Analysis of the guidance and predictive value of uterine artery flow parameters in patients with recurrent spontaneous abortion

To analyze the uterine artery blood flow parameters of patients with recurrent spontaneous abortion (RSA) at different gestational ages and to investigate the effects of aspirin and low molecular weight heparin (LMWH) on uterine artery blood flow parameters and pregnancy outcomes.


INTRODUCTION
The definition of recurrent spontaneous abortion varies by country. For example, the American Society for Reproductive Medicine defines recurrent spontaneous abortion as two or more failed pregnancies before 20 weeks of gestation 1 and the European Society of Human Reproduction and Embryology defines recurrent spontaneous abortion as loss of two or more pregnancies. 2 In contrast, the Royal College of Obstetricians and Gynecologists defines recurrent spontaneous abortion as three or more consecutive occurrences with the same Xiaolu Lian and Zhansheng Pan are co-first authors. sexual partner and pregnancy loss before 24 weeks of gestation. Recurrent spontaneous abortion in China, that is, in this study is defined as two or more consecutive pregnancy losses with the same spouse before 28 weeks of gestation. 3 Overall, the etiology of recurrent spontaneous abortion is very complex. Recent studies have found that an increasing number of patients with recurrent spontaneous abortion have increased uterine artery resistance, which affects endometrial receptivity, causing embryo implantation failure or difficulty in maintaining early pregnancy. It also influences placental blood perfusion, leading to local oxidative stress in the placenta, formation of microthrombi, and gradual apoptosis of trophoblast cells, ultimately causing pregnancy failure. 4 In addition to anticoagulant and antithrombotic effects, aspirin and LMWH have recently been shown to promote trophoblast cell proliferation, invasion and differentiation and to inhibit trophoblast apoptosis. 5 However, the effects of aspirin and LMWH on uterine artery blood flow have rarely been investigated. Therefore, this study investigated the effects of aspirin and LMWH on uterine artery blood flow parameters in patients at different gestational ages.

Subjects
This study analyzed clinical data for 254 patients treated at the First Hospital of Soochow University Reproductive Medicine Center from December 2020 to May 2021 using a retrospective cohort design. A total of 140 patients were ultimately enrolled through screening of basic conditions and clinical medications. The patients were divided into three groups on the basis of clinical medication regimens: 44 patients without a history of miscarriage were recruited for controls; 96 patients with a history of two or more consecutive spontaneous abortions with the same spouse before 28 weeks of gestation were enrolled in the RSA group and divided into the aspirin group (47 patients) and the aspirin combined with LMWH group (49 patients) ( Figure 1). This study was approved by the Ethics Committee of the First Affiliated Hospital of Soochow University (Approval No. 121(2022)).

Research methods
Aspirin group: Oral aspirin should be taken when serum HCG >5 after 4 weeks of pregnancy (aspirin entericcoated tablets, CENSUS Pharmaceutical Co., Ltd., 25 mg per tablet, orally two tablets/day).
Control group: normal pregnancy without aspirin and heparin treatment ( Figure 1).
Uterine arterial blood flow (RI, PI, S/D) was measured at the luteal phase and at 10, 12, 16, and 20 weeks of gestation using GE Voluson E8 ultrasound. Vaginal measurements were taken during the luteal phase and at 10 weeks of gestation, and abdominal measurements were taken at 12, 16, and 20 weeks of gestation. Measurement was first made on the right side of uterine artery blood flow; optimized images were made on blood flow images to obtain the best, longitudinal scan showing bent beam. Blood flow sampling was along the long axis of the blood vessel theta angle of <30 , which is the equivalent of the cervical level steady blood flow spectrum. After 3-5 consecutive stable cardiac cycles, the blood flow spectrum was assessed, and the RI, PI, and S/D values of the right uterine artery were recorded. This

Note:
The uterine artery blood flow parameters (mRI, mPI, mS/D) of the three groups were analyzed during the luteal phase, 10, 12, 16 and 20 weeks of gestation. (1) With the increase of gestational age, the uterine artery blood flow parameters of the three groups gradually decreased. (2) In the luteal phase, the uterine artery blood flow parameters of aspirin group and combination group were higher than those of control group. (3) The parameters of uterine artery blood flow at 10-20 weeks of gestation ranged from small to large as the combination group < aspirin group < control group. Comparison with aspirin group, □ p < 0.0001; Comparison with the combination group, ▲ p < 0.0001; Comparison with control group, ☆ p < 0.0001; Comparison with 12 weeks gestation, ◊p < 0.0001; Comparison with 16 weeks gestation, ◊◊p < 0.0001; Comparison with 12 weeks gestation, ◊◊◊p < 0.0001. Data were analyzed using one-way ANOVA.
procedure was repeated to measure the RI, PI, and S/D of the left uterine artery. All parameters were measured three times and averaged. All procedures were performed by the same senior sonographer.

