**Analysis of Circulating C19MC MicroRNA as an Early Marker of Hypertension and Preeclampsia in Pregnant Patients: A Systematic Review**

**Adrianna Kondracka 1, Ilona Jaszczuk 2, Dorota Koczkodaj 2, Bartosz Kondracki 3,\*, Karolina Fr ˛aszczak 4, Anna Oniszczuk 5, Magda Rybak-Krzyszkowska 6, Jakub Staniczek 7, Agata Filip <sup>2</sup> and Anna Kwa´sniewska <sup>1</sup>**


**Abstract:** Preeclampsia and hypertension complicate several pregnancies. Identifying women at risk of developing these conditions is essential to establish potential treatment modalities. Biomarkers such as C19MC microRNA in pregnant patients wopuld assist in defining pregnancy surveillance and implementing interventions. This study sought to analyze circulating C19MC microRNA as an early marker of hypertension and preeclampsia in pregnant patients. A systematic review was undertaken using the following registers: disease registries, pregnancy registries, and pregnancy exposure registries, and the following databases: PubMed, CINAHL, Web of Science, Scopus, and EMBASE. The risk of bias was assessed using the Cochrane technique. From the 45 publications retrieved from the registers and databases, only 21 were included in the review after the removal of duplicates, screening, and eligibility evaluation. All 210 publications had a low risk of bias and illuminated the potential use of circulating C19MC microRNA as an early marker of hypertension and preeclampsia in pregnant patients. Therefore, it was concluded that C19MC microRNA can be used as an early marker of gestational preeclampsia and hypertension.

**Keywords:** C19MC microRNA; early pregnancy biomarkers; hypertension; preeclampsia

### **1. Introduction**

MicroRNAs have been suggested as possible hypertension and pre-eclampsia indicators since they are crucial cell process regulators. Most investigations have conducted the primate-specific microRNA cluster on chromosome 19 (C19MC microRNA) profiling analysis on total serum samples or maternal plasma to treat the later incidence of pregnancy-related problems, such as gestational pre-eclampsia, hypertension, and fetal growth restriction. Exosomal nanoparticles released into the blood and extracellular space include microRNAs [1]. They allow communication between close-by and far-off cells. Over the past decade, interest in forming non-invasive modes of cell-free nucleic acid detection has been on an upward trajectory. They include microRNAs during maternal circulation [2]. The ability to diagnose via given molecular biomarkers alongside amalgamating them into current prognosis algorithms for issues linked to pregnancy is vital [2]. Small non-coding RNAs (sncRNAs) guide post-transcriptional gene expression by blocking messenger RNA targets from translation. This systematic literature review analyzes findings from different

**Citation:** Kondracka, A.; Jaszczuk, I.; Koczkodaj, D.; Kondracki, B.; Fr ˛aszczak, K.; Oniszczuk, A.; Rybak-Krzyszkowska, M.; Staniczek, J.; Filip, A.; Kwa´sniewska, A. Analysis of Circulating C19MC MicroRNA as an Early Marker of Hypertension and Preeclampsia in Pregnant Patients: A Systematic Review. *J. Clin. Med.* **2022**, *11*, 7051. https://doi.org/10.3390/jcm11237051

Academic Editors: Roland Axt-Fliedner and Yariv Yogev

Received: 10 September 2022 Accepted: 27 November 2022 Published: 29 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

sources on circulating C19MC microRNA as an early marker of hypertension and preeclampsia in pregnant patients. It establishes that circulating C19MC microRNAs may contribute to developing pre-eclampsia and prenatal hypertension in early pregnancy.

### **2. Materials and Methods**

The review followed the PRISMA rule to report the stepwise procedure used to retrieve information from various databases and registers. The PRISMA guidelines were also adhered to strictly to eliminate bias and ensure the successful completion of the systematic literature review. Figure 1 below shows the PRISMA chart demonstrating various phases of the review.

**Figure 1.** PRISMA chart showing the different stages of the systematic literature review. Ten records were retrieved from databases: PubMed, CINAHL, Web of Science, Scopus, and EMBASE. Thirty-five records were retrieved from registers: disease registries, pregnancy registries, and pregnancy exposure registries. Before the screening, 5 duplicate records were removed, 2 were marked as ineligible using RobotAnalyst, and 3 records were removed because they were written in languages other than English. Thirty-five records were screened, and one was excluded, as its abstract did not include all the crucial keywords required. Thirty-four records were sought for retrieval and only thirty-two were retrieved. Two records could not be retrieved. The 32 records were assessed for eligibility and 2 records were excluded because 1 had less than five participants, while the other focused on C19MC microRNA as an early marker of pregnancy-related problems other than pre-eclampsia and hypertension and nine were review. Therefore, 21 records were included in the review.

The inclusion criterion required using systematic reviews investigating circulating C19MC microRNA as an early marker of hypertension and pre-eclampsia in pregnant patients. Other inclusion requirements included any study, be it experimental, cohort, or case study, articles published in English between 1 January 2013, and 5 August 2022, and original research undertaken in any region of the world with a sample size of at least five participants. The exclusion criteria were papers with fewer than 5 cases, papers published before 2013, and review papers except for systematic reviews. The exclusion criteria required the removal of studies from the review, encompassing articles published in languages other than English and materials that did not concentrate on investigating circulating C19MC microRNA as an early marker of hypertension and pre-eclampsia in pregnant patients.

### *2.1. Information Sources*

The databases used in the review included PubMed, CINAHL, Web of Science, Scopus, and EMBASE. These databases were consulted simultaneously within one month (May 2022) to identify studies that could be included in the review. Similarly, registers such disease registries, pregnancy registries, and pregnancy exposure registries were also searched to identify publications aligned with the topic of interest. These registers were also searched simultaneously within one month (June 2022). The reference lists of the articles obtained from the databases and registers were also used to identify studies that focused on investigating various aspects of the topic of interest, circulating C19MC microRNA as an early marker of hypertension and pre-eclampsia in pregnant patients. The studies were also retrieved from the abovementioned databases and stored for subsequent processes and steps.

### *2.2. Search Strategy*

The scholarly materials retrieved from the databases and registers mentioned above were limited to those published between 1 January 2013, and 5 August 2022. The keywords included C19MC microRNA, early hypertension markers, early pre-eclampsia markers, hypertension in pregnant patients, pre-eclampsia in pregnant patients, and C19MC microRNA in pregnant patients. A manual search was conducted by reading the bibliographies of the review articles or materials that were retrieved from the reference lists, and frequently mentioned publications on gestational hypertension and pre-eclampsia were used to uncover papers that were not identified by the electronic search.

