Recurrent spontaneous abortion is a complex condition with multifactorial etiology, and its specific causes and pathogenesis have been a hot topic of research. Currently, many theories suggest that endothelial dysfunction and placental ischemia-hypoxia are important factors leading to spontaneous miscarriage in pregnant women [7, 8]. The function of endothelial cells is regulated mainly by proangiogenic factors and antiangiogenic factors. Numerous studies have shown that placental growth factor (PLGF) and soluble fms-like tyrosine kinase-1 (sFlt-1) play important roles in pregnancy and in adverse pregnancy outcomes. Placental growth factor (PLGF) is a member of the vascular endothelial growth factor (VEGF) family. It is mainly secreted by syncytiotrophoblasts and is also expressed in the villous stroma and villous vessels. It is an important angiogenic factor. Due to its high homology with VEGF, PLGF binds to VEGF receptor-1 (VEGFR-1) and activates signaling pathways through autophosphorylation to exert its biological effects. VEGF receptor-1 (VEGFR-1) is divided into membrane-bound and soluble forms. The soluble form, known as soluble fms-like tyrosine kinase-1 (sFlt-1), is expressed in the placenta. sFlt-1 is an endogenous antagonist of VEGF that blocks receptor tyrosine kinase activation and plays an important role in endothelial dysfunction. Excess sFlt-1 in the circulation binds with high affinity to VEGF and PLGF, neutralizing their effects and reducing their concentrations in the circulation.

In the normal process of pregnancy, syncytiotrophoblasts invade the spiral arteries of the uterus early in pregnancy, and syncytiotrophoblasts secrete proangiogenic factors, thereby promoting placental vascular formation and remodeling and facilitating the normal development of the placenta and embryo. In the 1970s, some scholars believed that the impairment of trophoblast invasion and spiral artery remodeling was the cause of uteroplacental circulation defects and subsequent placental ischemia [9]. In 2009, Burton et al. successfully simulated damage to the chorionic villi due to failure of spiral artery remodeling [10], demonstrating that true placental ischemia may only occur late in the disease. Most current theories [11, 12] have suggested that various stressors, such as oxidative stress, inflammation, and possibly mechanical shear stress, contribute to the release of sFlt-1. When excess sFlt-1 enters the maternal circulation, it antagonizes VEGF-A and PlGF, causing endothelial dysfunction. The impaired development of vascular endothelial cells leads to impaired formation and branching of placental blood vessels, resulting in poor formation of the placental vascular network and ultimately leading to placental ischemia-hypoxia. This condition makes it difficult to provide a favorable environment for fetal survival, leading to various adverse pregnancy outcomes, including miscarriage, intrauterine fetal demise, preterm birth, pregnancy-induced hypertension, preeclampsia, and intrauterine fetal growth restriction. Similarly, the antiangiogenic state induced by excess production of sFLT-1 from the placenta can be rescued by administration of VEGF-A and PlGF [13].

The uterine artery is the main blood supply to the uterus and placenta, and uterine artery blood flow parameters can directly reflect blood perfusion in the placenta and embryo. Trophoblast invasion and subsequent remodeling of the uterine spiral arteries are important processes in placental formation during pregnancy [14]. The downregulation of PLGF and upregulation of sFlt-1 leading to endothelial damage results in impaired development of the endometrium and the muscle layer near the endometrium. This leads to poor infiltration of trophoblast cells into the muscle layer arteries, causing impaired uterine artery perfusion, ischemia, and subsequent ischemia‒reperfusion, leading to a strong oxidative stress response in the embryo and resulting in impaired development, degeneration, stagnation, and, in severe cases, miscarriage. Second, ischemia‒reperfusion injury caused by impaired placental blood perfusion affects the generation and release of vascular active factors in the body, and some inflammatory cytokines enter the patient's systemic circulation through the villous gaps, leading to systemic inflammatory reactions in the patient and further damage and destruction of the vascular endothelium, ultimately leading to adverse pregnancy outcomes such as miscarriage, pregnancy-induced hypertension, and preeclampsia.

Clinically, uterine artery blood flow perfusion can be measured by uterine artery blood flow parameters (RI, PI, and S/D). Numerous studies have shown that uterine artery blood flow parameters are significantly greater in patients with recurrent miscarriage than in healthy pregnant women.

