Risk factor analysis for congenital heart defects in children

Congenital heart defects (CHDs) are the most common malformations, occurring in almost 1.0 in 100 births. We investigated an association between risk factors and CHDs, because epidemiological studies have reported conflicting results regarding risk factors and CHDs recently. The study of CHD frequency was conducted in Chernivtsi region (Northern Bukovina) on the basis of the medical genetic center. A retrospective method of research by studying registration genetic maps was used to analyze risk factors. 91 cards of infants suffering from CHD (47 boys and 44 girls) aged 0–1 living in the territory of Northern Bukovina were selected. In order to identify risk factors, 133 cards of healthy infants (77 boys and 56 girls) were used. The analysis of risk factors revealed that the female gender of a child is a risk factor for CHD development. The analysis of the ordinal number of pregnancy revealed that the second and the third pregnancies are probable risk factors for the development of this pathology. It was found in our study that folic acid intake during the first trimester prevented CHD development (OR 2.33). The study revealed that among stressful risk factors are: unplanned pregnancy (OR 3.13); out-of-wedlock pregnancy and stress during pregnancy. Maternal CHD increased the CHD development in offspring approximately by two times. Some factors, such as a woman doing hard physical work during pregnancy, having sedentary work during pregnancy, the mother being a housewife or having an incomplete secondary education (OR 3.61), the mother’s secondary education, the father’s incomplete secondary education (OR 18.62), the father serving in the army (OR 2.15) or being a student at the time of woman’s pregnancy (OR 2.97) were significant for CHD development in the fetal stage. A young age of the father (up to 43 years) was also considered as one of the risk factors. This article is expected to provide timely information on risk factors for CHD development to a wide range of medical staff, including pediatric and adult cardiologists, pediatricians, thoracic surgeons, obstetricians, gynecologists, medical geneticists, genetic counselors and other relevant clinicians.


Introduction
Congenital heart defects, also known as congenital heart disease (CHD) are among the most common serious congenital anomalies, accounting for about a third of all defects in development and are a common cause of miscarriage, stillbirth, neonatal and infant mortality, and a major cause of many medical abortions (Chen et al., 2014;Tanner et al., 2015;Kim et al., 2017). CHDs are among the most frequent malformations among all congenital anomalies, representing one of the most widespread global healthcare problems. Heart defects compose nearly thirty percent of all major congenital anomalies (Dolk et al., 2011). The global prevalence of CHD is between eight and ten cases per 1,000 live births (Saxena, 2005), but it varies considerably between countries. The prevalence of CHD in India is reported to be between 2.5-5.0 cases per 1,000 live births, but recent studies by Bhat et al. (2013) and Smitha et al. (2006) showed a prevalence of 8.5-13.6 cases.
The frequency of CHD at birth (sometimes called prevalence) depends on how the population is studied. Prior to the introduction of echocardiography the incidence rates ranged 5.0-8.0 per 1,000 live births, but a more accurate diagnosis revealed many more CHD cases, so current rates range 8.0-12.0 per 1,000 live births. Much depends on how early the diagnosis is made (Wren et al., 2012).
A heart ultrasound in all newborns shows that about 5% have minor defects in the muscular part of the interventricular septum, most of which close spontaneously within a year. Minor defects of the atrial septum, which also close spontaneously, make up an even more significant percentage. In some newborns, the arterial duct closes late. Therefore, it is better to consider only those heart defects that require treatment in childhood or are present in one-year-old infants. A more accurate indicator of CHD prevalence in newborns is about 10-12 per 1,000 live births. This number includes children with minor CHD, which close on their own within a year and patients with non-stenotic bicuspid aortic valves. They rarely cause problems in childhood, but in adulthood lead to the development of late arterial stenosis or regurgitation (Julien-Hoffman, 2013).
Approximately 30% of infants who die at birth show signs of CHD (Rosamond et al., 2007), and in some studies they are present in approximately 1.0% of live births (Bernier et al., 2010). A study in the UK found that of 700,000 live births, 6.4% had CHD, 15% of which were lifethreatening; only 70% of them were diagnosed before birth and after birth before discharge from the hospital, and 30% were found later with the onset of symptoms or after the death of the child (Wren et al., 2008).