### *2.3. Selection Process*

The selection process was undertaken using three crucial steps. First, reviewers who worked independently selected all articles that were retrieved from the databases and registers to reduce the chances of bias. Second, all the randomly selected publications were reviewed to determine their eligibility. Their abstracts and titles were screened against the eligibility criteria to determine whether the articles that met the inclusion criterion. RobotAnalyst was used as an automation tool during the eligibility screening. This second step enabled the removal of duplicate publications and ushered in the last phase of the selection criterion, which mainly handled the articles with titles and abstracts that did not give sufficient information regarding the study. The last step involved a full-content evaluation by the reviewer to assert whether those particular publications could be included in the review. This three-step process was undertaken independently to ensure that only the necessary publications were included in the review.

#### *2.4. Data Collection Process*

The search yielded thirty articles about circulating C19MC microRNA, gestational hypertension, or pre-eclampsia. The threshold index was assessed before synthesizing the data. The diagnostic index tests, including summary receiver operating characteristic (SROC), diagnostic odds ratio (DOR), negative or positive likelihood ratio (NLR or PLR), specificity (Spe), and sensitivity (Sen), were measured with a confidence interval of 95%.

### *2.5. Data Items*

The data items were manually extracted from the publications included in the review. The essential information from the selected reports was amassed and recorded in a table. Since this review focused on analyzing the circulating C19MC microRNA as an early marker of hypertension and pre-eclampsia in pregnant patients, the data collected from the selected articles included the author(s), titles, years of publication, and outcomes of the reports. The results segment of the chosen publications provided information concerning circulating C19MC microRNA as an early marker of gestational pre-eclampsia and hypertension. For all statistical studies, the statistical analysis tool or technique used and the statistical results were recorded under the data item and outcomes of the reports.

### *2.6. Study Risk of Bias Assessment*

The Cochrane technique was used to assess the risk of bias in all the publications included in the review. Cochrane is a standard risk appraisal instrument that uses judgments of unclear risks (?), high risk (-), and low risk (+) on different axes for studies such as systematic reviews, which may have biases in their decisions, results, strategies, and other aspects of interest. One reviewer independently assessed the risk of bias in each study using the Cochrane tool.

### *2.7. Synthesis Method*

The data in this review were synthesized using thematic analysis and grouping similar information, all presented in Table 1. The four columns included the publications' author(s), titles, publication years, and outcomes. Under the outcomes column, the studies' results were thematically presented, focusing on whether C19MC microRNA can be used as an early marker of gestational hypertension and pre-eclampsia. The rows comprised the heading row and the thirty studies included in the review.

### *2.8. Reporting Bias Assessment*

The bias risk assessment performed using the Cochrane technique was reported using the Cochrane bias risk assessment, as shown in Table 2, which has eight columns and thirty-one rows. The columns included the study of interest, selection bias (random sequence generation), selection bias (allocation concealment), performance bias, detention bias, attrition bias, reporting bias, and other biases. The first row provides the heading information, while the other thirty comprise the publications included in the review.


**Table 1.** Study

Characteristics.




**Table 2.** Cochrane Bias Risk Assessment.

(1) Selection Bias (Random Sequence Generation); (2) Selection Bias (Allocation Concealment); (3) Performance Bias; (4) Detention Bias; (5) Attrition Bias; (6) Reporting Bias; and (7) Other Bias. Symbols: unclear risks (?), high risk (-), and low risk (+).

#### **3. Results**

### *3.1. Study Selection*

The results of this review can be categorized according to PRISMA guidelines. The search, identification, and retrieval of articles resulted in a collection of fifty-five publications; ten were retrieved from the databases, and forty-five were from the registers. The fifty-five publications were then checked, and five duplicates were removed. Two articles were removed because of their ineligibility, as marked by RobotAnalyst. Three publications were also removed because they were written in languages other than English. Only thirty-five remaining reports were subjected to screening. The screening process involved checking and evaluating their abstracts and titles to determine their suitability to be included in the review. One publication was excluded because its abstract did not include all the crucial keywords required. The remaining thirty-four articles were sought for retrieval, but only thirty-two were obtained because two publications were inaccessible. The thirty-two reports were assessed for eligibility. Two did not meet the eligibility criteria because one had less than five participants, while the other focused on C19MC microRNA as an early marker of pregnancy-related problems other than pre-eclampsia and hypertension. The remaining thirty publications met the eligibility criteria and were included in the review.

### *3.2. Study Characteristics*

The study characteristics obtained in this review were divided into four groups: author(s), title, year of publication, and outcomes of the reports, as evident in Table 1. Table 1 shows that only one article was published by one author [2]. All other twenty-nine studies were published by two or more authors. All the publications had different titles directly associated with the research topic. Furthermore, they were published during different periods, as evident via their years of publication. Regarding year of publication, 3.33% of the articles were published in 2013 [1], 2014 [19], or 2015 [13], and 6.67% of the reports were published in 2016 [22,23] and 2019 [2,12]. In addition, 10% of the studies were published in 2017 [4,24,25] and 2018 [15,21,26], while 13.33% of the articles were published in 2021 [3,8,14,17] and 2022 [6,10,27,28]. Finally, 30% of the studies were published in 2020 [5,7,9,11,16,18,20,29,30]. This statistic shows the diversity of the documents' retrieval where their publication years are concerned.

### *3.3. Risk of Bias in Studies*

The results of the Cochrane bias risk assessment are evident in Table 2, which illuminates that all the materials have low reporting and selection bias risks and a highperformance bias risk. A significant number of the other Cochrane method measures underscored low bias risk. These results suggest that all the publications were of good quality and deserved to be included in the review.

### *3.4. Results of Individual Studies*

### 3.4.1. miRNAs of Different Stages of Preeclampsia

This review discovered informative facts about the role of C19MC microRNA as an early marker of gestational hypertension and pre-eclampsia. For instance, clinically confirmed hypertension and pre-eclampsia have been linked to changes in extracellular microRNA expression [13]. No distinction between fetal and normal pregnancies could be made on the basis of the levels of circulating microRNA expression. Furthermore, according to He and Ding, pre-eclampsia often appears after 20 weeks of pregnancy and is characterized by proteinuria and gestational or chronic hypertension [6]. The condition arises from a problem with placentation, which leads to insufficient uteroplacental blood perfusion and ischemia. Pre-eclampsia is an implantation condition, and its reasons are yet unclear. Its fundamental etiological theory assumes that placentation and insufficient trophoblast invasion are related to a poor adaptation of the local maternal immune system to extra-villous cytotrophoblast produced at the fetal–maternal interface [19].

