensuring inspection efficiency). It was found that each group must include at least 50 individuals. The study was approved by the Ethics Committee of the Fourth Hospital of Shijiazhuang (approval no: 20210002), and clinical specimens were collected in accordance with the Declaration of Helsinki of the World Medical Association. Informed consent and signed confirmation were obtained from the patients and their families.
2. Main reagents and instruments
The total tissue RNA extraction kit (CoWin Biotech Co., Beijin, China) was obtained from Beijing Kangwei Century Biotechnology Company (CoWin Biotech Co.). The polymerase chain reaction (PCR) reverse transcription into cDNA (PrimeScriptTM RT Reagent kit; Takara Bio Inc., Shiga, Japan) and PCR amplification kits (SYBR® Premix Ex TaqTM II; Takara Bio Inc.) were purchased from TaKaRa (Takara Bio Inc.). Additionally, the bicinchoninic assay (BCA) protein concentration assay kit (Beyotime Biotech Inc., Shanghai, China) was procured from Shanghai Biyuntian Biotechnology Company. The electrochemiluminescence color development kit was acquired from Invitrogen (Invitrogen, Carlsbad, CA, USA). Rabbit anti-VEGF polyclonal antibody (Abcam, ab2350), rabbit anti-HIF-1α polyclonal antibody (Abcam, ab228649), rabbit anti-MMP2 polyclonal antibody (Abcam, ab97779), and sheep anti-rabbit immunoglobulin G-horse radish peroxidase (HRP) secondary antibody (Cell Signaling Technology Inc., Danvers, MA, USA; cat: #7054) were utilized. Laboratory instruments such as electrophoresis tank and electrotransformer were obtained from Bio-Rad (Bio-Rad Laboratories Inc., Hercules, CA, USA). The ChemiDoc XRS chemically enhanced luminescence detection system (Bio-Rad Laboratories Inc.) and Gel-Doc gel imaging analysis system (Bio-Rad Laboratories Inc.) were used. The Image J image processing software (National Institutes of Health, Bethesda, MD, USA) was used for image analysis.
3. Hematoxylin and eosin (HE) staining
Placental villous tissues were washed with normal saline to remove blood, fixed with 4% paraformaldehyde for 48 hours, dehydrated with gradient alcohol, embedded in paraffin, and then cut into 4-mm thick slices. The sections were dewaxed in xylene and gradient alcohol, stained with hematoxylin for 5 minutes, and then immersed in hydrochloric acid for a few seconds. Following eosin staining for 2 minutes, the staining effect was observed under the microscope. Finally, xylene penetration and resin sealing were performed. All sections were observed under a microscope.
4. Sample collection and storage
Placental villous tissues were collected from both groups after abortion, rinsed with saline to remove all blood cells, and stored in freezing tubes at −80°C for subsequent total RNA and protein extraction.
5. Quantitative real-time polymerase chain reaction (qRT-PCR)
Placental villous tissue specimens were cryogenically ground, and subsequently, total RNA extraction was executed using a designated RNA extraction kit (CoWin Biotech Co.). The optical density (OD) 260/280 ratio was assessed to ascertain the content, integrity, and purity of the RNA. Subsequently, cDNA was synthesized from the extracted RNA serving as a template for the subsequent qRT-PCR reaction using a PCR reverse transcription kit (Takara Bio Inc.). The qPCR procedure followed the manufacturer’s protocol for the SYBR Green Master Mix (Takara Bio Inc.). The reaction system comprised a final volume of 20 μL, incorporating 10 μL SYBR Premix Ex Taq II (Tli RNaseH Plus; Takara Bio Inc.), 2 μL cDNA, 0.8 μL each of upstream and downstream primers, and 6.4 μL nucleasefree water. The primer sequences are presented in Table 1. The PCR reaction conditions included pre-denaturation at 95°C for 2 minutes, denaturation at 95°C for 30 seonds, annealing at 60°C for 1 minute, extension at 72°C for 15 seonds, all repeated for 40 cycles. Utilizing the glyceraldehyde 3-phosphate dehydrogenase gene as an internal reference, the target mRNA expression levels in the samples were calculated utilizing the 2−ΔΔCT method. All procedures were strictly adherent to the provided kit instructions, with PCR experiments being repeated thrice.
6. Western blotting
The Trizol kit (Invitrogen, Carlsbad, CA, USA) was used to extract total protein from the villous tissue. The total protein was quantified using the BCA method. Subsequently, the protein samples were standardized based on their measured content to ensure uniform protein quantities in equivalent sample volumes, thereby maintaining consistency. Then, the protein samples were subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis gel electrophoresis, polyvinylidene fluoride (PVDF) membrane transfer, and subsequent closure with Tris-buffered saline containing 0.5% (v/v) tween 20 solution containing 5% skimmed milk powder at room temperature for 2 hours. The PVDF membranes were later subjected to overnight incubation at 4°C in a primary antibody solution (dilution 1:500). On the subsequent day, the membrane was removed, the primary antibody was discarded, and the PVDF membrane was subjected to three 10-minute washes. It was then immersed in an incubation solution with HRP-labelled secondary antibody (dilution 1:50,000) and incubated at 37°C for 2 hours. After discarding the secondary antibody, the membrane was washed thrice for 10 minutes each time. Semi-quantitative analysis of the expressed bands was performed using the Image J medical image analysis software (National Institutes of Health), with the results expressed as mean optical density values OD values. The ratio of the OD of the target factor to the OD of the internal reference (β-actin) was employed to interpret the acquired data.
