Introduction

Pre-eclampsia (PE) is the leading cause of maternal mortality worldwide and is a significant cause of perinatal mortality. It is also an important cause of perinatal mortality, with a prevalence of approximately 5–7% in all pregnant women1,2. It is a severe stage of hypertensive disorder of pregnancy (HDP), which is often accompanied by organ or systemic dysfunction involving hepatic, renal, and cerebral dysfunction, and defective placentation may be an important pathogenic factors3,4,5. Therefore, early identification and active treatment, reducing disease severity, and delaying disease progression are important to improve maternal and infant outcomes.

The placenta is a unique exchange organ between the mother and baby, and is crucial for successful pregnancy and fetal health. Defective or absent transformation of the uteroplacental artery myometrial segment can lead to reduced maternal blood flow through the placenta in PE6. Although placental pathology has long revealed that placental villi are highly vascularized structures with hierarchical branches7,8,9, direct assessment of placental blood flow has always been limited because of the low blood flow velocity of placental villi. In recent years, with the continuous development of ultrasound diagnostic instruments, such as superb microvascular imaging10,11,12 and microvascular flow imaging13, the sensitivity of detecting low-speed blood flow has increased, making it possible to directly assess placental blood flow.

High resolution flow (HR-Flow) improves the spatial resolution of color Doppler by continuously sampling of flow velocities in combination with zone sonography technology14, and provides non-invasive visualization of placental blood flow to predict and diagnose pathological conditions. However, there is no consensus regarding the influence of placental villous vascularization on PE.

The aim of our study was to assess the value of HR-Flow ultrasonography in determining the vascularization of placental villi, such as the degree of placental villous branching, pulsatility index (PI), resistance index (RI), and ratio of peak systolic flow velocity to end diastolic flow velocity (S/D) of secondary villi in late pregnancy to predict PE.

Materials and methods

Study participants

In this retrospective case–control study, singleton pregnant women with HDP treated at the Affiliated Yixing Hospital of Jiangsu University from January 2022 to December 2023 were selected as the research group. According to the “Gestational Hypertension and Pre-eclampsia: ACOG Practice Bulletin, Number 222”15, patients were divided into the hypertension group (including gestational hypertension and pregnancy complicated with chronic hypertension) and the PE group (including PE and chronic hypertension accompanied by PE).

We used early collected PI data as a calculation reference and estimated that each group of PE and hypertension would require at least 12 (11.8) cases. Considering that the descriptive variables were presented as frequency and percentage, we selected 40 cases of HDP, with approximately 20 cases in the PE group and 20 cases in the hypertension group. Forty healthy pregnant women were matched based on gestational week as the control group during the same period. When there was more than one matched pregnant woman, the one with the closest age was chosen. This study included a total of 80 participants (Fig. 1).

Fig. 1
figure 1

Flow diagram of the participants. PE: pre-eclampsia; HDP: hypertensive disorder of pregnancy.

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The exclusion criteria for the women were as follows: (1) verification of gestational age < 28 weeks through the last menstrual period and nuchal translucency examination; (2) twins and above pregnancy; (3) abnormal placental morphology or position, such as velamentous cord insertion, accessory placenta, placental implantation, and placenta previa; (4) abnormal uterine morphology, such as unicorn uterus and mediastinal uterus; and (5) loss to follow-up. Clinical data and outcomes for mothers and neonates were obtained from the clinical electronic case records system.

Ethical considerations

All women participated in this study voluntarily. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Research Ethics Committee of the Affiliated Yixing Hospital of Jiangsu University (Data of approval: lunshen eighth Technology 2021). Informed consent was obtained from all the patients.

Ultrasound techniques

In this study, all placental screenings were performed by a single ultrasonographer with more than ten years of experience in ultrasound and participated in the National Birth Defects Prevention and Control Talent Training Project.

Mindary Resona 8S and Resona R9 ultrasound diagnostic instruments were used, and a 1–5 MHz convex probe was selected. Pregnant women were placed in the supine position and underwent placental HR-Flow examination after completing routine prenatal ultrasound to observe placental villous blood flow.

Identification of placental villous vascularization

Based on the pathological anatomy and vascular casting7,8,9, the blood flow analysis of placental villous branching in this study was as follows: (1) Primary branch: the umbilical artery enters the chorionic villus and continues to form the chorionic villous artery, which then centrifuges and branches into the placental parenchyma to form the thick stem chorionic villous artery, also considered as the primary branch. Each placenta has an average of approximately 4–9 branches, with a diameter of approximately 3.6–5.6 mm. (2) Secondary branch: each primary branch has an average of approximately 3–4 branches, with a diameter of 1.4–2.0 mm; (3)Third branch: each secondary branch has an average of approximately 3–4 branches, with a diameter of approximately 0.1 mm; (4) Fourth branch: the branches of the third villi, with a diameter of about 0.01 mm, is a bud-like expanding structure about 0.1 mm long. In this way, the 1–4 degree branches form a hierarchical branching villous tree-like structure that extends from the fetal placental side to the maternal placental side.