While about 30% of all CHDs may be attributed to chromosomal or monogenic disorders or be influenced by other known factors, the cause of the remaining 70% remains unknown (Jenkins et al., 2007).
Therefore, it is important to study possible etiological factors, especially where they can be changed; this can be a key step in implementing primary prevention. Over the last decade, a large number of epidemiological studies has shown that many environmental risk factors have significantly contributed to CHD development. For example, maternal alcohol consumption, smoking, medication intake and exposure to certain physical factors during pregnancy have been associated with an increased risk of developing CHD (Malik et al., 2008;Ou et al., 2016). On the other hand, taking folic acid can reduce the risk of developing these defects (Donofrio et al., 2014).
Over the last decade, there has been a deeper understanding of the hereditary causes of congenital heart defects, including detection of specific genetic inadequacies for certain types of defects (Jenkins et al., 2007). In addition, an analysis of literary sources has shown that a large number of non-hereditary risk factors were associated with a high risk of CHD development, including acute and chronic maternal diseases, influence of medications, environment and parental factors. Some known risk factors included the mother suffering from rubella; phenylketonuria; gestational diabetes; taking thalidomide, large doses of vitamin A or retinoids (Donofrio et al., 2014).
Numerous studies have examined the relationship between various maternal diseases, such as hypertension, diabetes, thyroid disease, and chronic upper respiratory tract infections during pregnancy with CHD ( Van der Lugt et al., 2012;Liu et al., 2013). There was evidence that maternal infection during pregnancy might be associated with miscarriage, stillbirth and birth defects in future generations (Kourtis et al., 2014;Wheeler et al., 2015).
As well as the most common diseases during pregnancy, the impact of colds and the mental state of pregnant women also deserve attention. The prevalence of antenatal depression or anxiety during pregnancy was noted to range 7.0-11.0 per 100 pregnant women in high-income countries (Gaynes et al., 2005;Steel et al., 2014) and 11.0-27.0 per 100 pregnant women in China (Liang et al., 2009). Psychosocial stress during pregnancy was considered a possible risk factor for chronic diseases in children (Tegethoff et al., 2008;Cookson et al., 2009).
However, most studies reported only acute infectious diseases and fever, not a specific disease, when analyzing the relationship between common diseases during pregnancy and CHD in children. It was difficult to differentiate independent and joint effects of the colds or fevers that a pregnant woman had (Ou et al., 2016). Regarding psychological factors, most studies have focused on psychological stress and anxiety (Janssen et al., 2016;Carroll et al., 2017). CHD is the leading cause of infant mortality due to congenital malformations. Several studies have shown a sharp decline in mortality and morbidity over the last century due to improvements in cardiac surgery, anesthesia and pediatric services (Moons et al., 2010;Erikssen et al., 2015). Mortality from CHD was found to have decreased significantly in the whole world between 1990 and 2017, regardless of gender, age and region of residence. However, the mortality level has varied substantially around the world, primarily due to economic development and health care (Wu et al., 2020).
The highest mortality rates from CHD were in low-income countries in Africa and Asia. Moreover, the tendency to increased CHD mortality occurred mainly in countries with underdeveloped economies, such as North Korea and Pakistan. On the contrary, CHD mortality in developed countries decreased significantly over the same period as reported in previous studies (Jortveit et al., 2016;Mandalenakis et al., 2016). This inequality indicates that in low-income countries, more attention should be paid to primary prevention and timely diagnosis of CHD to alleviate the burden of these defects. In addition, more than 80% of CHD deaths occurred in children under the age of 5, underscoring the importance of CHD prevention and early CHD screening.
Although, in recent decades advances in cardiovascular medicine and surgery have dramatically reduced mortality and allowed most patients to reach adulthood, CHD remains the leading cause of death from congenital defects and is a heavy burden worldwide (Bouma & Mulder, 2017;Liu et al., 2019). Thus, our objective was to study the risk factors for CHD development in children of Northern Bukovina.