Czernek and Duchler examined the C19MC microRNA gene expression in simple and complex pregnancies. According to their assertion, there are 56 microRNA genes in the chromosome 19 microRNA cluster. The research focused primarily on microRNAs previously indicated to be substantially present in placental tissues and those reported to be uniquely expressed in them. The research examined C19MC microRNA gene expression in connection with established risk factors for worse perinatal results. Analysis of C19MC microRNA gene expression connected to the degree of clinical symptoms, the doppler ultrasonography parameters, and the delivery date was conducted to determine the severe condition. Complex pregnancies and controls generally have distinct expression profiles for C19MC microRNAs. Pre-eclampsia patients had down-regulation of C19MC microR-NAs more often in specific subgroups of pregnancy-related disorders. Further results demonstrated that one C19MC microRNA (miR-517-5p) was changed in pre-eclampsia, necessitating abortion before the nine month gestation period, while five C19MC microRNAs (miR-26b-5p, miR-7-5p, miR-181a-5p, hsa-miR-486-1-5p, and hsa-miR-486-2-5p) in severe preeclamptic pregnancies were dysregulated. The findings imply that the microRNAs have a role in pre-eclampsia's pathophysiology [29].

Additionally, moderate pre-eclampsia that lasted for a few weeks and was closely followed from the time of diagnosis to birth was shown to have a similar expression pattern of placental-specific microRNAs [22]. However, the down-regulation of placental-specific microRNAs seemed to be more profound the longer the pregnancy-related illness persisted. This implies that certain C19MC microRNA dysregulation may represent a compensatory strategy rather than the disease process itself. In another study, Whigham et al. discovered

maternal plasma C19MC microRNAs that distinguish between healthy pregnancies and non-pregnancies [20]. More recent research has shown that established pre-eclampsia is characterized by an increase in the circulating miR-526a, miR-525, miR-520a-5p, miR-517- 5p, and miR-516-5p genes. While the group of patients with hypertension alongside the control did not have different plasma levels of microRNAs, elevated levels were seen in the group of participants with developed pre-eclampsia [20].

Whigham et al. also note the capacity of extracellular C19MC microRNAs to distinguish between complicated and normal pregnancies at the beginning of pre-eclampsia with or without fetal growth restriction, which was verified using both relative and absolute quantification methods. Unfortunately, there is a lack of information comparing extracellular C19MC microRNA levels in abnormal and normal pregnancies [20]. The study's findings disagree with MacDonald et al., who noted an increase of extracellular miR-520h in four pre-eclampsia patients. The findings prompted an additional investigation into the relationships between circulating C19MC microRNAs and illness severity concerning the degree of delivery needs and clinical symptoms [28].

Furthermore, no correlation among the gene, plasmatic expression, and risk factors for lower C19MC microRNA neonatal levels was found in the association investigation. In both pregnancies with moderate and severe pre-eclampsia, the microRNA plasmatic and gene expression levels were comparable. According to the study's findings, many pathological and physiological processes depend heavily on microRNAs and are to blame for pregnancy-related problems. Consequently, circulating C19MC microRNAs may have a role in the etiology of pre-eclampsia [7]. The research involved increased circulating C19MC microRNAs that characterize the intriguing discovery that developed pre-eclampsia.

### miRNAs in the Normal and Abnormal Pregnancies

Even though C19MC microRNAs appear to be down-regulated in the placental tissues in response to a variety of pregnancy-related disorders, including pre-eclampsia and hypertension, as noted above, upregulation of the specific microRNAs appears only in the maternal circulation in pre-eclampsia cases [3]. The contradictory result may be interpreted in several different ways. This recent research by Ali et al. showed that, compared with gestation-matched controls, numerous microRNAs regulated by hypoxia were upregulated in pregnancies affected by significant preterm fetal growth limitation [3]. However, most studies focused on examining pregnancy-related microRNAs whose genes are not found in the miRNA clusters on the chromosome.

A large number of the physiological changes that occur during a typical pregnancy are caused by an acute-phase reaction that is triggered by an inflammatory response. The proximal source of the issues is the placenta [4]. Apoptotic bodies or syncytiotrophoblast microparticles, which include fetus and placenta extracellular nucleic acids, are released by the placenta while it undergoes continuous remodeling throughout normal pregnancy. Cronqvist et al. contended that circulating syncytiotrophoblast exosomes contribute to some syndrome symptoms and maternal inflammation. Pre-eclampsia (PE), hypertension, and other diseases' clinical characteristics may be explained by increased inflammationrelated symptoms that are reportedly present in healthy pregnancies at term [4].

According to Jin et al., pre-eclampsia with a clinically confirmed diagnosis is linked to changes in extracellular microRNA expression. However, after the evaluation of circulating microRNA, there was no difference between normal and abnormal pregnancies [8]. Špaˇcková found that several hypoxia-regulated microRNAs were complicated by extremely preterm fetal growth restriction [2]. Nonetheless, most researchers concentrated on investigating microRNAs connected with pregnancy whose genes are not included in the chromosome 19 miRNA clusters or C14MC.

Additionally, Qin et al. further discovered that C19MC microRNAs are present in maternal plasma and have been shown to distinguish between healthy pregnancies and nonpregnancies. They observed increased extracellular C19MC microRNA levels in regularly developing pregnancies. The findings of a follow-up study demonstrated that the upregulation of the C19MC microRNAs is a defining feature of pre-eclampsia that has already developed [17]. Srinivasan et al. indicated the ability of extracellular C19MC microRNAs to distinguish between individuals at risk of later developing placental-sufficiency-related issues and normal pregnancies at the onset of gestation. The findings emphasized the necessity for further investigation of extracellular microRNAs in maternal circulation with the goal of regular evaluation in daily practice and identification as possible indicators for pregnancy complications linked to placental insufficiency [18].

In addition, Jing et al. asserted that even though fetal growth restriction and preeclampsia may be detected using separate serum indicators or maternal plasma, combination screening tests are presently employed in clinical settings to determine the likelihood of developing pre-eclampsia. Pregnancy-associated plasma protein-A, placental growth factor, and, together with maternal blood biomarkers, uterine artery Doppler and maternal risk factors may detect roughly 95% of patients with early-onset pre-eclampsia with a 10% false-positive rate. [9] To enhance the prediction of problems associated with placental insufficiency, additional studies are required to find additional biomarkers with higher diagnostic performance. Jing et al. examined 754 miRNAs and found no predictive value for early pre-eclampsia in first-trimester maternal blood miRNA evaluation. According to miRNA profiling using the high-throughput Open Array TM technology, seven microRNAs have a distinct abundance profile in early pre-eclampsia. Hence, there were no discernible changes between pre-eclampsia and controls after validation by real-time quantitative analysis [9].

