Correlation of Anxiety and Uterine Artery Doppler Flow in Pregnant Women with High Risk of Down syndrome: A Prospective Cohort Study

Background: Maternal anxiety may lead to unfavorable pregnancy outcomes, but the underlying mechanism is unclear. This study aimed to evaluate the correlation between maternal anxiety and uterine blood ow index in pregnant women with high risk of Down syndrome undergoing amniocentesis. Methods: This prospective cohort study was conducted at 15-18 weeks of gestational age on 199 pregnant women with high-risk of Down syndrome, candidates for amniocentesis, and 176 pregnant women at low-risk of Down syndrome in the control group from 2017 to 2019. Anxiety state by Spielberger’s State-Trait Anxiety Inventory (STAI) and uterine artery blood ow indices were assessed at baseline and two weeks follow-up visits (immediately before amniocentesis and after receiving the karyotype results in the amniocentesis group). Results: The mean±SD age of participants was 33.11 ± 5.96 years. There were 176 negative and 23 positive results for Down syndrome in the amniocentesis group. State-Trait Anxiety Inventory scores were signicantly different between the amniocentesis and control groups at baseline and follow up (p = 0.033 and p=0.003 respectively) and between baseline and follow-up assessments in the amniocentesis-negative group (p = 0.001, with lower follow-up scores). A signicant decrease was observed between baseline and follow-up uterine PI in amniocentesis group (p<0.05), and between baseline and follow-up uterine RI in both amniocentesis and control groups (p <0.001). There was a signicant but weak correlation between uterine RI and State-Trait Anxiety Inventory scores at follow-up (r=0.137, p=0.008). Conclusions: Maternal anxiety may decrease uterine artery blood ow in pregnancy. Trial were used to present categorical variables. The study parameters were compared between amniocentesis and control groups at baseline and follow-up using relevant tests after checking the normality of data. Correlation between study variables was assessed using Pearson correlation. The level of statistical signicance was considered as p < 0.05.


Background
Stress is a non-speci c reaction to psychological, physical, and perceptional stimuli in the environment (1). Although the relationship between stress and various diseases have been con rmed, the underlying mechanisms for this association are not determined (2). Increased blood pressure, peripheral arterial tonometry, microvascular reactivity, pulse wave velocity, and serum catecholamine levels have been observed in stressful situations (2,3).
Anxiety is de ned as hyperarousal, fear, and worry at a counterproductive and debilitating level, which is manifested in response to stressors (4).
Pregnancy is a physiologic condition in life but also considered as a stressful life event experienced by women (5). The observed changes in hormones, physical appearance, abilities, and social roles of a pregnant woman may result in increased stress during pregnancy (6). One of the stressful events in pregnancy is undergoing amniocentesis due to abnormal aneuploidy screening test results (7)(8)(9). Therefore, prenatal screening tests may be considered as psychological stressors in pregnancy leading to anxiety and discomfort in pregnant women and her relatives.
Various studies have shown that anxiety and stress during pregnancy are associated with unfavorable pregnancy outcomes, including low birth weight, preterm labor, and intrauterine growth restriction (10)(11)(12). This may be related to the release of catecholamines, which results in placental hypoperfusion and consequent decreased oxygen and nutrients delivery to the fetus, leading to fetal growth impairment and/or preterm delivery. Due to the similarities in the pathophysiology of intrauterine growth abnormalities and maternal stress and anxiety, it could be hypothesized that anxiety in pregnancy may result in unfavorable uterine artery blood ow changes and adverse pregnancy outcomes if it continues (13). Fetal growth disturbances are linked to abnormalities in uterine artery blood ow, which is partly due to endothelial dysfunction (14).
In a recent study performed by Shirazi et al, different levels of maternal stress could affect uterine artery blood ow alterations (9). The study did not include healthy control subjects and therefore the relationship between maternal anxiety level and uterine blood ow could not be assessed (9). Regarding the fact that stress is a transient condition, anxiety might have a more signi cant and persistent effect on circulation (15,16). Uterine artery blood ow can be assessed non-invasively using Doppler ultrasonography. Although the hypothesis for the underlying effect of maternal anxiety on uterine artery blood ow seems straight forward, the ndings of previous studies are controversial (9,17). This study was conducted to assess the relationship between maternal anxiety and uterine artery blood ow indices among pregnant women candidates for amniocentesis due to abnormal aneuploidy screening test results, as we clinically observed a high level of anxiety among this group (9).