Materials and methods
The study of CHD frequency was conducted in Chernivtsi region (Northern Bukovina) on the basis of the medical genetic center. Reports of the Chernivtsi Regional Diagnostic Center of the Ministry of Health of Ukraine were used, specifically form No. 49-health. "Report on the provision of medical and genetic care", approved by order No. 141 of the Ministry of Health of Ukraine dated 16.06.1993 (2007-2016). Also data contained in the statistical yearbooks of Chernivtsi region (2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014)(2015)(2016)(2017)(2018)(2019) concerning the number and morbidity of children in the region were analyzed. To analyze risk factors for CHD development we used a retrospec-tive method of research by studying registration genetic maps between 2000 and 2019, which were approved by order of the Ministry of Health of Ukraine dated 13.12.1999. 91 cards of children suffering from CHD (47 boys and 44 girls) aged 0-1 living in the territory of Northern Bukovina were selected. In order to identify risk factors, 133 cards of healthy infants (77 boys and 56 girls) were used. The control group was formed on a population basis, as only those children whose parents lived permanently in Northern Bukovina were subject to registration.
The following data were collected from the parents: age (number of years) at the time of pregnancy, social status, the parents' bad habits, level of education of both mother and father, place of residence and place of work; somatic morbidity, ordinal number of pregnancy, placental insufficiency, threats of miscarriage, polyhydramnios or oligohydramnios, nuchal cord occurrence; a woman's gynecological health, history of abortion and miscarriages in the anamnesis; planned or unplanned pregnancies; folic acid intake in the first trimester of pregnancy, presence of stress; presence of TORCH infection during pregnancy; taking contraceptives and other medications.
The frequency of CHD cases was calculated as the ratio of CHD cases registered by the medical and genetic service during this period of time to the number of births and multiplied by 1000. The strength of association of the analyzed signs was determined using the value of the odds ratio (OR), which was carried out according to the standart formula.
A confidence interval (CI) was calculated for OR at 95% significance level. If the odds ratio was less than 1, then the risk decreased, if = 1, then there was no risk, if more than 1, then the risk was present (Fletcher et al., 1998). All data were analyzed by nonparametric methods of variational statistics using a computer program MedCalс (2006).

Results
The analysis was conducted concerning CHD prevalence in Northern Bukovina for two periods: between 2000 and 2019 ( Table 1). The frequency dynamics of CHD in newborns between 2000 and 2009 was viewed as a sinusoid with the highest rate in 2008 (7.72‰), which exceeded the average by two times. The lowest rate in the first observation period was in 2000, which may be due to improved prenatal diagnosis. In the next observation period between 2010 and 2019 the maximum number of CHDs occurred in 2019, which exceeded the average by 1.5 times, and the minimum in 2017. Risk factors in children with congenital heart defects were analyzed. The most important risk factors for CHD development are presented in Table 2. The analysis of risk factors revealed that female gender of the child is a risk factor for CHD development (OR 1.29). The analysis of the ordinal number of pregnancy revealed that the second (OR 1.59) and the third (OR 1.74) pregnancies are probable risk factors for the development of this pathology. Previous miscarriages, unplanned pregnancies in the anamnesis and out-of-wedlock pregnancies are also risk factors for CHD development.

Discussion
Our study found that in maternal age groups from 26 to 35 years and under 18 years, the risk of CHD development in their children was higher than in the group aged over 35 years. In the maternal age group from 26 to 35 years the probability of CHD presence was 1.44 (95% CI 0.59-3.53), 525 while in the group under 18 years the probability of CHD presence was 1.46 (95% CI 0.09-23.57). These data are slightly different from the general population. Thus, a study conducted by Miller et al. showed that mothers aged over 35 years were associated with an increased risk of CHD (2011). Cancer in the fourth generation on the mother's side 1.16 0.55-2.42 -Diabetes in the fourth generation on the mother's side 10.19 4.03-25.75 <0.001 Diseases of the cardiovascular system in the third generation on the mother's side 2.93 1.28-6.67 <0.05 Cancer in the third generation on the mother's side 5.11 1.91-13.66 <0.001 Diabetes in the third generation on the mother's side 0.79 0.22-2.82 -CHD in the third generation on the mother's side 12.53 0.58-267.1 -Thyroid diseases in the third generation on the mother's side A meta-analysis by Hackshaw et al. (2011) assessed the effect of the mother's smoking on the spectrum of congenital defects, including heart defects (OR 1.09; 95% CI 1.02-1.17). A meta-analysis of studies published between 1971 and 2011 (23 case-control studies, 5 cohort studies, and 5 cross-sectional studies) found a positive correlation for all combined CHDs (OR 1.11; 95% CI 1.02 -1.21). Our study found an effect of maternal smoking before pregnancy (OR 1.42; 95% CI 0.63 -3.38) (Lee & Lupo, 2013).