#### 3.4.2. miRNA as a Biomarker of Gestational Hypertension

To determine whether a combination of miR-518b and miR-520h biomarkers or a single plasmatic miR-520h biomarker offers valuable tools in the risk assessment for gestational hypertension, several large-scale, multi-center studies, including individuals from various demographics, are required [25]. The rise in extracellular C19MC microRNAs during the first trimester of pregnancy may be related to the down-regulation of various hormones and proteins studied as potential early markers for pre-eclampsia and hypertension. Various diseases, such as gestational hypertension, are associated with placental exosomes' content during pregnancy [27]. Wommack et al. focused on examining the role of miRNAs as signaling molecules. The authors discovered that miRNAs operate as co-regulated groups of signaling molecules to coordinate infant outcomes and gestation length [21].

However, using quantitative RT-PCR to test 30 non-placental microRNAs in maternal mononuclear cells from peripheral blood, Mavreli et al. accurately predicted late preeclampsia and miscarriage during the first trimester of pregnancy. The results were assessed with the help of a designed system, awarding points to each participant whose microRNA quantification outcome fell within the top eight. Findings were arranged from the highest to the lowest Ct value for each microRNA. Each patient's unique pregnancy risk score was calculated once the findings of all microRNAs were added together. Four microRNAs had very low values; therefore, they were deemed technically unfit for analysis and were not included in the score [26]. Similarly, Špaˇcková used microarray analysis to find 19 mature miRNAs that were differentially expressed in the blood of pregnant women who later had acute pre-eclampsia. Of them, 12 were upregulated, and 7 were down-regulated during the early gestational phases. In the blood of women who eventually experienced acute pre-eclampsia, mir-1233 was the most overexpressed [2].

In addition, to identify C19MC microRNAs with extracellular placental specificity in maternal circulation as possible biomarkers for problems associated with placental insufficiency, Li et al. suggested the necessity for a more thorough investigation of the microRNAs. Using the absolute and relative quantification methods, the ability of extracellular C19MC microRNAs to distinguish between normal pregnancies and people predisposed to develop intrauterine growth restrictions and pre-eclampsia in early pregnancy was described [11]. The pilot study involved six pregnant women with one early IUGR, four late pre-eclampsias, and one early pre-eclampsia [5]. As the findings revealed that the females

who subsequently had severe pre-eclampsia had upregulated placental-specific miR-520a in their sera at 12–14 weeks of pregnancy, Li et al. considerably contributed to validating the findings by Munjas et al. [14]. From the stem-loop and miR-520a\* combined, mir-520a (miR-520a-3p) is produced. Akin as well as Li et al. and Hornakova et al. found that circulating miR-517\* was upregulated in preeclampsia-prone early pregnancy [11,14,30].

Consequently, subsequent large-scale investigations are required to evaluate the positive predictive value, specificity, and sensitivity of C19MC microRNAs for hypertension or pre-eclampsia. The effectiveness of placental-specific microRNAs for diagnosing disease severity should be assessed in connection with Doppler ultrasonography characteristics, delivery needs, and clinical symptoms [10]. The research produced intriguing results, showing that up-regulation of circulating C19MC microRNAs is a hallmark of early pregnancy, which is predisposed to developing issues linked to gestational hypertension and placental insufficiency. In addition, elevated plasmatic levels of miR-516-5p, miR-518b, and miR-520h in the first trimester alone are strong indicators of future gestational hypertension. One C19MC placental-specific microRNA biomarker may be used to screen for the start of hypertension in the first trimester of pregnancy [23]. Alternately, miR-518b and miR-520h, both placental-specific C19MC microRNA prediction biomarkers, may be combined to forecast the incidence of prenatal hypertension.

### 3.4.3. Possibilities of Using miRNA in Clinical Diagnostics

Although combinations of second-trimester biochemical indicators, markers, and ultrasonography have been proposed, none has yet shown findings that are sound enough to be used in a therapeutic setting [19]. In the first trimester of maternal serum/plasma samples collected from patients with hypertension or pre-eclampsia, a number of the hypothesized targets of C19MC microRNAs in which the current study was interested were previously shown to be enhanced [10]. Several miRNAs may control the same gene. It is feasible to fully pinpoint the ones responsible for regulating specific genes of interest. Unfortunately, it is difficult to directly interpret experimental outcomes since complex networks typically govern the routes. Most of them target many genes for repression and collectively control them [16]. Hence, as mentioned earlier, pregnancy-related difficulties are caused by several pathological and physiological processes in which microRNAs play a vital role. The literature shows that circulating C19MC microRNAs may contribute to developing pre-eclampsia and prenatal hypertension in early pregnancy.

Furthermore, a different study by Légaré et al. did not find any information about "C19MC microRNA profiling in maternal plasma exosomes during the first trimester of pregnancy" [10]. Instead, it discovered that placental tissues generated from individuals with gestational hypertension and pre-eclampsia after childbirth had the same C19MC microRNA expression profile as first-trimester circulating plasma exosomes. Four of the fifteen C19MC microRNAs that were examined showed down-regulation in placental tissues when GH patients were present [15]. It is similar to the results in placental tissues taken from individuals with gestational hypertension at birth in patients with subsequent occurrences of hypertension. It was discovered after examining the first-trimester maternal plasma exosome C19MC microRNA expression profile of pregnancy, with the selection of only those with diagnostic potential.

In addition, eleven of the fifteen C19MC microRNAs evaluated showed down-regulation in pre-eclampsia patients. Légaré et al. discovered lower levels of miR-525-5p, miR-520a-5p, and miR-517-5p in individuals who subsequently developed pre-eclampsia, broadly matching the expression patterns reported in afflicted placental tissues [10]. The finding are in line with what was revealed in the researcher's previous study. The microRNAs were tested for their diagnostic potential during the first trimester in maternal plasma exosomes of pregnancy. However, the findings are at odds with those of He and Ding, which showed that circulating C19MC microRNAs in maternal plasma were upregulated in the first trimester and accurately predicted the eventual onset of pre-eclampsia or gestational hypertension [6]. From 12 to 14 weeks of pregnancy, other researchers noticed elevated

levels of several C19MC microRNAs in sera of women who eventually had severe preeclampsia [10]. Hence, a combination of variables, including those resulting from several different circumstances, may affect the different expression patterns of C19MC microRNAs between maternal plasma and their exosomes. At the very least, a representation of a specific C19MC microRNA expression in maternal plasma can be seen in placental cells from different regions undergoing apoptosis [24]. It releases placental debris into the mother's bloodstream and actively secretes exosomes that promote intercellular communication.

