Maternal periconceptional folic acid supplementation and risk for fetal congenital genitourinary system defects

ABSTRACT BACKGROUND Taking folic acid supplementation could reduce the risk of neural tube defects for offspring in the maternal periconceptional period, but the relationship between folic

acid use and other birth defects remains unclear, such as genitourinary system birth defects. METHODS The data from a Prenatal Health Care System and Birth Defects Surveillance System in

Tongzhou, Beijing, China, were collected from 2013 to 2018. We adjusted for differences in characteristics between comparison groups using propensity score inverse probability weighting and

assessed associations with Poisson regression modeling. RESULTS A total of 65,418 live births and stillbirths were included, and there were 194 cases with congenital genitourinary defects

among them. The prevalence of genitourinary system birth defects was 29.2 (34.9) per 10,000 for FA/MMFA users (nonusers). Compared to nonusers, FA/MMFA users had a lower risk for

genitourinary system birth defects (adjusted risk ratio [aRR] 0.81, 95% confidence interval [CI] 0.67, 0.98), and for hypospadias (aRR 0.55, 95% CI 0.40, 0.76). CONCLUSIONS FA or MMFA

supplementation during the maternal periconceptional period could reduce the risk for genitourinary system birth defects in offspring. More mechanisms should be explored for the protective

effect. IMPACT * Folic acid (FA) or multiple micronutrients containing folic acid (MMFA) supplementation during the maternal periconceptional period could reduce the risk for genitourinary

system birth defects in offspring. * Maternal FA/MMFA supplementation during the periconceptional period may reduce the risk for hypospadias. You have full access to this article via your

institution. Download PDF SIMILAR CONTENT BEING VIEWED BY OTHERS MATERNAL B-VITAMIN INTAKE AND B-VITAMIN SUPPLEMENTATION DURING PREGNANCY IN RELATION TO NEONATAL CONGENITAL HEART DEFECTS: A

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Article Open access 10 June 2021 EFFECTS OF MATERNAL FOLATE AND VITAMIN B12 ON GESTATIONAL DIABETES MELLITUS: A DOSE-RESPONSE META-ANALYSIS OF OBSERVATIONAL STUDIES Article 01 February 2022

INTRODUCTION Genitourinary system birth defects include malformations of the urinary system and anomalies of the reproductive system, like hypospadias, renal agenesis, duplex kidneys,

cryptorchidism, and so on.1 In the clinical study, malformations of the kidney and urethra are called congenital anomalies of the kidney and urinary tract (CAKUT).2 Genitourinary system

birth defects affect children’s quality of life and are risk factors for severe disease. Urinary malformations, such as hypospadias, can affect patients’ social interactions in childhood and

their sexual behavior and reproductive ability in adulthood.3 CAKUT is one of the most common causes of end-stage kidney disease in children and adolescents.4 Although genitourinary system

birth defects can be partially corrected with multiple reconstructive surgeries, the outcomes of such operations are not encouraging.5 The prevalence of congenital genitourinary

malformations varies worldwide, ranging from 24.3 to 130.1 per 10,000 births.6,7 In China, hypospadias is the fourth most prevalent birth defect in perinates, according to the National Birth

Defect Surveillance System.8,9 In Beijing, the prevalence of CAKUT increased from 8.3 per 10,000 in 2008 to 16 per 10,000 in 2014.10 Hypospadias and CAKUT are ranked in the five most common

birth defects in some districts of Beijing, such as Dongcheng from 2011 to 201211 and Pinggu from 2013 to 2016.12 The causes of genitourinary system birth defects are complex, as both

genetic and environmental factors are thought to come into play.13,14 Adult polycystic kidney disease and infantile polycystic kidney disease are caused by autosomal abnormalities, but the

etiologies of other genitourinary system birth defects are unknown.15 Variants in ALX4 are believed to be related to genitourinary defects.16 PAX2 and HNF1B may be associated with CAKUT.17