Study subjects
This prospective cohort study was conducted on pregnant women referred to Yas Hospital, a liated to Tehran University of Medical Sciences, Tehran, Iran between October 2017 and April 2019. The study protocol was approved by the Institutional Review Board of Tehran University of Medical Sciences (Registration code: IR.TUMS.IKHC.REC.1397.174). Case and control subjects with a gestational age of 15-18 weeks were included. The case group was selected among patients who were candidates for amniocentesis due to abnormal sequential screening test results while the control group included pregnant women with normal sequential screening test results referred for a routine prenatal visit. Subjects were excluded if they had a recent history of exposure to stressful events, comorbid diseases including hypertension or diabetes, positive aneuploidy culture, history of cigarette or alcohol consumption, or history of major psychological disorders. Written informed consent was obtained from included subjects prior to conducting the trial.

Sample Size
The sample size was calculated based on the ndings of a study conducted by Aksoy et al (18), which reported the mean uterine artery PI of 0.95 ± 0.53 among cases and 0.7 ± 0.36 among the control group. Considering the power of 80% and type I error of 0.05 the sample size was calculated as 81 subjects in each of amniocentesis and control groups. Considering the 30% response rate (19) for phone invitation and 20% dropout, the sample size was calculated to be 166 subjects in each of the study groups.

Instruments
The level of the anxiety of the subjects was assessed using the Spielberger's State-Trait Anxiety Inventory (STAI). State anxiety re ects the psychological and physiological transient reactions directly related to adverse situations in a speci c moment. In contrast, the term trait anxiety refers to a trait of personality, describing individual differences related to a tendency to present state anxiety (20). The Spielberger's State-Trait Anxiety Inventory included 40 questions and rated based on a four-point Likert scale based on the intensity of the feelings of the respondent (21). Scores more than 40 suggested high anxiety and scores that range from 15 to 20 indicate no anxiety. A cut point of 39-40 had been suggested to detect clinically signi cant symptoms for the scale (22). The Persian version of this questionnaire was validated with a Cronbach's Alpha coe cient of 0.72 (23).

Doppler ultrasound assessment
All subjects underwent a Doppler ultrasound assessment performed by the same perinatologist using the ACUSON Sequoia 512™ (Siemens Healthcare GmbH, USA) with a convex multi-frequency transducer (3.0 to 5.0 MHz). Uterine artery Doppler assessment was performed trans-abdominally. The uterine artery pulsatility index (PI) and resistance index (RI) were measured for both sides at the origin of the uterine arteries and the mean values for PI and RI were calculated. All follow-up, Doppler assessments were repeated two weeks later.

Study procedure
Subjects in both groups were recruited based on inclusion and exclusion criteria. Subjects in the study group underwent baseline Doppler ultrasound immediately prior to amniocentesis, and the follow-up Doppler assessment was performed two weeks later when they had already received their amniocentesis results. Subjects in this group were categorized into two sub-groups of amniocentesis-positive and amniocentesis-negative according to the positive and negative karyotype results for Down syndrome. In the control group, baseline Doppler study was performed in the rst visit between 15-18 weeks, and a follow-up Doppler study was performed two weeks later. In both groups, the Spielberger's State-Trait Anxiety Inventory questionnaires were lled prior to the baseline and the follow-up Doppler study. All data were collected, recorded, and analyzed among groups and subgroups ( Fig. 1).