### **4. Discussion**

Research by Hromadnikova et al. revealed no correlation between microRNA and a history of hypertension among individuals with pre-eclampsia that had already developed. The expression of microRNA genes in placental tissues did not alter Doppler ultrasonography parameters linked to worse outcomes in pre-eclampsia. The elevation of pertinent proteins involved in the direction of critical biological processes, including hemocoagulation, apoptosis, stress response, and angiogenesis, may result from the lowered amounts of C19MC microRNAs in placental tissues [1]. Lv et al. further argued that ischemia, hypoxia, insufficient uteroplacental blood perfusion, and defective placental angiogenesis may all lead to blood coagulation–fibrinolysis system failure, aberrant placental trophoblast apoptosis, and, lastly, the emergence of a widespread maternal inflammatory response. Predicted targets of C19MC microRNAs are elevated in placental tissue samples from women who had problems during pregnancy [12].

However, the observed down-regulation of C19MC microRNAs is at odds with the lower levels of several proteins found in the tissue of the placenta in patients with problems in their pregnancies. According to Miura et al., the microRNAs are anticipated to be targeted. There exist modes of fully identifying miRNAs that guide specific genes of value [13]. However, their routes are often complicated control networks that are hard to grasp. Moreover, it complicates the straightforward interpretation of experimental results [27]. Many target several genes for suppression, and they seem collaboratively regulated.

Lastly, the previous theory that exosomes discharged into the body's circulation serve as a non-invasive and singular source of signaling molecules, whose abnormal expression profile mimics that of the parent cells, was supported by this review. It lends credence to the hypothesis that those produced by the placenta may be used in first-trimester screening to detect a sizable fraction of women who may later develop pre-eclampsia or gestational hypertension [21]. The only drawback of the strategy is that since the downregulation of the same biomarkers begins early in pregnancy, the screening of C19MC microRNAs in plasma exosomes cannot distinguish between the women who will later develop hypertension and those who will have pre-eclampsia during the first trimester of pregnancy. Nevertheless, it may one day lead to the discovery of new microRNA biomarkers that may distinguish between women at risk for gestational hypertension or pre-eclampsia, allowing for the early determent of pre-eclampsia with earlier delivery of low-dose aspirin.

#### **5. Conclusions**

In conclusion, this literature review showed that circulating C19MC microRNAs may contribute to developing pre-eclampsia and prenatal hypertension in early pregnancy. In individuals with subsequent occurrences of gestational hypertension and pre-eclampsia, C19MC microRNAs were shown to be down-regulated. The circulating C19MC microRNA expression profile from the first trimester was identical to that in placental tissues collected from individuals with hypertension and pre-eclampsia. Expression analysis of maternal plasma exosomes, as opposed to entire maternal plasma samples, increased the first trimester C19MC microRNA screening's prediction accuracy for detecting hypertension and pre-eclampsia. The results require further confirmation by large-scale investigations. More first-trimester plasma samples must be gathered to achieve a sufficient number of

individuals who may later suffer pregnancy-related problems, making conducting the study very difficult.

**Author Contributions:** Conceptualization, A.K. (Adrianna Kondracka), B.K., D.K., M.R.-K., J.S. and A.K. (Anna Kwa´sniewska); methodology, A.K. (Adrianna Kondracka), B.K., I.J., K.F., A.O., A.F. and A.K. (Anna Kwa´sniewska); software, B.K., I.J., K.F. and J.S.; validation, A.K. (Adrianna Kondracka), A.O., D.K., M.R.-K., A.F. and A.K. (Anna Kwa´sniewska); writing—original draft preparation, A.K. (Adrianna Kondracka), B.K., I.J. and A.F.; writing—review and editing, D.K., K.F., A.O., M.R.-K., J.S. and A.K. (Anna Kwa´sniewska); visualization, A.K. (Adrianna Kondracka) and B.K.; supervision, A.K. (Anna Kwa´sniewska) and B.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** The APC was funded by Medical University of Lublin.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


**Xiaona Xu 1,2,†, Baoying Ye 3,†, Min Li 1, Yuanqing Xia 1, Yi Wu 1,2,\* and Weiwei Cheng 1,2,4,\***


**Abstract:** Background: Congenital heart disease/defect (CHD) is one of the most common congenital disabilities. Early diagnosis of CHD can improve the prognosis of newborns with CHD. The aim of this study was to evaluate the relationship between the factors and the onset of fetal congenital heart disease by measuring fetal umbilical artery (UA) Doppler index, maternal HCY, and Cys C levels during pregnancy. Methods: This retrospective study analyzed 202 fetuses with CHD, including 77 cases (39.1%) of simple CHD and 120 cases (60.9%) of complex CHD. Singleton pregnant women who were examined at the same time and whose malformation screening did not suggest any structural abnormalities in the fetus were assigned to the control group (*n* = 400). The UA Doppler index, plasma HCY, and Cys C levels were compared among the pregnant women across the three groups, and logistic regression analysis was performed on statistically significant markers. The ROC of UA S/D, PI, RI, HCY, and Cys C were plotted, and the area under the ROC (AUC) was calculated. Results: The UA S/D, PI, and RI in the complex CHD group were significantly higher than those in the control group (*p* < 0.05). The levels of HCY and Cys C in the CHD group were significantly higher than those in the control group (*p* < 0.05). HCY and S/D revealed a positive correlation (r = 0.157), and the difference was statistically significant (*p* < 0.001). Cys C and S/D were positively correlated (r = 0.131), and the difference was statistically significant (*p* < 0.05). The levels of UA Doppler indices, maternal plasma HCY, and Cys C were elevated in fetuses with CHD. The AUC of the combined test of the UA index, HCY, and Cys C was higher than that of each individual test. Conclusions: Elevated levels of the UA doppler indices, HCY, and Cys C during pregnancy are positively associated with the development of congenital heart disease in offspring. The combination of HCY and Cys C was the most efficient test for the diagnosis of CHD. We are the first to report that plasma Cys C levels of women pregnant with fetuses with CHD were higher than those of women pregnant with normal fetuses.

**Keywords:** congenital heart disease; homocysteine; cystatin C; the UA Doppler index

### **1. Introduction**

Congenital heart disease/defect (CHD) is one of the most common congenital disabilities, with a prevalence rate of 1 in 100 live births. It is one of the leading causes of perinatal mortality [1]. With the rapid development of prenatal imaging technologies, the application of fetal echocardiogram to detect CHD has advanced, and the use of color Doppler noninvasive detection of fetal hemodynamic changes is also becoming gradually common. Several studies have reported [2–4] that the increase in maternal plasma homocysteine (HCY) level is closely associated with cardiac malformations in their offspring.