Maternal exposure to endocrine disruptors may increase the risk for hypospadias.18 Fetal exposure to angiotensin-converting enzyme inhibitors and cocaine may also lead to CAKUT.19 A study of

the interaction between genes and the environment showed that different single-nucleotide polymorphism rs5000770 genotypes have varying susceptibilities to low-dose bisphenol A, which may

lead to hypospadias.20 The development of the kidney and urinary tract begins in the intermediate mesoderm, and the entire urinary system is completely developed at 32–36 gestational

weeks.21 Apoptosis of metanephric mesenchyme, suppression of ureteric branching, and DNA methylation play vital roles in developing abnormalities of the genitourinary system.22 Recently,

researchers used animal experiments and in vitro experiments to explore the relationship between folic acid (FA), DNA methylation, and kidney development. In rat models, nutritional

restriction (including restricting protein) induced renal dysplasia, which was attenuated by FA supplementation, and the in vitro experiment showed that DNA methylation is indispensable for

the development of metanephros and that FA is a significant methyl donor.23 Whether FA supplementation has a protective effect on congenital genitourinary malformations is inconsistent in

population studies. A case–control study reported that FA supplementation in the maternal periconceptional period can decrease the risk for hypospadias.18 Nevertheless, other studies have

documented that taking in nutrients containing FA does not affect the risk for birth defects in the genitourinary system.24,25 A case–control study in the Netherlands even showed that taking

FA alone during the gestational period may increase the risk for CAKUT.26 Maternal FA supplementation can prevent fetal neural tube defects.27 In 2009, the Chinese government implemented a

public health program called Folic Acid Supplementation to Prevent Neural Tube Defects. This program provided all women of childbearing age who planned to become pregnant with free FA pills

(0.4 mg/pill) for 6 months.28 The prevalence of neural tube defects decreased by 70% in Beijing after the program was initiated.29 Yet, to the best of our knowledge, no study has evaluated

the impacts of this program on the prevalence of genitourinary system birth defects in China. Thus, we investigated the relationship between maternal periconceptional supplementation with FA

or multiple micronutrients containing FA (MMFA) and the risk for genitourinary birth defects in offspring based on retrospective cohort data collected from 2013 to 2018 in Beijing. METHODS

STUDY POPULATION AND DATA COLLECTION The data from the Prenatal Health Care System (PHCS) and Birth Defects Surveillance System (BDSS), were collected from January 1, 2013, to December 31,

2018, in Tongzhou district, Beijing, China. A total of 65,418 fetuses and their mothers were registered in the PHCS and BDSS during the research period. Fetuses were delivered in the

hospital in Tongzhou district. Because of missing information on maternal FA/MMFA supplementation, 258 (0.4%) records were deleted. Ultimately, 65,160 mother–child pairs were included in the

study. The analysis tree on how the analysis data set has developed is showed in Fig. 1. The PHCS is a networked pregnancy healthcare system in all community and obstetric hospitals in

Beijing. Every woman who lives in Beijing and who becomes pregnant needs to register with the system in her 6th–13th week of pregnancy at the community hospital closest to her residence.

Through face-to-face questionnaire interviews, doctors or nurses collect information on sociodemographic characteristics, reproductive history, and FA supplementation during the

periconceptional period. Then, the woman can choose an obstetric hospital for her prenatal healthcare and delivery. All information collected during prenatal healthcare and delivery is

uploaded to the PHCS by doctors in the obstetric hospital. The Maternal and Child Health Care Hospital in each district in Beijing is responsible for administering the system and ensuring

quality control. In October and November of each year, doctors in the Maternal and Child Health Care Hospital randomly select 400 individuals to participate in a telephone survey to verify

the accuracy of the information collected in the community hospital at registration.30 The BDSS is a hospital-based surveillance system that includes all hospitals with departments of

gynecology and obstetrics in Beijing, Maternal and Child Health Care Hospitals in each district, and the Beijing Maternal and Child Health Care Hospital (municipal Maternal and Child Health