Statistical analysis
Data were analyzed using the statistical package for social sciences (SPSS) software version 22 (IBM, Inc, Chicago, IL, USA). Mean and standard deviation (SD) were used to present continuous variables while frequency and percentage were used to present categorical variables. The study parameters were compared between amniocentesis and control groups at baseline and follow-up using relevant tests after checking the normality of data. Correlation between study variables was assessed using Pearson correlation. The level of statistical signi cance was considered as p < 0.05.

Results
A total of 375 subjects (199 cases and 176 controls) participated in the study. The mean ± SD age of the subjects was 33.11 ± 5.96 years. The demographic characteristics of amniocentesis and control groups are presented in Table 1. Although there was a statistically signi cant difference between case and control groups in terms of age (p = 0.016), this difference was not clinically signi cant. After receiving the karyotype results for Down syndrome, the mean ± SD age of pregnant women was 34.09 ± 8.19 and 33.77 ± 5.59 years in amniocentesis-positive and amniocentesis-negative groups respectively. The mean state and trait Anxiety Inventory scores were signi cantly different between amniocentesis and control group at baseline (p = 0.003 and p = 0.033 respectively). In the amniocentesis group, mean state and trait anxiety scores were > 40 (42.79 ± 11.02 and 41.37 ± 10.8 respectively), indicating high levels of anxiety that mothers experienced ( Table 2, Fig. 2).
There was no signi cant difference at baseline mean uterine artery PI and RI between case and control groups ( There was no difference in anxiety trait scores, anxiety state scores, mean uterine PI, and mean uterine RI among amniocentesis-positive, amniocentesis-negative, and control groups in follow-up assessment (p = 0.85, p = 0.71, p = 0.7, p = 0.5 respectively) ( Table 3). Uterine PI was signi cantly decreased in the amniocentesis group; there was a signi cant difference between baseline and follow-up mean uterine artery PI in both amniocentesis-positive (p < 0.001) and amniocentesisnegative (p = 0.04) groups (case group). No signi cant difference was observed between baseline and follow-up uterine PI in the control group (p = 0.4). On the other hand, RI was decreased in all subjects in amniocentesis and control groups (p = 0.001) ( Table 4). In terms of anxiety trait scores, there was a signi cant difference between baseline and follow-up in both amniocentesis-positive (p = 0.017) and amniocentesis-negative group (p < 0.001) ( Table 4 and Fig. 3). Anxiety state scores were signi cantly different between baseline and follow-up assessments only in the amniocentesisnegative group (p < 0.001) (Fig. 4).
Pearson correlation test revealed a signi cant but weak correlation between mean uterine RI at follow-up and both anxiety trait and anxiety state score. There was no more signi cant correlation between baseline mean uterine RI and PI and anxiety scores. These results were also similar after being controlled for maternal age (Table 5).