**Citation:** Xu, X.; Ye, B.; Li, M.; Xia, Y.; Wu, Y.; Cheng, W. The UA Doppler Index, Plasma HCY, and Cys C in Pregnancies Complicated by Congenital Heart Disease of the Fetus. *J. Clin. Med.* **2022**, *11*, 5962. https://doi.org/10.3390/ jcm11195962

Academic Editor: Alexander H. Maass

Received: 6 September 2022 Accepted: 7 October 2022 Published: 10 October 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The maternal plasma cystatin C (Cys C) is a biomarker of early kidney injury [5] and also a novel cardiac biomarker [6] independently associated with the risk of cardiovascular anomalies and mortality. The relationship between Cys C level during pregnancy and the occurrence of fetal CHD has not been reported yet. In this study, we aimed to determine the relationship between the factors and the onset of fetal congenital heart disease by measuring fetal umbilical artery (UA) Doppler index, maternal HCY, and Cys C levels during pregnancy.

### **2. Materials and Methods**

### *2.1. Patient Cohort*

The present study is a retrospective study. Fetal cases diagnosed as CHD by fetal cardiography in International Peace Maternity and Child Health Hospital from January 2015 to December 2021 were enrolled in the CHD group. Fetal cases with normal anomaly scans were enrolled in the control group. Electric clinical medical charts were reviewed to obtain demographic information on maternal age and pregestational body mass index (BMI). Serological screening results of maternal plasma HCY and Cys C level at 12–14 weeks of gestation were also collected from the medical charts.

The inclusion criteria were as follows: fetuses with CHD were included in the CHD group. The control group was normal fetuses without structural malformation matched with maternal age and gestational age, and a 1:2 ratio was used. The exclusion criteria were as follows: fetuses with CHD combined with extracardiac anomalies, multiple pregnancies, and pregnancies with maternal obstetric complications.

This study was approved by the Medical Ethics Committee of the International Peace Maternity and Child Health Hospital (GKLW 2019–24). Written informed consent was obtained from the pregnant women involved in our study.

CHD was diagnosed by ultrasound and clinical diagnosis. Based on the codes of the International Classification of Diseases, Ninth Revision, Clinical Modification, complex CHD (CCHD) refers to all types of CHD except for simple CHD (SCHD). The main types of SCHD included ventricular septal defect (VSD), aortic valve stenosis, pulmonary stenosis, patent ductus arteriosus, and secundum atrial septal defects The fetal heart defects that were not included in the study were persistent left superior vena cava, simple right aortic arch, aberrant right subclavian artery, and heart tumors. The main types of CCHD include tetralogy of Fallot (TOF), tricuspid atresia and stenosis, coarctation of the aorta, hypoplastic right heart syndrome, tricuspid valve dysplasia, atrioventricular septal defect, single ventricle, interruption of aortic arch, pulmonary atresia, persistent truncus arteriosus, hypertrophic cardiomyopathy, vascular ring, hypoplastic left heart syndrome, transposition of great arteries, and double outlet right ventricle [7,8].

### *2.2. Fetal Echocardiogram*

The gestational age range for echocardiography was 18–28 weeks. Detection of fetal echocardiography was performed based on the Guidelines of the International Society of Ultrasound in Obstetrics and Gynecology (ISUOG) [9]. Ultrasound examination was performed by radiologists with prenatal ultrasound diagnostic qualifications using GE Voluson E10 (GE Healthcare, Zipf, Austria), Philips iU Elite (Philips, Copenhagen, Denmark), or Philips iE33 (Philips, Copenhagen, Denmark). Subjects were kept a flat, lying position and exposed their lower abdomen. The number of fetuses was routinely checked, multiple pregnancies were excluded, and the fetal orientation was determined. The transverse and four-chamber heart section of the fetal abdomen was determined along with the position of the internal organs, the heart, and the cardiac axis. Based on this, the left and right ventricular outflow tract, long axis and short axis section, three-vessel section or three-vessel tracheal section, aortic arch section, ductus arteriosus arch section, and the vein-atrial connection section determined the fetal atrioventricular connection relationship. The left and right atrioventricular valves determined the fetal ventricular–artery connection relationship. The interrelationship between the great arteries, the ratio of the inner diameter

of the aortic arch and the ductus arteriosus, and finally the measurement results were collected and stored.

All fetuses with CHD were diagnosed by two experienced fetal echocardiographists. Postnatal results were obtained from the newborn's examination records at the hospital or directly from the parents. For CHD cases, neonatal echocardiograpy was performed by another examiner (approximately 2–4 days after birth) prior to discharge. A clinical examination by an experienced pediatrician included auscultation of the heart murmur and oxygen saturation of the upper and lower extremities. The newborn/fetus was considered normal if no abnormalities were suspected or found.

### *2.3. The UA Doppler Index at 22–24 Weeks of Pregnancy*

When ultrasound fetal malformation screening was performed at 22 to 24 weeks of pregnancy, after detecting the free segment of the umbilical cord, the umbilical artery blood flow spectrum was obtained. When measuring the umbilical artery spectrum, pregnant women had to hold their breath for 3–5 s to facilitate the acquisition of a stable umbilical artery spectrum. The indices of systolic/diastolic ratio (S/D), resistance index (RI), and pulsatility index (PI) were measured and recorded.

### *2.4. Blood Sampling and Biochemical Characteristics at 12–14 Weeks of Pregnancy*

Pregnant women underwent fasting after 22:00 on the day before the first antenatal examination in the 12th to 14th weeks of pregnancy. A total of 3 mL of venous blood was drawn on an empty stomach at 08:00–10:00 on the day of examination. The serum was routinely separated, and the levels of Hcy and Cys C were measured. The automatic biochemical analyzer was used for detection, and the detection of various indicators was performed according to the instructions given in the kit.

### *2.5. Statistical Analysis*

SPSS 25.0 statistical software was used for analysis. The measurement data following the normal distribution were expressed as a one-way analysis of variance. The data are expressed as mean ± standard deviation. The comparison of the counting data is expressed by the Wilcoxon rank sum test, and the data are expressed as frequencies. Pearson's (r) correlation was performed to analyze the correlation between maternal plasma HCY, Cys C level, and the UA doppler indices, and the logistic regression analysis of the various influencing factors of fetal congenital heart disease was performed. A *p*-value < 0.05 indicated that the difference was statistically significant.