Care Hospital). The BDSS is organized and led by an expert team that includes individuals from Beijing Maternal and Child Health Care Hospital, the department of child healthcare in each

district Maternal and Child Health Care Hospital responsible for birth defect surveillance in their district, dedicated staff members from each hospital responsible for birth defect

surveillance, all doctors in departments of gynecology or obstetrics or pediatrics (who act as reporters), and sonographers and pathologists who deal with birth defect diagnosis. The BDSS

covers birth defects, including external anomalies, severe visceral malformations, congenital metabolic diseases, and chromosomal abnormalities. Pregnant women undergo a series of screening

and diagnostic examinations for fetal birth defects, including biochemical examination and ultrasound, beginning in the 13th gestational week. In the second trimester, fetal systematic

ultrasonography is also used to diagnose birth defects and test amniotic cell chromosomes if needed. If a congenital abnormality is found, the pregnant woman is referred to a specialized

hospital qualified to diagnose birth defects for a confirmed diagnosis. Once diagnosed, the woman can choose whether to continue with the pregnancy. After delivery or termination of the

pregnancy, a comprehensive physical examination of the baby is carried out by an obstetrician and pediatrician or pathologist. Malformations of the kidney or urinary tract are diagnosed by

ultrasound. Once a birth defect, including a genitourinary system malformation, is diagnosed, doctors complete a Birth Defects Case Report Form and report it to the BDSS via paper and direct

online reporting. The BDSS data are subject to tertiary quality control inspection, namely, hospital self-examination; secondary quality control in the district Maternal and Child Health

Care Hospital; and tertiary quality control in the municipal Maternal and Child Health Care Hospital. Staff in the district Maternal and Child Health Care Hospital or hospitals with

departments of gynecology or obstetrics receive standardized training each year from the expert team at Beijing Maternal and Child Health Care Hospital. At the hospital level, the doctor who

makes the diagnosis checks the information on the birth defect case report form against the clinical record in the hospital information system after completing the form. Moreover, a

dedicated staff member checks the number of birth defect cases, the diagnosis, birth outcome, and other information on the mother and the child against maternal gynecology or obstetrics

records and pediatric clinical records in the hospital information system monthly or quarterly. For secondary quality control, every year, staff of the district Maternal and Child Health

Care Hospital randomly select at least half the hospitals in the district to check the information in each case report form against the hospital information system. Each year, the team at

Beijing Maternal and Child Health Care Hospital randomly selects 10–15% of hospitals in the whole city to check information from each birth defect case report form against the hospital

information system. EXPOSURE AND OUTCOME We defined the periconceptional period as the time from 3 months before the last menstrual period to the end of the first trimester. Three questions

were designed to collect information on FA/MMFA supplementation: (1) whether the woman supplemented with FA/MMFA during the periconceptional period (yes or no), (2) what formula was used for

supplementation (FA only or MMFA), and (3) how many days out of 10 supplements were taken (high frequency was defined as 8 days or more out of 10, and low frequency was defined as fewer

than 8 days out of 10). Information was collected through face-to-face surveys in the first trimester at the community hospital when the woman registered in the PHCS. In China, the most

common FA tablet contains 0.4 mg FA.31 According to a survey in Jiangsu Province, China, 71% of MMFA users took a formula that contained 0.8 mg FA.32 The outcome of the study was whether

babies were diagnosed with genitourinary system birth defects. Diagnoses of live births were verified by professionally trained obstetricians and sonographers, and stillbirths were diagnosed

by pathologists. Hypospadias was diagnosed by physical or ultrasound examinations. Hydronephrosis and other kidney and ureteral malformations were diagnosed by ultrasound examinations.

Hypospadias and hydronephrosis were coded Q54.0~Q54.3, Q62.0, respectively, according to the International Classification of Diseases, 10th Revision. Other kidneys and ureteral malformations

included renal agenesis (Q60.0~Q60.2), renal hypoplasia (Q60.3~Q60.5), duplex kidneys (Q63.01) and congenital malformations of ureter (Q62.801). In analysis, we defined hydronephrosis,

renal agenesis, renal hypoplasia, duplex kidneys, hydronephrosis and congenital malformations of ureter as CAKUT. STATISTICAL ANALYSIS Participants were divided into three pairs of

comparison groups based on FA supplementation: FA/MMFA users or nonusers, FA users or MMFA users, and high-frequency users or low-frequency users. We described their basic characteristics.