7. Statistical analysis
Statistical analysis was conducted using the SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). Data were presented as the mean±standard deviation and analyzed using the t-test. The correlation between VEGF, HIF-1α, and MMP2 mRNA and protein expression in placental villous tissue was analyzed using Pearson’s method. A P-value of <0.05 was considered statistically significant.
Results
1. Comparison of general information of pregnant women between the two groups
General information such as age (years), gestational duration (days), and body mass index was assessed separately, and the analysis revealed no statistically significant differences between the groups (P>0.05) (Table 2).
2. Pathological morphological changes of placental villi in patients with RSA using HE staining
The histological morphology of placental villi in both cohorts was examined using HE staining. As illustrated in Fig. 1, compared to the control group, the placental villi in the RSA group exhibited variability in size, irregular villous outlines, and an increased presence of interstitial cells within the villi. Additionally, trophoblast cells displayed polarity, and certain villi showed signs of interstitial edema.
3. Comparison of VEGF, HIF-1α, and MMP2 mRNA levels between the placental villous tissues of two groups
Relative to the control group, the mRNA levels of VEGF in the placental villous tissue decreased significantly among patients with RSA (P<0.05). Conversely, the mRNA expression levels of HIF-1α and MMP2 were observed to be relatively high in individuals with a history of RSA (P<0.05) compared to the control group (Table 3, Fig. 2).
4. Comparison of VEGF, HIF-1α, and MMP2 protein levels between in the placental villous tissues of two groups
Compared to the control group, the protein levels of VEGF were significantly reduced in the placental villous tissue of the patients in RSA group (P<0.05), consistent with the mRNA analysis results. Similarly, the protein levels of HIF-1α and MMP2 were significantly elevated in the RSA group patients (P<0.05) compared to those in the control group (Table 4, Fig. 3).
5. Correlation of VEGF, HIF-1α, and MMP2 expression between the placental villous tissues of patients with RSA
Pearson analysis revealed a lack of significant correlation between the mRNA expression of VEGF, HIF-1α, and MMP2 in the placental villi of the patients in the RSA group (Fig. 4A-C). Conversely, the western blot results indicated that the levels of VEGF protein were inversely correlated with those of MMP2 in the placental villous tissue (r=−0.375; R2=0.141; Fig. 4D), while the levels of HIF-1α protein exhibited a positive correlation with MMP2 (r=0.350; R2=0.123; Fig. 4F). Consistent with the mRNA findings, no significant correlation was observed between VEGF and HIF-1α expression at the protein level (Fig. 4E). Detailed results of the correlation analysis are presented in Table 5.
Discussion
RSA is a prevalent complication during pregnancy but the mechanisms involved have not been fully elucidated. Given the challenging clinical treatment and high medical costs involved, it significantly impacts the physical and mental well-being of women and their families. In a normal pregnancy, neovascularization and the proliferation of trophoblast cells occurs within the placental villous tissue [12] with VEGF acting as a specific mitogen and survival factor for endothelial cells to mediate angiogenesis under both physiological and pathological conditions. VEGF stimulates the proliferation and differentiation of vascular endothelial cells but abnormalities in angiogenesis and/or its function may promote the onset of a spontaneous abortion [13,14].
The significant decrease in VEGF mRNA and protein expression in the chorionic and metaphase tissues of patients who experienced an RSA [15] suggests that reduced VEGF expression plays a vital role and is therefore a potential target for drug development. The N6-methyladenosine modulator plays a central role in stromal/immune cell interactions at the maternal/fetal interface, potentially inducing RSA by regulating VEGF-mediated stromal cell-macrophage interactions [16]. This is consistent with our findings indicating that VEGF mRNA and protein expression are significantly lower in the placental villous tissue of patients who had experienced an RSA. However, Pang et al. [16,17] reported that soluble Fms-like tyrosine kinase (sFlt-1) and VEGF protein expression were significantly higher in the serum and chorionic villous tissues of patients with unexplained RSA compared to those who experienced a healthy early pregnancy abortion. Similarly, Xu et al. [18] noted that the activation of the Fas/FasL signaling pathway promotes trophoblast apoptosis through oxidative stress and is associated with inhibition of the NOTCH1 signaling pathway and upregulated sFlt-1 and VEGF expression. Therefore, the present study was designed to explore and compare VEGF expression to that of HIF-1α and MMP2 in the placental villous tissue of patients with RSA.