### **3. Results**

A total of 197 fetuses with CHD were assessed, which included 77 cases (39.1%) of simple CHD and 120 cases (60.9%) of complex CHD. The type of CHD is depicted in Table 1. The demographic characteristics of the patients in the CHD groups and the healthy control groups are depicted in Table 2. The mean maternal age and pregestational BMI were not significantly different between the CHD and control groups (*p* > 0.05; Table 2).

### *3.1. The Comparison of the UA Doppler Indices at 22–24 Weeks of Pregnancy between the CHD and Control Groups*

The UA S/D, PI, and RI in the complex CHD group were significantly higher than those in the control group (3.43 ± 0.84, 1.18 ± 0.19, 0.70 ± 0.06 vs. 3.21 ± 0.57, 1.12 ± 0.16, 0.68 ± 0.05; *p* < 0.05, respectively). No statistically significant difference was noted in the UA Doppler indices between the simple CHD group and the complex CHD group. In addition, no statistically significant difference was noted in the UA Doppler indices between the simple CHD group and the control group (Table 3).


**Table 1.** Antenatal cardiac finding in all cases of CHD.

VSD, ventricular septal defect; COA, coarctation of the aorta; PS, pulmonary stenosis, TOF, tetralogy of Fallot; TGA, transposition of great arteries; HLHS, hypoplastic left heart syndrome; HRHS, hypoplastic right heart syndrome; DORV, double outlet of right ventricle; TVD, tricuspid valve dysplasia; AVSD, atrioventricular septal defect; SV, single ventricle; IAA, interruption of aortic arch; PA, pulmonary atresia; TA, tricuspid atresia; PTA, persistent truncus arteriosus; HCM, hypertrophic cardiomyopathy.

**Table 2.** Maternal characteristics of the case and control group.


**Table 3.** Comparison of the UA Doppler indices at 22–24 weeks of pregnancy.


*3.2. The Comparison of Maternal Plasma HCY and Cys C Levels at 12–14 Weeks of Pregnancy*

The levels of HCY and Cys C in the simple CHD group were significantly higher than those in the control group (*p* < 0.05). The HCY and Cys C levels in the complex CHD group were significantly higher than those in the control group, and the difference was statistically significant (*p* < 0.05). However, the difference in the HCY and Cys C levels between the simple and complex CHD groups was not statistically significant (Table 4).


**Table 4.** Comparison of HCY and Cys C levels at 12–14 weeks of pregnancy.

### *3.3. Correlation between the Plasma Biochemical Indicators and the UA Doppler Indices*

Pearson's correlation was applied to analyze the maternal plasma HCY and Cys C levels and the UA Doppler indices. The results revealed that HCY and S/D had a positive correlation with PI (r = 0.157, 0.088; *p* < 0.05, respectively). Cys C and S/D, PI, and RI were positively correlated (r = 0.131, 0.118, 0.118; *p* < 0.05, respectively) (Tables 5–7).

**Table 5.** Pearson correlation of UA S/D with HCY and Cys C.


**Table 6.** Pearson correlation of UA PI and HCY with Cys C.


**Table 7.** Pearson correlation of UA RI and HCY with Cys C.


### *3.4. Analysis of the Influencing Factors of Fetal Congenital Heart Disease*

Multifactorial logistic regression equations were constructed by including age, pregestational BMI, UA S/D, UA PI, UA RI, and maternal plasma HCY and Cys C levels with the presence or absence of CHD as the dependent variable. The results showed that the effect of UA S/D, PI, RI, and the level of maternal plasma HCY and Cys C on fetal CHD were statistically significant (*p* < 0.05). The ORs for UA RI and Cys C were especially high (69.55 and 24.75) (Table 8).

**Table 8.** Logistic regression analysis.


The ROC of UA S/D, PI, RI, HCY, and Cys C were plotted, and the area under the ROC (AUC) was calculated. The binary logistic regression analysis was used to obtain the cut-off value of the combined prediction probability of each index to evaluate the sensitivity and specificity of the individual and combined tests for the diagnosis of CHD. The value of the individual and combined tests for the diagnosis of CHD was analyzed. The results showed that the AUC of the combined test of HCY and Cys C was higher than that of each individual test, and its optimal cut-off value was 0.371, and the sensitivity and specificity for the diagnosis of CHD were 45.7% and 78.5%, respectively (Table 9, Figure 1).


**Table 9.** The area under the ROC.

**Figure 1.** ROC Curve.

### **4. Discussion**

The umbilical cord is the only channel through which any material can be transmit ted between the mother and the fetus. Therefore, the umbilical artery blood contains a large amount of fetal–placental circulation information, and the UA S/D, PI, and RI reflect the placental resistance and the fetal intrauterine condition. In the first trimester of pregnancy, the resistance of umbilical artery blood flow is high. With the progression of pregnancy, to ensure the blood supply of normal fetal development, the placenta gradually matures, the villus increases and thickens, the resistance of the placental blood vessels decreases, and the blood flow increases. If the placenta function is poor, the placental vascular spasm, infarction, edema, and other conditions make the lumen narrow, which results in increased fetal–placental circulatory resistance and reduced umbilical artery blood flow. This affects the development and growth of the fetus [10]. Previous studies [11,12] have reported that in cases of isolated major fetal heart defects, maternal serum placental growth factor (PIGF) decreases at 11–13 weeks of gestation, which indicates that placental angiogenesis was impaired in the first trimester of pregnancy. Moreover, the overexpression of vascular endothelial growth factor A, soluble FMS-like tyrosine kinase-1 (sFlt-1), and soluble endocrine in fetuses with congenital heart disease were observed in their heart tissue and cord blood. Maternal blood PIGF levels were decreased, and sFlt-1 levels were increased at 18–37 weeks of gestation. This indicated that placental angiogenesis is affected when the fetus has congenital heart disease [10]. A study [13] reported that increased UA PI is associated with CHD in the fetus. Another study [14] reported that regardless of the type of congenital heart disease, the UA PI increased as the pregnancy progressed, which suggested that the degree of placental damage increased with the progression of pregnancy. We found that UA S/D, PI, and RI of the fetuses with CHD were higher than those of normal fetuses, and UA S/D, PI, and RI were particularly increased in the fetuses with complex CHD. UA hemodynamic changes are a good indicator of changes in placental functions [15], and the increase in UA Doppler indices in the second trimester in the CHD group in this study indicated possible placental function impairment in the second trimester of pregnancy, reflecting that CHD occurrence is associated with placental hypofunction, followed by placental ischemia and hypoxia and fetal ischemia and hypoxia, ultimately leading to adverse pregnancy outcomes. Based on previously reported studies, echocardiography is recommended in fetuses with increased UA Doppler indices in the second trimester to detect fetal heart defects as soon as possible and improve the prenatal diagnosis rate of fetal CHD. In case fetal echocardiography indicates a fetus with CHD, ultrasound should be performed regularly to measure the blood flow index of the UA to assess the growth and development of the fetus, monitor the intrauterine safety of the fetus, terminate the pregnancy on time, and ensure that newborns with CHD receive timely treatment.