Maternal demographics were categorical variables. _χ_2 tests were used to compare differences in general characteristics between the comparison groups. The prevalence of genitourinary system

birth defects, CAKUT, hypospadias was calculated as the number of cases per 10,000 births. Hypospadias was studied only in boys. We used the multiple imputation method to impute maternal

parity and education. The specific process is described in Supplementary Materials. Because there were only 23 cases of genitourinary system birth defects in the non-exposure group, and

three variables needed to be adjusted, we used propensity score inverse probability weighting to balance confounding variables in baseline characteristics between the exposure and

non-exposure groups. This technique combines multiple variables into one variable, which reduces dimensionality and simplifies the analysis. Weights were defined as the inverse of the

predicted probability of receiving the exposure factor. Specifically, propensity scores were calculated with logistic regression models that included maternal age, education level, and

parity to estimate the probability of a pregnant woman being exposed. The estimated propensity scores were used to allocate weights by inverse probability weighting. Weights were applied to

all study subjects to create a pseudo-population of exposure and non-exposure groups that were balanced on the confounding variables. Then standardized mean differences were used to evaluate

the balancing effect of the confounding variables. A standardized mean difference <10% indicated that the distribution of the variable was balanced between the comparison groups after

adjusting. Standardized mean differences before and after propensity score adjustment are shown in Supplementary Table 1. Poisson regression models were used to calculate crude and adjusted

risk ratios (RRs) and 95% confidence intervals (95% CIs) for the association between FA/MMFA supplementation in pregnancy and genitourinary system birth defects. We used clustered regression

analyses using mothers’ identification numbers as the cluster variable in all analyses. The weighted results were used in the Poisson regression models. All analyses were performed in R

version 4.0.5 (R Foundation for Statistical Computing, Vienna, Austria). SENSITIVITY ANALYSIS To test the robustness of our results after multiple imputations for covariates, we conducted a

sensitivity analysis for complete cases. Moreover, we trimmed the non-overlapping regions of propensity scores between the exposure group and the non-exposure group and retained the overlap

in the scores for analysis. RESULTS Of the 65,160 children, 58,564 (89.5%) had a mother who took FA or MMFA supplements during the periconceptional period, and 6595 (10.5%) did not. Of the

mothers who took supplements, 27,083 (46.6%) took FA and 31,048 (53.4%) took MMFA. The general characteristics of the mothers and children are shown in Table 1. A total of 51.8% of fetuses

were male. More than 86.5% of mothers were older than 25 years, and 35.4% of mothers had at least a college degree. Finally, 59.3% of mothers were primiparous. Birth year, maternal age,

maternal education, and maternal parity differed between the three pairs of comparison groups. Compared to nonusers, FA/MMFA users were older (>25 years: 79.2% vs. 81.3%) and more

educated (more than upper secondary: 88.5% vs. 71.5%) and were more likely to be primiparous (60.8% vs. 44.6%). In addition, compared to FA users, MMFA users tended to be older (>35

years: 11.9% vs. 10.2%) and more educated (more than upper secondary: 90.3% vs. 86.5%), and were more likely to be multiparous (41.3% vs. 36.4%). Finally, compared to women who supplemented

less frequently, those who did so highly were younger (>35 years: 11.9% vs. 10.2%) and more educated (more than upper secondary: 90.3% vs. 87.0%) and were more likely to be primiparous

(68.3% vs. 53.8%). Table 2 shows the diagnoses of genitourinary system birth defects among the 194 cases. Among these 194 fetuses, 134 cases of CAKUT (69.1%), 60 cases of hypospadias

(30.9%). According to the detailed diagnostic information, the number of urinary system malformations combined with congenital heart disease was 9. In addition, urinary birth defects are

combined with other defects, such as limb defects (_n_ = 5) and anorectal atresia/stenosis (_n_ = 4). The association between FA or MMFA supplementation and congenital genitourinary birth

defects is shown in Table 3. Maternal periconceptional supplementation with FA or MMFA decreased the risk for total genitourinary system birth defects (aRR 0.81, 95% CI 0.67, 0.98). This

trend was also found for hypospadias (aRR 0.55, 95% CI 0.40, 0.76), but not in CAKUT (aRR 0.90, 95% CI 0.71, 1.14). MMFA supplementation did not show a better preventive effect than FA

supplementation for genitourinary system birth defects, CAKUT, or hypospadias: aRRs were 0.90 (95% CI 0.73, 1.11), 1.00 (0.74, 1.35), and 0.90 (0.73, 1.11), respectively (Table 4). Compared

to low-frequency FA/MMFA use (<8 out of 10 days), high-frequency use (≥8 out of 10 days) did not show a better preventive effect on total genitourinary system birth defects (aRR 1.07, 95%