Statistical analysis
Statistical analysis was performed using SPSS 22.0. Count data are expressed as "n%" and were F I G U R E 2 Changes of uterine artery blood flow parameters in three groups at different periods. The uterine artery flow parameters in the control group, the aspirin group and the combination group were gradually decreased in the mid-luteal phase, at 10, 12, 16, and 20 weeks of pregnancy, and the uterine artery flow parameters in the RSA group were significantly greater than those in the control group during the luteal phase, and the uterine artery flow parameters in the RSA group were significantly smaller than those in the control group after pregnancy medication. compared using the chi-square test. Measurement data are expressed as (x ± SD), and one-way ANOVA was used for comparisons among groups. Bonferroni(B) test was used when the variance was homogeneity and Tamhane's T2(M) test was used when the variance was not homogeneity. Differences were considered statistically significant at p < 0.05. MATLAB 2016 was employed for function fitting to predict the changing trend of uterine artery blood flow parameters, and Ori-gin2020 was used for plotting. k-Nearest Neighbor (KNN) classification algorithm, as the simplest machine learning algorithm, can be used to fill missing data. The KNN method in MATLAB 2020 was used to fill in missing data.

Comparison of basic information of the enrolled patients
Age and body mass index (BMI) in the three groups were not significantly different (p > 0.05). There was also no significant difference in the number of spontaneous One minute Apgar score 9.84 ± 0.6 9.9 ± 0.4 9.9 ± 0.6 0.866 Ten minutes Apgar score 10 10 10 -Note: The live birth rates of control group (95.5%) and combination group (91.8%) were higher than that of aspirin group (74.5%), and the difference was statistically significant (p < 0.05). In pregnancy complications, the hypothyroidism rate in aspirin group was higher than that in combination group, the difference was statistically significant (p < 0.05), while there were no significant differences in other pregnancy complications, gestational age at delivery, postpartum blood loss, neonatal length, neonatal weight, and neonatal Apgar score among the groups. Data were analyzed using chi-square tests、one-way ANOVA. a Adjusted p-value: Logistic analysis was performed to clear out the confounding effects of Age/BMI and number of spontaneous abortions.
abortions between the aspirin and combination groups (p > 0.05), as shown in Tables 1 and S1.
Comparison of uterine artery blood flow parameters in the middle luteal phase before treatment in the three groups The uterine artery blood flow parameters (mPI, mRI, and mS/D) in RSA group were significantly higher than those in the control group, and one-way analysis of variance (ANOVA) showed significant difference (p < 0.0001). While in the RSA group, there was no significant difference in uterine artery blood flow parameters between the aspirin group and the combination group. (p > 0.05, Tables 2, S2, and Figure 2).

Comparison of uterine arterial blood flow parameters at 10-20 weeks of gestation in patients receiving different drug regimens
The mRI, mPI, and mS/D of the women in the three groups were compared based on gestational age. The above uterine artery blood flow parameters followed the order of combination group < aspirin group < normal pregnancy group. One-way ANOVA indicated the results to be significant (p < 0.0001, Tables 2, S3, S4, and Figure 2).
Trends for uterine artery blood flow parameters in the three groups of patients 1. An exponential function with the sum of squares error (SSE) function was used to fit mPI, and the results were as follows: normal pregnancy group: y ¼ 12:19 Ã x À0:7673 (SSE = 0.00518); aspirin group: y ¼ 14:75 Ã x À0:9161 (SSE = 0.00510); and combination group: y ¼ 17:11 Ã x À1:061 (SSE = 0.00048). (x denotes the number of weeks of gestation and y denotes the mPI value.) The derivative of the function is shown in Figure 3a. mPIs in the three groups were similar. The mPIs provided in Figure 3a were transformed, with that for the combination group shifting upwards by 0.26 to nearly overlap with that for the aspirin group and upwards by 0.52 to nearly overlap with that for the normal pregnancy group. (Figure 3b). 2. Functional fitting of mRI was performed, and the results were as follows: normal pregnancy group: y ¼ 1:777 Ã x À0:3384 (SSE = 0.0003); aspirin group: y ¼ 2:221 Ã x À0:4528 (SSE = 0.0009); and combination therapy group: y ¼ 2:866 Ã x À0:6047 (SSE = 0.0001). (x denotes the number of weeks of gestation and Y denotes the mRI value.) The derivation of the function is shown in Figure 3c. The decreasing rate of mRI in the combination group was greater than that in the aspirin group and the normal pregnancy group. 3. Function fitting of mS/D was performed, and the results were as follows: normal pregnancy group: y ¼ 51:3 Ã x À0:9715 (SSE = 0.06343); aspirin group: y ¼ 40:36 Ã x À0:9648 (SSE = 0.01625); and combination group: y ¼ 33:36 Ã x À0:9798 (SSE = 0.0076). (x denotes the number of weeks of gestation and y denotes the mS/D value.) The derivation of the function is given in Figure 3d. The decreasing rate of mS/D in the normal pregnancy group was greater than that in the aspirin and combination groups.