When clicking the HR-Flow button on the instrument screen to start the examination, the ultrasonographer scans the entire placenta in order from left to right and from top to bottom, selects a primary branch in the middle, left, and right regions of the placenta as the observation starting point, and counts the degree of placental vascular branching respectively.

The counting results of the middle part of the placenta were used for statistical analysis, whereas the counting results of the left and right sides were used to analyze the placental villous blood flow. Samples were then taken from the secondary branch of placental villous blood flow with a sampling volume of 2 mm, a speed of 5–7 cm/s, and an angle of 0–60°. When 3–5 repeated continuous waveforms were obtained, the images were frozen, and the RI, PI, PSV, and EDV were measured. Each parameter was measured three times (once in the left, middle, and right regions), and the average value was calculated (Fig. 2).

Fig. 2
figure 2

Analysis of the placental vascularization. (A) The gross observation of the umbilical artery to the chorionic artery, and the thick stem chorionic villous artery which continues as the primary branch in the placenta. (B) The primary branching arteries continuously branch to form a tree like structure. (C,D) The HR-flow and waveforms from a PE pregnant woman at gestational age of 37W. (E,F) The HR-flow and waveforms from a gestational hypertension pregnant woman at gestational age of 33W. Double headed arrow: chorionic artery; White solid arrows: thick stem chorionic villous artery, and the primary branch within the placenta; Orange solid arrow: secondary branch; Orange humble arrow: third branch; White humble arrow: fourth branch; W: week; PE: pre-eclampsia; HR-Flow: high resolution flow.

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Statistical analysis

Statistical software (SPSS 19.0) was used to analyze the data. Visual (histogram and probability plots) and analytical methods (Kolmogorov–Smirnov/Shapiro–Wilk test) were used to analyze whether the variables were normally distributed. The quantitative data with normal distribution were expressed as mean ± standard deviation (x ± s). Differences between groups were tested using analysis of variance and the least significant difference method. For numerical data with non normal distribution, descriptive analysis was conducted using median and quartiles (Q1–Q3). The Mann–Whitney U tests were performed to compare these parameters among the groups. Descriptive analyses were conducted using frequency and percentage for the categorical variables. Analyze the relationship between categorical variables using the Chi-square test or Fisher’s exact test (when chi-square test assumptions do not hold due to low expected cell counts). The value of each indicator for predicting PE was determined using a receiver operating characteristic (ROC) curve. The sensitivity, specificity, and area under curve (AUC) value were calculated when a significant cut-off value was observed. P < 0.05 was indicative of a significant difference.

Results

In our study, 80 pregnant women were ultimately included in the statistical analysis, including 18 cases in the hypertension group with 13 cases of gestational hypertension and 5 cases of pregnancy complicated with chronic hypertension, 22 cases in the PE group with 13 cases of PE and 9 cases of chronic hypertension complicated with PE, and 40 cases in the control group.

Comparison of general clinical data among three groups of pregnant women

There were no statistically significant differences in general clinical data among the three groups of pregnant women, such as advanced age (≥ 35 years), multiple pregnancies (≥ 3 pregnancies), and multiparity (≥ 3 deliveries) (all p > 0.05) (Table 1). In addition, there were 10, 6, and 6 cases of anterior wall placenta, posterior wall or bottom wall placenta, and lateral wall placenta in the PE group; 9, 5, and 4 cases in the hypertension group; and 23, 9, and 8 cases in the control group, respectively. There were no statistically significant differences in the distribution of the placental positions (p > 0.05). There was no statistically significant difference in the degree of the villous branching in the middle, left, and right regions of the placenta (p > 0.05).

Table 1 Comparison of clinical characteristics among three groups of pregnant women.
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Comparison of placental villous blood flow in three groups of pregnant women

There was a statistically significant difference in the degree of placental villous branching among the three groups of pregnant women (Χ2 = 132.684, p < 0.001), and there was a trend change among the PE, hypertension, and control groups. The control group showed a higher degree of branching of the placental villi, the PE group had sparse branches, and the hypertension group was in the middle. This difference was statistically significant (linearΧ2 = 33.983, p < 0.001) (Table 2). In addition, all 22 cases in the PE group showed primary branches, 19 cases showed secondary branches, and both primary and secondary branches were observed in the hypertension and control groups. There were no statistically significant differences in the display rates of the primary and secondary branches among the three groups (all p > 0.05).

Table 2 The degree of placental villous branches in three groups of pregnant women.
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There were no statistically significant differences in PI, RI, and S/D between the PE group and hypertension group (all p > 0.05), but they were all higher than those in the control group, and the differences from the control group were statistically significant (all p < 0.05) (Table 3).

Table 3 Comparison of blood flow parameters of the placental secondary branch among the three groups of pregnant women. Data are presented as mean ± standard deviation.
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Comparison of pregnancy outcomes among three groups of pregnant women

The body mass of newborns in the PE group was significantly lower than that in the hypertension and control groups (p < 0.05). The premature birth and cesarean section rates were higher than those in the hypertension and control groups (both p < 0.05). However, there were no statistically significant differences in neonatal body mass, premature birth rate, or cesarean section rate between the simple hypertension and control groups (all p > 0.05) (Table 4).