CI 0.87, 1.33), CAKUT (aRR 0.98, 95% CI 0.70, 1.25), or hypospadias (aRR 1.25, 95% CI 0.85, 1.84; Table 4). In sensitivity analysis, the association between taking supplements and

genitourinary system birth defects was similar to the overall results (Supplementary Table 2) based on a complete case analysis. In addition, we trimmed the non-overlapping region for

propensity score, and the preventive effect on hypospadias still existed (Supplementary Table 3). DISCUSSION We investigated the association between maternal supplementation with FA or MMFA

and the risk for genitourinary system birth defects in a retrospective cohort study. The results showed that supplementation with FA or MMFA may decrease the risk for genitourinary system

birth defects, but the association was not found for CAKUT. An association between FA/MMFA supplementation and genitourinary system birth defects has also been reported in other studies. A

case–control study showed that women exposed to FA antagonists, such as dihydrofolate reductase inhibitor and antiepileptic drugs, in early pregnancy have an increased risk for urinary

system birth defects in offspring.33 The use of a multivitamin with FA may reduce the risk conferred by exposure to dihydrofolate reductase inhibitor. In subgroup analyses, the use of FA and

MMFA showed the same protective effect. A large cohort study in Norway showed a protective effect of FA on the genital system but not the urinary system.6 In that study, the main urinary

system birth defect was hydronephrosis (64.8%), whereas the main genital system birth defect was hypospadias (87.5%). In subgroup analyses in our study, FA/MMFA supplementation had a

protective effect on hypospadias (a genital system birth defect) but not on CAKUT (urinary system birth defects). This is consistent with the results of Norwegian study. However, a

population-based survey found that FA supplementation may not reduce the risk for genitourinary system birth defects.34 The number of cases (_n_ = 46) may have been too small to detect the

association. Our findings also showed that supplementing with FA/MMFA may have a preventive effect on hypospadias. This corresponds well with the literature.18,35 A case–control study showed

that FA supplementation was associated with a reduced risk for isolated hypospadias in a dose-dependent manner.36 Moreover, the use of the folate antagonist valproic acid may increase the

risk for hypospadias.37,38 Another study suggested that supplementing with FA is not associated with hypospadias.39 However, in that study, the phenotypes of hypospadias were moderate and

severe. Our hypospadias cases also included mild phenotypes (34/62), and this may be one of the reasons for the difference in results. This inconsistency suggests that the relationship

between FA/MMFA supplementation and specific subtypes of hypospadias needs to be explored with larger sample sizes. The development of the urinary system begins at 3rd week gestation and

ends at 36th–38th weeks of gestation.40 The development of the fetal genitals begins in the 4th week of pregnancy and completes at the 29th week of gestation.41 Hypospadias occurs during the

embryologic period of 7–14 weeks gestation when the urethral folds fail to fuse completely, resulting in abnormal urethra development and opening.42 Exposure to endocrine disruptors,

abnormal gene expression, and signal pathway regulation may increase the risks for hypospadias.43 Richard et al. used genome-wide DNA methylation profiling to compare the methylation level

for hypospadias and controls.44 Their findings suggested that the hypomethylation of VAV3, TASPAN9-PRMT8-EFCAB4B, and WWC2-CLDN22-CDKN2AIP-ING2 may be involved in the etiology of

hypospadias. FA metabolism is important for DNA synthesis and repair, and converse homocysteine to methionione by the action of 5-methyltetrahydrofolate (5-methyl-THF).45 Folate deficiency

can cause a decrease in 5-MTHF levels, which in turn leads to a decrease in S-adenosylmethionine, resulting in the inhibition of DNA methyltransferase activity and DNA hypomethylation.46