HIF-1α, as a hypoxia-inducible transcription factor, governs the adaptive transcriptional response to hypoxia via interaction with hypoxia response elements. Research demonstrates that VEGF serves as a target gene of HIF-1α, whereby HIF-1α, particularly in hypoxic conditions, fosters vascular neogenesis by enhancing VEGF expression [19]. A recent study has shown that the expression of HIF1α mRNA increases in placental tissue samples of patients with recurrent miscarriage [20]. Notably, the expression of HIF-1α in the endometrium of patients with RSA was significantly higher than that of normal fertile women and positively correlated with microvascular density (MVD), suggesting that endometrial hypoxia and altered neovascular MVD could contribute to recurrent miscarriage [21]. Our results corroborated these findings, showing that the expression levels of HIF-1α mRNA and protein in the placental villous tissue of patients with RSA were markedly higher than those in the control group, signifying a hypoxic state in the villous tissue of patients with RSA, thus aligning with prior research. Similarly, a study by Liu et al. [22] showed that the levels of HIF-1α mRNA in the chorionic tissue of patients in the missed abortion group were higher than those in the normal pregnancy group, and the levels of VEGF mRNA were lower than those in the normal pregnancy group. Moreover, Pearson correlation analysis showed that the expression of p53 was positively correlated with HIF-1α and negatively correlated with VEGF in the missed abortion group. However, another study showed that VEGF and HIF-1α expression in chorionic villi was significantly higher in patients with ≥4 miscarriages than in patients with ≤3 miscarriages, and Spearman correlation analysis revealed that the expression of miR-29a was positively correlated with VEGF and HIF-1α expression [23]. Our study is consistent with that by Liu et al. [22]; the results show that mRNA and protein levels of VEGF decreased significantly, while HIF-1α was higher in the chorionic tissues of patients than in those of the normal pregnancy group. Interestingly, there was no significant correlation between VEGF and HIF-1α in the chorionic villous tissue of patients in the RSA group; however, this result should be validated and confirmed using a larger sample.
MMPs constitute a class of zinc ion-dependent proteases that actively participate in the degradation and remodeling of the ECM, playing a partial role in angiogenesis. MMPs are associated with invasive diseases and angiogenesis, with MMP2 and MMP9 identified as key enzymes facilitating the invasion of the endometrium by trophoblasts during early pregnancy [24,25]. The balanced expression of MMP2/9 and their inhibitors (tissue inhibitors of matrix [TIMP] 1/2) is necessary to sustain a successful pregnancy [26]. Upregulated MMP2 and MMP9 in the endometrium of women affected by infectious RSA is accompanied by an imbalance of MMPs/TIMPs, implying that infection triggers excessive degradation of the endometrial matrix, leading to miscarriage [27]. Nissi et al. [28] reported a significant increase in the levels of MMP9 and the MMP2/TIMP-2 complex in the serum of patients with RSA. Furthermore, a recent meta-analysis provided evidence of a significant association between the MMP2-735T and MMP9-1562T alleles and the risk of RSA [29]. A case-control study in the Chinese Han population also revealed a significant association between MMP2 and MMP9 polymorphisms in the promoter region and the risk of RSA [30]. The findings of this study were consistent with the aforementioned ones, demonstrating significantly elevated levels of MMP2 mRNA and protein in placental villous tissues of patients with RSA compared to those in the control group. These results informed the hypothesis that MMP2 overexpression induces excessive degradation of the endometrial matrix, consequently inhibiting the invasion and migration of villous tissue, ultimately resulting in recurrent miscarriage. The negative correlation between VEGF and MMP2 expression and the positive correlation between HIF-1α and MMP2 expression suggests the involvement of these elements in the development and progression of RSA but further studies are required to fully elucidate the mechanism.
This study has certain limitations. The relatively small sample size may have limited the reliability and accuracy of the results, thus necessitating larger multicenter studies in the future. Second, RSA is inherently heterogeneous and encompasses a spectrum of etiologies, clinical presentations, and pathophysiological mechanisms. This heterogeneity is evident in its diverse underlying causes, including chromosomal abnormalities, genetic anomalies, autoimmune disorders, endocrine dysfunction, anatomical abnormalities, infectious agents, and idiopathic factors [31]. Particularly, chromosomal abnormalities in embryos are a common cause of RSA. Anomalies in the chromosomal structure or quantity may compromise normal fetal development, leading to a natural miscarriage. In the future, we will conduct embryo chromosome testing and basic etiological screening for all enrolled patients with recurrent miscarriage. For patients with recurrent miscarriage whose etiology is unclear or whose embryos have chromosomal abnormalities, the expression of VEGF, HIF-1α, and MMP2 in the placental villi will be detected, expecting to provide a theoretical basis for the pathogenesis of patients with recurrent miscarriage.
In conclusion, our study demonstrated low VEGF expression in the placental villous tissue of patients with RSA, concurrent with the elevated expression of HIF-1α and MMP2. Although all three elements appear to play crucial roles in the mechanism of RSA development, further investigation is warranted to delineate their specific molecular regulatory mechanisms and pathways.
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