The Baltimore-Washington Infant Study concluded that the mother smoking more than one pack of cigarettes a day led to certain CHDs: ventricular septal defect and pulmonary artery stenosis (women over 34 years) (Correa-Villaseñor et al., 1993). The study by Polifka & Fried-526 man (2002) reported a link between maternal cigarette smoking and specific CHD (ventricular septal defect, tetralogy of Fallot).
The Nicoll study as well as later studies that showed a link between CHD and maternal smoking did not show a high OR for all CHDs; in fact, higher OR rates were found for parental or passive smoking (2018). A study conducted in the United States showed that the effects on the mother caused by secondhand smoking at home and at work led to the development of atrial septal defects with OR 1.37 (95% CI 1.09 -1.72) (Hoyt et al., 2016), while maternal and paternal smoking resulted in an additive effect with OR 4.5 (95% CI 2.5-8.3) (Cresci et al., 2011). Parental smoking of more than 14 cigarettes a day more than doubled the risk of CHD (OR 2.1 95% CI 1.3-3.5) (Baardman et al., 2012). This and other studies cannot explain why the risk of CHD increases with secondhand or parental smoking. These data may indicate that mothers often conceal the fact of smoking. A hospital case-control study of nicotine levels in the hair revealed a dose-response effect with OR rising for all CHDs due to increase of nicotine levels in the hair .
The study revealed that the intake of folic acid during the first trimester prevented CHD development (OR 2.33; 95% CI 1.31-4.13, P < 0.05). A Hungarian randomized trial (Botto et al., 2000) and the Atlanta casecontrol study showed a similar correlation in which CHD risk in offspring was reduced by 60% and 25% for mothers who took folic acid in the first trimester of pregnancy (Scanlon et al., 1998). The analysis of pregnant women who had SARS during the first trimester found that this indicator was not a risk factor for CHD development (OR 0.72, 95% CI 3.13-24.31, P < 0.001). However, the study of Shi et al. (2014) showed that mothers who had a history of SARS in the first trimester had an increased chance of CHD developing in their children (OR 3.717; 95% CI 1.63-8.50) (Abquari et al., 2016). Our study demonstrated that CHD probability in children was increased with mothers who had a history of previous miscarriages (OR 1.53, 95% CI 0.65-3.67), gestosis in the second half of pregnancy (OR 2.17, 95% CI 1.10-4.29, P < 0.05), chronic fetoplacental insufficiency (OR 1.13, 95% CI 0.41-3.15), polyhydramnios (OR 1.16, 95% CI 0.45-2.99) and dehydration (OR 3.38, 95% CI 1.09-10.5, P < 0.05). Thus, regular fetal screening is strongly recommended, especially in high-risk pregnancies. However, some researchers Hasan et al. (1997) did not find such a correlation.
In a study by Chou et al. (2016) maternal congenital heart defects increased risk of CHD development in offspring by about three times, which is consistent with other studies  as well as with our data (OR 2.25, 95% CI 0.20-25.21). Apparently genetic predisposition plays an important role.
Taking medication during pregnancy, especially in the first trimester increased the risk of CHD in offspring by 1.5 times (OR 1.54, 95% CI 0.68-3.49). A study by Li et al. (2018) found that women of childbearing age who had taken cold medications, antibiotics, drugs containing salicylic acid, antifungals, and other drugs during early stages of pregnancy increased the risk of their children developing CHD.