Studies have shown that both aspirin and LMWH can reduce trophoblast apoptosis. During pregnancy, these agents can reduce uterine artery resistance parameters, significantly improve pregnancy outcomes, and reduce miscarriage rates [15]. Aspirin inhibits platelet aggregation and increases prostaglandins, achieving a good anticoagulant effect [16]. LMWH inhibits the activity of coagulation factors [17, 18], reduces blood viscosity, improves blood circulation, prevents microthrombosis, reduces local oxidative stress reactions in the placenta, regulates the intrauterine environment, increases blood supply to the placenta and embryo, reduces miscarriage and fetal death, and improves pregnancy outcomes [19]. Recent studies have also shown that aspirin and LMWH, in addition to their good anticoagulant effects, can promote trophoblast proliferation, invasion, and differentiation; inhibit trophoblast apoptosis; protect vascular endothelium; and promote placental formation [20, 21]. This study analyzed the uterine artery blood flow parameters and serum PLGF and sFlt-1 levels of patients with recurrent spontaneous abortion treated with aspirin alone, aspirin combined with LMWH, or no medication to evaluate their relationship with pregnancy outcomes and explore the impact of medication on patients with recurrent spontaneous abortion.

Uterine artery blood flow parameters

The results of this study revealed changes in uterine artery blood flow parameters between 30 and 32 weeks of pregnancy. There were no significant differences in the data between the aspirin group, combined drug group, and control group, while the non-drug group continued to have higher uterine artery blood flow parameters (including the pulsatility index, resistive index, and peak systolic velocity/end diastolic velocity) than did the other three groups. This may suggest that after 30 weeks of pregnancy, the use of aspirin or aspirin combined with low molecular weight heparin in women with recurrent spontaneous abortion is no different from that in normal pregnant women. However, further research is needed to determine whether discontinuing drug treatment at this time leads to changes in uterine artery blood flow parameters and pregnancy outcomes. On the other hand, in the non-drug group, from before pregnancy to 32 weeks of pregnancy, uterine artery blood flow parameters were all greater than those in normal pregnant women, and there was no change at the 30-week pregnancy mark. This indicates that early and timely treatment is crucial for patients with recurrent miscarriage.

Serum placental growth factor (PLGF) and soluble fms-like tyrosine kinase-1 (sFlt-1)

There are reports in the literature that the serum level of placental growth factor (PLGF) in nonpregnant patients is extremely low. After pregnancy, the PLGF level slowly begins to rise. In normal pregnant women, the serum PLGF concentration gradually increases with gestational age, peaking at approximately 28-30 weeks of gestation, after which it gradually decreases to 55-65 pg/ml in late pregnancy. This is mainly because PLGF is mainly secreted by the syncytiotrophoblast cells of the placenta. In early pregnancy, the placenta is immature, the placental circulation is incomplete, and the placenta is in a hypoxic state, leading to lower PLGF levels. As the placental blood flow circulation improves, the placental vasculature gradually changes from a branching structure to a nonbranching structure, and PLGF regulates the formation of nonbranching blood vessels. When the placenta is most mature, the PLGF also peaks. After 30-32 weeks of gestation, as the placenta matures and begins to age, the PLGF gradually decreases.

On the other hand, soluble fms-like tyrosine kinase-1 (sFlt-1) levels do not change significantly in early to mid-pregnancy but gradually increase in late pregnancy. Therefore, the sFlt-1/PLGF ratio is lowest in mid-pregnancy [22, 23]. This study selected the mid-pregnancy period, specifically at 30-31+6 weeks, when the PLGF levels were the highest and the sFlt-1/PLGF ratio was the lowest, to monitor the serum PLGF and sFlt-1 levels and compare the sFlt-1/PLGF ratios. The results showed that the serum PLGF level in the nondrug group was significantly lower than that in the other three groups, while there was no significant difference in the serum PLGF level among the other three groups. The serum sFlt-1 level was significantly greater in the nondrug group than in the other three groups, with no significant difference among the three groups. Studies have shown that monitoring decreased serum PLGF and increased sFlt-1/PLGF in early pregnancy is associated with an increased risk of miscarriage [24]. Similarly, monitoring decreased serum PLGF and increased sFlt-1/PLGF in mid-pregnancy is associated with a significantly increased risk of developing gestational hypertension and preeclampsia. In this study, there was no significant difference in the serum PLGF and sFlt-1/PLGF levels between the aspirin group and the combined therapy group, while the untreated group showed decreased serum PLGF and increased serum sFlt-1 and sFlt-1/PLGF levels, which may indicate that both single-agent aspirin and aspirin combined with low molecular weight heparin can correct these levels to those of normal pregnant women, achieving normal serum factor levels.