Comparison of pregnancy outcomes of the three groups of patients
In this study, the pregnancy outcomes of women in the three groups were tracked, and the live birth and miscarriages rate were analyzed. The live birth rates for the normal pregnancy, aspirin, and combination groups were 95.5%, 74.5%, and 91.8%, respectively. The live birth rate in the combination group was significantly greater than that in the aspirin group, with a significant difference (p < 0.05). The incidence of hypothyroidism in the combination group was significantly lower than that in the aspirin group (p < 0.05). However, there were no significant differences in other pregnancy complications, gestational age at delivery, postpartum blood loss, neonatal length, neonatal weight, or neonatal Apgar score among the groups (Table 3).

DISCUSSION
The etiology and pathogenesis of recurrent spontaneous abortion are complex and have always been hot research topics. Studies have shown that nearly 30% of patients with recurrent spontaneous abortion have increased uterine artery blood flow resistance. 6 The uterine artery is the main blood vessel that supplies nutrients to the uterus and fetus. Increased resistance of uterine artery blood flow hinders development of the endometrium, affecting endometrial receptivity, and leading to implantation failure and eventual miscarriage. 7,8 After pregnancy, uterine artery blood flow is transferred to the intervillous space through the spiral arteries and ultimately supplies the placenta and embryo with nutrients and oxygen. 9 After 10 gestational weeks, trophoblast cells secrete proteolytic enzymes that invade the spiral artery, causing rupture and vascular remodeling of the spiral artery to change the morphology from flexural to upright, which decreases the elasticity and resistance of the spiral artery and causes a large influx of blood into the placental intervillous space, supplying the placenta and embryo with nutrients.
In patients with recurrent spontaneous abortion, because trophoblasts cannot normally invade the spiral artery and blood flow resistance in the spiral artery cannot be reduced normally, the placenta and embryo cannot receive an effective and sufficient blood supply. Thus, the placenta and embryo undergo ischemia and hypoxia, leading to formation of microthrombi, local oxidative stress in the placenta, and gradual apoptosis of trophoblast cells, ultimately causing adverse pregnancy outcomes. 10,11 Studies have shown that both aspirin and LMWH can attenuate apoptosis in trophoblast cells, 12 reducing uterine artery resistance, significantly improving pregnancy outcomes, and reducing miscarriage rates. 13 Aspirin exerts anticoagulant effects by inhibiting platelet aggregation and increasing prostaglandins. 14 LMWH inhibits the activity of coagulation factors, 15,16 reduces blood viscosity, improves blood circulation, prevents microthrombosis, reduces the local oxidative stress response in the placenta, and regulates the intrauterine environment; these effects increase the blood supply to the placenta and embryo and reduce the occurrence of miscarriage and stillbirth, improving pregnancy outcomes. 17 In recent years, studies have shown that in addition to having good anticoagulant effects, aspirin and LMWH promote trophoblast proliferation, invasion and differentiation, inhibit trophoblast apoptosis, protect the vascular endothelium, and promote placenta formation. 18,19 This study showed that the uterine artery blood flow parameters in the normal pregnancy, aspirin, and combination groups all gradually decreased with increasing gestational age; all these results were statistically significant. Before the luteal phase, the uterine artery flow parameters were significantly greater in the recurrent spontaneous abortion group than in the normal pregnancy group, results that were also statistically significant. In contrast, there was no statistically significant difference between the aspirin and combination groups. In the aspirin group, mRI decreased from 0.82 ± 0.05 to 0.54 ± 0.03, mPI decreased from 2.25 ± 0.42 to 0.91 ± 0.13, and mS/D decreased from 6.05 ± 1.41 to 2.18 ± 0.27; these differences were statistically significant (p < 0.0001). In the combination group, mRI decreased from 0.83 ± 0.04 to 0.46 ± 0.02, mPI decreased from 2.25 ± 0.33 to 0.70 ± 0.12, and mS/D decreased from 6.19 ± 1.28 to 1.82 ± 0.24, also significant difference (p < 0.0001) and consistent with most previous studies. 