This may be one of the reasons why FA deficiency leads to hypospadias. In addition, hypospadias occurs when the urethral opening is incorrectly positioned at the tip of the penis because of

the developmental arrest of urethral fusion. Therefore, the pathogenesis of hypospadias is similar to that of deformities associated with a failure to close, such as neural tube defects and

cleft lip and palate.36 FA supplementation may reduce the risk for these birth defects.27 So, the preventive effect of FA on hypospadias may have a similar mechanism to the prevention of

NTDs. In our study, there was no association between FA/MMFA supplementation and CAKUT. A case–control study in The Netherlands showed that FA use may increase the risk for CAKUT, especially

a duplex collecting system or vesicoureteral reflux, and supplementation with MMFA could decrease the risk for CAKUT.26 However, when the researchers explored the relationship between

dosage and risk for CAKUT, they only adjusted for one confounding factor (birth year) in the model. Overall, the inconsistent results suggest that the relationships between FA/MMFA

supplementation and various subtypes of CAKUT should be explored further. STRENGTH AND LIMITATIONS We identify three main strengths of our study. First, it is a cohort study. We recorded FA

supplementation at the time it occurred so there was less recall bias and information on exposure was collected before the outcome. Second, quality control in the BDSS is strict, and the

data were reliably collected. Third, the BDSS covers all children diagnosed with birth defects in the second or third trimesters (over 13 gestational weeks) and early neonatal period. Yet

our research also has the following limitations. First, we did not measure maternal blood folate. Second, mothers’ unhealthy lifestyle choices, such as smoking, may also cause malformations

of the fetal genitourinary system, but we did not collect information on these variables.47 We only adjusted for three covariates in the analysis, which is insufficient for genitourinary

system birth defects with complex etiology. Third, not all newborns have an ultrasound after delivery. Thus, some diagnoses of CAKUT may have been missed. CONCLUSIONS FA or MMFA

supplementation during the maternal periconceptional period could reduce the risk of genitourinary system birth defects in offspring, especially for hypospadias. Further research with large

sample sizes and randomized controlled studies is needed to evaluate the preventive effects. DATA AVAILABILITY The datasets generated during and analyzed during the current study are

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FUNDING This study was supported by the National Key R&D Program of China, 2021YFC2701000 and 2021YFC2701001. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Epidemiology and

Biostatistics, School of Public Health, Peking University, Beijing, 100191, China Zixi Pang, Jie Zhang, Di Wang & Lei Jin * Institute of Reproductive and Child Health, National Health

Commission Key Laboratory of Reproductive Health, Peking University, Beijing, 100191, China Zixi Pang, Jie Zhang, Di Wang & Lei Jin * Tongzhou Maternal and Child Health Hospital of

Beijing, Beijing, 101100, China Lei Jin & Wenying Meng Authors * Zixi Pang View author publications You can also search for this author inPubMed Google Scholar * Lei Jin View author

publications You can also search for this author inPubMed Google Scholar * Jie Zhang View author publications You can also search for this author inPubMed Google Scholar * Wenying Meng View

author publications You can also search for this author inPubMed Google Scholar * Di Wang View author publications You can also search for this author inPubMed Google Scholar * Lei Jin View

author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS L.J. and Z.P. conceptualized and designed the study, drafted the manuscript, performed the

analysis and revised the manuscript critically for important intellectual content. L.J., J.Z. and W.M. contributed to the acquisition and interpretation of data and revised it critically for

important intellectual content. All authors approved the final manuscript as submitted and agreed to be accountable for all aspects of the work. CORRESPONDING AUTHOR Correspondence to Lei

Jin. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ETHICS APPROVAL AND CONSENT TO PARTICIPATE This study was approved by the biomedical ethics committee

of Peking University (no. IRB00001052-18010). Informed consent of all participants is exempted. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to

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version of this article is solely governed by the terms of such publishing agreement and applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Pang, Z., Jin, L.,

Zhang, J. _et al._ Maternal periconceptional folic acid supplementation and risk for fetal congenital genitourinary system defects. _Pediatr Res_ 95, 1132–1138 (2024).

https://doi.org/10.1038/s41390-023-02808-7 Download citation * Received: 29 March 2023 * Revised: 06 June 2023 * Accepted: 14 June 2023 * Published: 15 September 2023 * Issue Date: March

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