Table 4 Pregnancy outcomes of three groups of pregnant women.
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The predictive value of placental villous blood flow parameters for PE

The ROC curve showed that the degree of placental vascular branching, PI, RI, and S/D of the secondary villi had moderate predictive value for PE (Table 5). The sensitivity of PE was predicted to be the highest (95.5%) when the parallel PI value was ≥ 0.625 with branch degree ≤ 2.5, and the specificity of PE was predicted to be the highest (93.1%) when the series RI value was ≥ 0.485 with branch degree ≤ 2.5.

Table 5 ROC curves of the parameters for predicting PE.
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Discussion

In this study, we investigated the hemodynamic changes of the placenta in late pregnancy with HDP by using high resolution flow ultrasonography. The main finding was that: the PI, RI, and S/D of the secondary placental villi in HDP were higher than those in normal controls, regardless of PE or simple hypertension, however, in PE, the degree of the placental villous branches was significantly lower.

HR-Flow imaging is a low-velocity flow color Doppler application produced by the Mindary ultrasound equipment manufacturer, providing a new method for revealing the flow in small vessels. In this study, we used HR-Flow imaging to evaluate the placenta and found significant changes in placental villus hemodynamics during Doppler ultrasound examination in singleton pregnant women with hypertension in the late stage of pregnancy. Among them, the RI, PI, and S/D of pregnant women with simple blood pressure elevation were higher than those in the control group, indicating that the placental villous artery was in a high resistance state. This may be related to vascular spasm caused by hypertension, leading to increased placental vascular resistance16. Although adverse pregnancy outcomes in pregnant women with hypertension were slightly higher than those in the control group, the difference was not statistically significant. This may be related to the fact that there was no statistical difference in placental branch perfusion between the pregnant women with hypertension and the control group. This suggests that if pregnant women with HDP show a simple blood pressure elevation, placental branch perfusion is not significantly reduced. When PE occurred, the third and fourth degree branches of the placental villi were significantly reduced, perfusion was significantly decreased, and the RI value of the second villi did not further increase, but the PI value was significantly increased. Simultaneously, flow velocity indicators such as PS, ED, and TAMAX decreased. This may indicate that pregnant women with HDP gradually experience occlusion of placental villi from far to near as the disease progresses or the course of the disease is longer, and the decrease in placental perfusion leads to a series of complications such as hypoxia, infarction, or premature abruption. This may be related to the incomplete erosion of the spiral artery by trophoblast cells during placental formation, resulting in insufficient recasting of the uterine spiral artery17,18,19. Studies have shown that the circular RNA hsa_circ_0005579, which inhibits the proliferation, migration, and invasion ability of trophoblast cells, is highly expressed in the placenta of PE patients20. Some scholars have pointed out that vascular damage caused by PE may be more pronounced in early gestational age, as the new angiogenic biomarker PFN1, is significantly higher in the PE group, and especially higher in the preterm birth subgroup with PE21. In addition, it may also be related to maternal immune imbalance, maternal self-state, higher inflammatory parameters, abnormal glucose and lipid metabolism, etc., which can cause or exacerbate maternal fetal damage22,23,24,25,26,27,28.

There was no statistically significant difference in the display rates of the primary and secondary branches among the three groups in this study, which is consistent with the literature. Peker et al.8 also reported no significant difference in the diameter and number of cotyledons contained in the primary and secondary branches of placental villi in pregnant women with PE using the vascular casting technique on placental specimens. This may be related to the supporting role of the primary and secondary branches of the placenta7. In this study, three patients had HR-Flow only primary branch display, including two cases of severe PE and one case of fetal intrauterine distress and neonatal asphyxia. This suggests that displaying only the primary branch indicates the need for emergency clinical treatment; however, such cases are rare and require further research.

In addition, this study found that, compared with RI and S/D, PI predicted a larger area under the PE curve, indicating a higher diagnostic efficiency of PI. This may be related to the fact that PI can not only reflect the peak systolic and diastolic flow velocities but also the average flow velocity of the entire cardiac cycle, which can better represent the overall situation of the blood flow waveform. Additionally, compared with other indicators, there was a gradient increase in PI in the control, hypertension, and PE groups, indicating a positive correlation between the PI value of the secondary branch of the placental villi and HDP severity. Villi branching is negatively correlated with HDP severity, therefore, serial or parallel evaluation of different indicators can achieve high specificity or sensitivity, which may provide the possibility for direct ultrasound evaluation of PE after 28 weeks of pregnancy.

This study has certain limitations: (1) it was a single-center study; and (2) the number of cases was relatively small, so there may be some selection bias. Future research team should expand the number of cases and comprehensively evaluate the diagnostic efficacy of various examinations in combination with other clinical parameters.

Conclusions

Ultrasound HR-Flow detection of placental villous blood flow parameters can predict PE and can be used for screening and evaluating PE in pregnant women with HDP in late pregnancy.