A study Rappazzo et al. (2016) examined the effects of particular chemicals on pregnant women and found an increased risk of developing certain CHDs, the same data were found in a study by Carmichael (2014). Our study showed that the influence of chemical factors during pregnancy on CHD development was significant (OR 1.92, 95% CI 0.11-3.33, P < 0.05).
There is a high risk of CHD associated with a low level of maternal education, which persisted after adjusting for other risk factors, suggesting that certain environmental factors associated with maternal education (possibly due to individual socioeconomic status) are relevant and require further research. Studies in the United Kingdom (Vrijheid et al., 2000) and Canada (Agha et al., 2011) found a higher risk of ATC associated with parents' low socioeconomic levels.
The study by Su et al. (2015) did not find a general connection between the father's age and the prevalence of combined CHD in offspring. The young age of the father (under 43 years) was a risk factor in our study (OR 3.28,.06, P < 0.05). A real explanation for the effect of young age is that unhealthy lifestyles and the presence of bad habits such as smoking and alcohol consumption may predominate among young couples (Kazaura et al., 2004).
Mother's usage of alcohol during pregnancy is associated with the development of congenital defects in children (Polifka & Friedman, 2002;Brent, 2004). Adverse effects of alcohol on the fetus include a range of structural anomalies and behavioural disorders (Autti-Ramo et al., 2006) and lead to an increase in the number of newborns with fetal alcohol syndrome (Shillingford & Weiner, 2001). Shillingford & Weiner (2001) reported that atrial septal defects were the most common heart defects these newborns had.
The study by Martinez-Frias et al. (2004) conducted in Spain found that the highest risk of developing CHD was in the group with the highest prenatal alcohol exposure (absolute alcohol intake was over 92 g per day). Cigarette smoking is a well-known teratogenic risk factor for congenital defects and can affect the development of certain organ systems (Lee et al., 2013). Nicotine can strongly affect sperm activity and cause chromosomal aberration, which can affect fetal development and lead to heart defects (Correa et al., 2015). In addition, parental smoking can cause passive smoking by the mother, which can also increase the risk of CHD development (Liu et al., 2017). Our study showed the father's smoking as a risk factor for developing CHD (OR 1.58, CI 0.92-2.69).
Risk factors for CHD development from the father's side were: social status of the father as a worker (OR 2.21, CI 1.23-3.99, P < 0.05); hard physical work of the father before conception (OR 2,27, CI 1.31-3.96, P < 0,05); influence of chemical factors on the father before conception (OR 1.41,. Studies that evaluated the effects of chemical agents or drugs on the father of a child with CHD were analyzed. The effect of chemicals or drugs on parents was found to be closely associated with an increased risk of CHD (OR 2.15, CI 1.53-3.02) (Fleming et al., 2014). Certain occupations of fathers, such as factory workers, janitors, painters and plywood factory workers may increase the risk of development of CHD (Fleming et al., 2014;Rufer et al., 2010). However, conflicting results have been shown in studies on the connection between the father being a firefighter and CHD risk in the offspring (Selevan et al., 2000).
In addition to the abovementioned risk factors, there are also several studies on other factors from the father's side, such as chronic diseases, viral infections etc., which revealed that the father having a viral infection (OR 2.46, or taking antibiotics (OR 10.04, 95% CI 1.28-78.45) might also increase CHD risk (Nie et al., 2013;Li et al., 2017). The presence of chronic diseases in the father (OR 2.40, 95% CI 1.22-4.72), especially chronic diseases of the cardiovascular system (OR 30.13, 95% CI 4.06-321.10) is shown to be among risk factors for CHD development. The following factors in parents are largely associated with an increased risk of CHD: family history of CHD (OR 5.23), the mother having chronic diseases (OR 4.19), environmental pollution at the mother's place of residence (OR = 3.63), chronic disease of the father (OR 4.87) (Jiayu Peng et al., 2019). Our study also identified certain risk factors that provoked CHD development, i.e. the mother suffering from other 527 chronic diseases (OR 2.67, 95% CI 1.07-6.67, P < 0.05). The pedigree analysis identified CHD risk factors associated with previous generations having the following diseases: diabetes in the fourth generation on the mother's side (OR 10.19, 95% CI 4.03-25.75, P < 0.001), diseases of the cardiovascular system in the third (OR 2.93, 95% CI 1.28-6.67, P < 0.05) and the fourth generations (OR 1.96, 95% CI 0.86-4.46) on the mother's side, cancer in the third generation on the mother's side (OR 5.11, 95% CI 1.91-13.66), diseases of the cardiovascular system in the third generation on the father's side (OR 1.22, 95% CI 0.48-3.09), congenital heart defects on the father's side (OR 4,47, 95% CI 0,21-96.41), congenital heart defects from previous pregnancies (OR 3.94, 95% CI 0.21-75.29), thyroid diseases on the mother's side (OR 2.27, 95% CI 0.85-6.07) and on the father's side (OR 13.58, 95% CI 3.91-47.8).