Pregnancy outcomes

This study examined the rates of live birth, miscarriage, and pregnancy complications. The miscarriage rates in patients ranked from highest to lowest were as follows: non-drug group > aspirin group > combined drug group > control group. Subgroup comparison analysis revealed a significant difference in the miscarriage rate between the aspirin group and the combined drug group. This demonstrates that aspirin combined with low molecular weight heparin (LMWH) can effectively reduce the miscarriage rate in patients with recurrent spontaneous abortion, and the effect is superior to that of aspirin alone. This result is consistent with the findings of Peter Clark [25] and YU X M [26], who reported that the live birth rate in the combined medication group was greater than that in the aspirin group, suggesting that the combination of LMWH and aspirin is more effective at improving the miscarriage rate than aspirin alone.

For pregnancy complications, only gestational hypertension and preeclampsia differed among the four groups. The incidence of other pregnancy complications, including liver dysfunction, gestational diabetes, preterm birth, fetal growth restriction, placenta previa, hypothyroidism, placental abruption, and neonatal asphyxia, did not significantly differ among the four groups. The incidence of gestational hypertension and preeclampsia in the nondrug group was greater than that in the other three groups, while there was no significant difference in incidence among the other three groups. This finding is consistent with our findings for mid-pregnancy serum PLGF, sFlt-1, and uterine artery blood flow parameters. Patients with low PLGF, high sFlt-1, and high uterine artery blood flow parameters had a correspondingly increased incidence of gestational hypertension and preeclampsia.

Previous studies by Liu Xiaoning and others have shown that pregnant women with preeclampsia have significantly lower serum PLGF levels than women in the control group in early pregnancy (11-13+6 weeks of gestation), and their uterine artery blood flow parameters are significantly greater than those of women in the control group [27,28,29,30]. Hasko et al. [31] studied serum PLGF, sFlt-1, and sFlt-1/PLGF levels in 164 women with preeclampsia and 36 women with gestational hypertension and reported that the sFlt-1/PLGF ratio in the preeclampsia group was significantly greater than that in the normal control group; thus, the sFlt-1/PLGF ratio could be used as a good diagnostic tool for distinguishing preeclampsia in pregnant women. These results are consistent with the findings of our study.

The predictive value of uterine artery blood flow parameters, serum placental growth factor (PLGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and the sFlt-1/PLGF ratio for pregnancy outcomes

In this study, PLGF showed poor predictive efficacy for predicting hypertensive pregnancy disorders, while sFlt-1 demonstrated good predictive value, with the sFlt-1/PLGF ratio showing even better predictive value than sFlt-1 alone. When uterine artery blood flow parameters (mRI, mPI, mS/D) were combined with serum PLGF, sFlt-1, and sFlt-1/PLGF, the predictive value for pregnancy-induced hypertension and preeclampsia was significantly improved. Reports in the literature indicate that the sFlt-1/PlGF ratio is a better screening tool than serum PLGF or sFlt-1 alone [32,33,34,35,36]. Studies by Liu Tingting and others have shown that combining uterine artery ultrasound with PLGF and other indicators can effectively improve the accuracy of diagnosing preeclampsia in early pregnancy and mid-pregnancy [37, 38]. This is consistent with the results of this study.

One of the primary limitations of our study is its retrospective nature, which inherently carries the risk of selection bias. Patients were not randomly assigned to treatment groups; instead, treatment decisions were made based on clinical indications, physician judgment, and patient preferences. While this reflects real-world practice, it introduces the possibility that unmeasured confounding factors could influence the observed outcomes. We have taken this into account in our interpretation of the results, and we advise caution in generalizing these findings. Future prospective studies are warranted to further explore and confirm the efficacy of these treatment strategies in patients with recurrent spontaneous abortion.