Discussion
In this study, the mean age of the amniocentesis-positive group was 34.09 ± 8.1 years. It was previously shown that the risk of Down syndrome signi cantly increases with an increase in maternal age over 35 years old (24). This relationship might be signi cant at lower ages in the case of consanguineous marriage (25). This study did not assess the consanguinity of the couples but regarding the high incidence of consanguineous marriage in Iran (26), which may justify the lower age of the amniocentesis-positive group to some extent. This study also found a signi cant age difference between high-risk and control groups. Although this difference was of statistical signi cance, the mean difference was nearly one year, which was not of clinical signi cance.
The ndings of this study revealed that the mean anxiety state and trait scores in all participants were higher compared to the normal range (scores less than 39 indicate less anxiety) (9). This nding was in line with the ndings of previous studies that indicated a positive antenatal screening test may result in increased anxiety in pregnant women (9,(27)(28)(29)(30). On the other hand in our study, the control group also exhibited a higher anxiety level compared to the normal women in a similar age group. Investigations have shown a higher prevalence of antenatal depression and anxiety disorders in Iranian pregnant women compared to the world average (31)(32).
Animal based studies have shown a signi cant relationship between stress and fetal growth. In a study, anxiety and chronic stress were found to increase serum cortisol and corticotrophin-releasing hormone in both mother and fetus resulting in dysregulations in the hypothalamic-pituitary-adrenal (HPA) axis in the fetus that in part causes structural abnormalities in the hippocampus, frontal cortex, amygdala, and nucleus accumbens (32). Further animal studies have also revealed a signi cant decrease in uterine blood ow due to stress (33)(34)(35). Human studies have also hypothesized a similar effect for stress and anxiety on uterine blood ow (12,17). Previous studies have shown that anxiety may be correlated to reduced uterine artery blood ow (9,17).
The ndings of this study revealed a signi cant reduction in uterine artery RI over time, indicating that regardless of being high-risk or low risk for Down syndrome, the mean RI signi cantly reduced from baseline through the follow-up assessments, while PI was decreased signi cantly only in the amniocentesis group. It was previously shown that the uterine artery pressure and resistance decrease as the gestational age increases, which is mainly due to the formation and expansion of placental vascular bed (36). Therefore, one reason for the reduced uterine PI and RI could be the physiological changes in pregnancy. On the other hand, one might attribute the degree of mean uterine artery changes to the level of anxiety in both amniocentesis-positive and amniocentesis-negative groups, as they both were exposed to a stressful event which might have resulted in an increased baseline uterine artery PI and RI. Unfortunately, this hypothesis could not be tested in this study as there were no anxiety-free subjects in this study. The ndings of this study also revealed no signi cant group effects for uterine PI and RI, which indicate that the mean uterine PI and RI changes were not signi cantly different between amniocentesis and control groups at the baseline and the follow-up assessments.
The ndings of this study revealed a signi cant reduction in trait anxiety scores over time in both amniocentesispositive and amniocentesis-negative groups while state anxiety score was signi cantly reduced over time in the amniocentesis-negative group. This indicates that the trait and state anxiety scores changed differently in the amniocentesis-positive and negative groups during the two weeks follow-up period. This nding might be due to the reduction of anxiety after observing a negative result or might show that a two week period could provide an opportunity for mothers to cope with their anxiety. This nding indicates that higher baseline uterine PI and RI may be related to higher baseline levels of anxiety in the amniocentesis group. As the anxiety decreased by coping with the condition, the uterine artery PI and RI also decreased in the amniocentesis-positive group. The presence of high baseline levels of anxiety in the control group may justify the lack of signi cant difference in uterine PI and RI between groups.
The ndings of the study revealed high baseline levels of anxiety in both amniocentesis and control groups, which made it impossible to have an anxiety-free arm in our study to assess the effect of anxiety on the uterine blood ow. The other limitation of this study was the short follow-up duration. Two weeks follow-up duration was chosen to decrease the high risk of losses to follow-up as the study was conducted in a tertiary hospital with a substantial number of patients being referred from other cities and provinces. Therefore, longer cohort studies are suggested to screen uterine vascular indices and pregnancy outcomes among pregnancies complicated by anxiety.

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The ndings of this study revealed that high maternal anxiety may result in decreased uterine artery blood ow. It seems necessary to develop stress management training for pregnant women and to provide stronger emotional support, especially for those who have been recognized to be at high risk of Down syndrome by prenatal aneuploidy screening tests.

Declarations
Ethics approval and consent to participate: This manuscript was performed in accordance with Helsinki declaration. All patient's data were kept con dential. This study was approved by Institutional Review Board of Tehran University of Medical Sciences (Registration code: IR.TUMS.IKHC.REC.1397.174) and 96-03-91-36165. Consent for publication: Written consent was signed upon admission by all patients included in this study to use their information in research studies and publication in public (Persian version) and is available for review.
Availability of data and materials: The datasets used during the current study are available from the corresponding author on reasonable request.
Competing interests: The authors report no con ict of interest All authors are agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature. Changes in anxiety trait scores during the study duration as per study groups.  Changes in anxiety trait scores during the study duration in amniocentesis subgroups