The mother's having pregestational diabetes increases the risk of CHD. Maternal diabetes is usually associated with a wide range of CHDs: transposition of the great vessels, non-chromosomal atrioventricular septal defects, double outlet right ventricle, tetralogy of Fallot, hypoplastic left heart syndrome and cardiomyopathy. Recent studies have shown that long-term maternal diabetes increased the risk of congenital cardiovascular disorders (Nielsen et al., 2005).
The teratogenic mechanism of maternal diabetes has not been fully studied and is probably multifactorial (Hornberger, 2006;Nold & Georgieff, 2004). It is possible that presence of increased osmolarity and abnormal levels of ketones, imbalance of amino acids and fatty acids may contribute to CHD development. High glucose levels in the blood can cause congenital malformations, inhibiting glycolysis, i.e. the main process of energy production during embryogenesis (Nold & Georgieff, 2004). Hyperglycemia has a direct effect on the proliferation and migration of neural crest cells, which are important for heart development. A recent study by Roest et al. (2007) showed a high incidence of cardiovascular malformations in embryos whose cardiac neural crest cells experienced elevated glucose levels. Another study reported that abnormal glucose levels in embryos disrupted the expression of Pax-3, a developmental control gene that is an important factor in the transcription of cardiac neural crest cells (Epstein et al., 2000).
The effects of preconception diabetes begin during embryonic development in the first trimester; and continues to affect the fetus in the second and third trimesters and the newborn in the perinatal and neonatal periods (Hornberger, 2006).
The risk of developing CHD can be significantly reduced due to a good control of blood glucose levels. On the contrary, the mother having a long-term type 1 diabetes as well as poor glycemic control before and during pregnancy increase the risk of congenital malformations (Kalaidzhieva et al., 2003).

Conclusions
The risk factors for CHD development in offspring may vary, namely the mother having chronic diseases, CHDs, chronic stress, the mother's age (under 18 years and between 26 and 35 years), social status (incomplete secondary and secondary education), exposure to chemical factors, medication intake during pregnancy, smoking before and during the pregnancy, complicated pregnancy (dehydration, previous miscarriages, gestosis during the second half of pregnancy) or the family living near highways.
The analysis of non-genetic risk factors for CHD development on the father's side revealed the high impact of incomplete secondary education, place of work before pregnancy (worker, serving in the military, the father being a student), the habit of smoking, hard physical work, influence of chemical factors before conception, the father suffering from chronic diseases, especially of the cardiovascular system. Also, such risk factors as pedigree analysis showing presence of diseases of the cardiovascular system, thyroid diseases both on the mother's and father's sides, cancer on the mother's side and congenital heart defects from previous pregnancies should be taken into account.
The results of this study can be used in family counseling before conception and in identifying high-risk pregnant women. Women with relevant risk factors should eliminate them as much as possible before conception to minimize the risk of congenital heart defects in their children.
It is important to encourage fertility at a certain age, to form healthy lifestyle habits, starting with quitting smoking and drinking and trying to avoid exposure to chemical and environmental factors.
Prenatal screening is recommended for high-risk pregnant women, if necessary, using invasive methods. Early detection of congenital heart defects also allows medical staff to optimally prepare and provide care during pregnancy, childbirth and puerperium.