4,[20][21][22] Luo et al. 23 reported that mRI gradually decreases with increasing gestational age during early pregnancy. In this study, uterine artery blood flow parameters at different gestational weeks in the normal pregnancy group were analyzed, with mRI decreasing from 0.80 ± 0.05 to 0.63 ± 0.04. Liang et al. 24 measured umbilical artery blood flow in 4326 pregnant women in the third trimester, and the results showed that umbilical artery blood flow resistance gradually decreased with increasing gestational age. The findings indicated that the embryo may need more nutrients as gestational age increases and that more maternal nutrients are needed, with uterine artery and umbilical artery blood flow resistance indirectly reflecting whether the embryo or fetus is receiving enough nutrients. It has been shown that uterine artery flow parameters are significantly greater in patients with recurrent spontaneous abortion than in normal subjects, 25 which is consistent with the results of this study. It can be concluded that patients with high uterine artery flow parameters are more likely to have miscarriage than patients with normal flow parameters and that uterine artery flow parameters can predict the probability of miscarriage in patients to some extent.
This study showed that the use of aspirin and LMWH significantly reduced uterine artery blood flow parameters and that the combination of the two drugs had a synergistic effect. At the 10th week, the mRI, mPI, and mS/D of the women in the combination group were approximately 0.06, 0.28, and 0.81 lower than those of the women in the aspirin group and 0.11, 0.59 and 2.11 lower than those of the women in the normal pregnancy group, respectively. At the 20th week, the mRI, mPI, and mS/D of the women in the combination group were approximately 0.08, 0.21, and 0.36 lower than those of the women in the aspirin group and 0.17, 0.5, and 0.99 lower than those of the women in the normal pregnancy group, respectively. This study analyzed the effect of medication regimen on the rate of change in mPI and found that both aspirin and LMWH effectively reduced this parameter in patients with recurrent spontaneous abortion. However, the rate of decrease in the mPI of the three groups of patients was similar, indicating that aspirin and LMWH did not accelerate a decrease in mPI and that mPI is not a good indicator for the effect of drugs on uterine arteries. This study also assessed the rate of change in the mRI, and the results showed that the rate of decrease in mRI in the combination group was greater than that in the aspirin group and that aspirin combined LMWH increased the rate of mRI decrease and the live birth rate. Therefore, mRI may be an independent predictor for patients with recurrent spontaneous abortion, a finding that is generally consistent with the results of the study by Nakatsuka et al. 26,27 By accelerating the reduction in mRI, aspirin and LMWH further increase the blood supply to the placenta and embryo, thereby increasing the live birth rate and reducing the miscarriage rate. This study also found that the live birth rate for women in the combined group was higher than that for women in the aspirin group, indicating that the combination of aspirin and LMWH may effectively improve pregnancy outcomes, consistent with the findings of Clark 28 and Yu. 29 The live birth rate in the aspirin group in this study was 74.5%; live birth rates in patients with recurrent spontaneous abortion without medication of 66.67% 30 and 63.33% 31 have been reported. The results suggest that the use of single-agent aspirin may improve the live birth rate in patients with recurrent spontaneous abortion, but that it is not as effective as the combination of aspirin and LMWH.
In summary, this study analyzed the effects of aspirin and LMWH on uterine artery blood flow in patients with recurrent spontaneous abortion at different gestational ages, monitored uterine artery blood flow parameters at different gestational weeks, and compared rates of decrease in uterine artery blood flow parameters at different gestational weeks and final pregnancy outcomes. The results showed that uterine artery blood flow parameters decreased gradually with increasing gestational age; both aspirin and LMWH were effective in reducing uterine artery blood flow parameters and had a synergistic effect. It is hypothesized that mRI may be an independent predictor for patients with recurrent spontaneous abortion. This study also has some limitations. Because the data were only from one center and the sample size was small, there may be bias in the data. The results need to be further verified through studies with large sample sizes. Due to the difficulty obtaining clinical data during follow-up, coagulant data, including for anti-phospholipid antibody and lupus anticoagulant, were not examined in this study, and thus evaluation of aspirin and LMWH for thromboprophylaxis was not performed.