This study also showed that the HCY of the simple CHD group was higher than that of the control group, and the difference was statistically significant (*p* < 0.05). The HCY level of the complex CHD group was significantly higher than that of the control group (*p* < 0.001). Previous studies [4,16,17] have shown that hyperhomocysteinemia in pregnant women may be associated with CHD occurrence in their offspring, and the present study also confirmed that high maternal HCY levels are associated with fetal cardiac malformation. HCY can pass through the placental barrier and exert cytotoxic effects, inducing excessive apoptosis in early embryonic cells, impairing placental functions, and causing embryonic malformations [18]. The abnormal differentiation of neural crest cells at high HCY concentrations may lead to neural tube defects and abnormal cardiac development [19]. If the maternal plasma HCY level can be determined in the first trimester of pregnancy, hyperhomocysteinemia can be detected as early as possible, and early screening for fetal congenital heart disease in pregnant women with hyperhomocysteinemia can be performed. Such women can be supplemented with an appropriate diet and nutrition.

Cys C is a non-glycosyl alkaline protein belonging to cysteine protease inhibitors. Previous studies showed [20,21] that Cys C can be reabsorbed and completely degraded at the proximal tubule after glomerular filtration; hence, Cys C is less affected by factors such as age, sex, weight, diet, lipid metabolism, and inflammation. Cys C levels may be associated with cardiovascular disease. Cys C impairs the cardiovascular system by affecting smooth muscle cell functions, coagulation, lipid peroxidation, and endothelial cell functions [22]. We found that Cys C levels of the subjects who were pregnant with fetuses with CHD were significantly higher than those of the normal control group (*p* < 0.05). Infants and children with CHD have increased Cys C levels, and Cys C can be used as a biomarker to predict postoperative complications of acute kidney injury after undergoing cardiac surgery [23,24]. The relationship between maternal plasma Cys C and fetal CHD has not been reported. To the best of our knowledge, we are the first to report that plasma Cys C levels of women pregnant with fetuses with CHD were higher than those of women pregnant with normal fetuses. Future studies are necessary for establishing a correlation

between newborn plasma Cys C, placental Cys C, and maternal plasma Cys C levels in CHD for better understanding.

A strong positive correlation exists between Cys C levels and HCY. Cys C is a strong independent predictor of long-term all-cause death and major adverse cardiac events in elderly patients with acute myocardial infarction. The combined detection of Cys C and HCY further improves the predictive value [25]. Cys C can inhibit HCY decomposition, increase its concentration in blood, and interact with factors including plasma HCY and histones, thereby increasing the risk of hypertension and gestational diabetes in pregnant women and further increasing the risk of premature birth, miscarriage, and fetal intrauterine growth restriction [26]. The accumulated HCY in the body passes through the placental barrier; if the endothelial function of the blood vessels in the placenta is impaired, the vascular resistance in the placenta increases, which damages placental functions, affecting the blood flow motility of the UA and eventually increasing UA blood flow resistance. This study showed that increased UA Doppler indices and maternal plasma HCY and Cys C levels affected the fetuses suffering from CHD. A positive correlation was observed between maternal plasma HCY and Cys C levels and UA Doppler indices. From the clinical perspective, fetal echocardiography is a routine part of prenatal screening and is only added if a possible abnormality in heart development is detected during fetal systemic ultrasound or if there are high-risk factors for CHD, so some congenital heart diseases are not detected prenatally and are detected after birth due to presence of cardiac symptoms. The early detection of high HCY and Cys C levels in the first trimester of pregnancy or the high UA Doppler indices in the second trimester of pregnancy will help assist fetal echocardiography in fetal CHD diagnosis. This will improve the detection rate of fetal CHD, thereby increasing the rate of neonatal treatment and reducing neonatal mortality.

A limitation of our study was that not all heart malformations can be diagnosed by ultrasound in the fetus, which might lead to a possible statistical bias in our results. Fetal cardiac structure screening mainly relies on prenatal echocardiography. For those simple CHDs, such as isolated ventricular septal defect, the diagnostic accuracy of fetal echocardiography is not very high. This is mainly because some defects would close spontaneously during pregnancy or after birth. Echocardiography is not routinely performed on newborns after birth, but only newborns diagnosed with CHD in the fetal period require an echocardiogram after birth and a clinical examination of each newborn by an experienced pediatrician, including auscultation of heart murmurs and oxygen saturation of the upper and lower extremities. Another limitation of the present study was that the blood testing and UA Doppler were not performed in the same time, which could also cause some biases in the results. In the subsequent study design, we would consider performing the blood sampling and ultrasound in the same gestational age.

### **5. Conclusions**

To summarize, UA S/D, PI, and RI values of the fetuses with CHD, especially complex CHD, were significantly higher than those of normal fetuses. The maternal plasma HCY and Cys C levels of women pregnant with fetuses with CHD increased significantly. Maternal plasma HCY, Cys C, and UA Doppler indices were positively correlated, which were increased in fetal CHD. The detection of increased HCY and Cys C in the first trimester of pregnancy suggests an increased risk of fetal CHD, and fetal echocardiography is required in the second trimester, indicating the screening value of increased HCY and Cys C levels in the first trimester of pregnancy. In this study, we only analyzed the relationship between maternal plasma HCY and Cys C levels in the first trimester of pregnancy, UA Doppler indices, and fetal CHD. Prospective application of these results in a validation study is still required to evaluate the clinical utility of these markers or a combination of these markers. In future large-sized studies, researchers need to investigate maternal blood indicators in the first, second, and third trimesters of pregnancy and make longitudinal comparisons to explore the correlation between various indicators and fetal CHD. A few studies on the correlation between maternal plasma Cys C and fetal CHD are available. In the future, the

effect of plasma Cys C on fetal CHD can be analyzed in depth by conducting basic trials combined with prospective clinical studies, which can help to detect congenital disabilities as early as possible and reduce the incidence of adverse outcomes.

**Author Contributions:** Supervision, W.C.; writing—original draft, X.X.; writing—review and editing, Y.W. and B.Y.; software, Y.X.; visualization, M.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Shanghai Committee of Science and Technology, China, grant number 18411963500.

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of IPMCH (GKLW 2019-24).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are openly available in FigShare at 10.6084/m9.figshare.21300045.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

