EnvironmEntal risk factors associatEd with biliary atrEsia in rio GrandE do sul , brazil

Biliary atresia (BA) seems to be a multifactorial disorder in which environmental factors interact with the patient’s genetic constitution. This study aimed to analyze information concerning environmental risk factors associated with BA in southern Brazil. A case-control study with mothers of patients with BA and mothers of patients with cystic fibrosis (CF) was conducted. Inquiry included questions related to exposition to environmental risk factors during the periconceptional and gestational (second and third trimesters) periods. Mothers of BA patients had smoked during pregnancy more frequently in comparison with the mothers of CF patients, but no significant difference was found in a multivariate analysis. There was no between group difference in terms of seasonality, but the multivariate analysis showed a significant difference within the BA group between date of conception in winter compared to other seasons. In conclusion, smoking during pregnancy seemed to increase the risk of BA while date of conception in winter decreased it.

Biliary atresia (BA) is a disease that begins exclusively in infancy and consists in the complete obstruction of extrahepatic and intrahepatic bile ducts 1,2 .Regardless of a timely surgical treatment, BA induces cirrhosis and cholangiopathy and constitutes the leading cause of pediatric liver transplantation 3,4 .The etiology of biliary obstruction and chronic cholangiopathy is not fully understood, and their association with genetic/chromosomal alterations, immune and immunogenetic abnormalities, environmental factors and maternal diseases, such as gestational diabetes, has been proposed 1,[5][6][7] .An immune disorder induced by viral infection seems to be part of BA etiology 8 .Distinct variants of the disease, such as BA splenic malformation (BASM), isolated BA, cystic BA, and cytomegalovirus IgM-positive BA, suggest distinct patterns of acquisition 3,7 .BA seems to be a multifactorial disorder in which environmental factors interact with the patient's genetic constitution.Environmental risk factors associated with the development of BA have been seldom investigated 5,[9][10][11][12] .In the present study, we collected and analyzed information concerning family history and environmental risk factors in the gestational and periconceptional periods associated with the occurrence of BA in the state of Rio Grande do Sul, southern Brazil, aiming to elucidate the etiology of this disease.

METHODS
This was a case-control study in which the evaluated sample included mothers of patients with the isolated form of BA.

RESULTS
Each group (BA and CF) consisted of 27 mothers.Fifteen mother of BA patients (55.6%) were female, and at the time of inquiry, they had a median age of 5 (2-9) years.Among mothers of CF patients, 17 (63%) were female, and their median age was 4 (2-9) years.There were no significant differences in relation to sex (P =0.782) nor age at inquiry (P = 0.986).
Mothers of BA patients had been smokers during pregnancy more frequently than mothers of CF patients (P = 0.050, table 1), but the multivariate analysis (table 2) showed no significant difference (P = 0.063; odds ratio, OR = 8.48; 95% CI: 0.89-81.0).The following variables showed significant associations with BA: parents' age; pregnancy of non-smokers living with smokers or with a pregestational smoking habit; consumption of alcohol or illicit drugs before, during, or after pregnancy; consumption of erva-mate or teas; use of medications, skin creams, or condoms during an unsuspected pregnancy (table 1).
There was no between-group difference in relation to seasonality at conception and at birth (P = 0.086 and P = 0.591, respectively) (table 1).However, considering specifically the BA group, there was a significant difference (P = 0.030) in the multivariate analysis between date of conception in winter compared to other seasons (OR = 0.19, 95% CI: 0.04-0.85)(table 2), demonstrating a lower risk of developing BA when conception occurs in winter.There was no significant association between the presence of BA and infections during the time periods of interest.Moreover, mothers of BA and CF patients did not report any intestinal infection during the second and third trimesters of gestation.

DISCUSSION
In terms of etiology, biliary atresia seems to be a heterogeneous condition in which environmental factors can act both in the early intrauterine period (embryonic form) and in the perinatal period (isolated form) [13][14][15][16] .
This study showed that the conception of a patient with BA was 84% less likely if this condition was diagnosed in winter, suggesting a seasonal influence on BA etiology, although season at birth was not associated with the occurrence of BA.Seasonality suggests that environmental factors can trigger BA, but conflicting results have been described on this topic [9][10][11][17][18][19] . In Texs, a higher number of BA patients were born during summer and spring in the countryside, suggesting that the noxious agent could be an agricultural toxin 17 .In New York, babies born in spring were at a higher risk of suffering from BA 18 .The NS et al. observed that conception during spring was more associated with the occurrence of BA than conception during winter 9 .Other groups from different countries have found no association of BA with seasonality 5,[10][11][12]19 .Different investigation methods, samples sizes, and environmental or weather conditions in different regions may explain these conflicting results.
In the present study, mothers of BA patients were smokers during pregnancy more frequently than controls, but there was only a tendency of difference in the multivariate analysis.Fischler et al. obtained similar results for pregnant women consuming more than 10 cigarettes/day 11 .Another study did not find association between active or passive smoking habit during pregnancy and BA 9 .The putative association of maternal smoking and BA is interesting, since the livers of BA patients have a molecular profile suggestive of hypoxia-ischemia possibly attributable to vascular disorders 7 .
The present study did not find any relation between the occurrence of BA and the other investigated variables.The absence of relation between BA and genitourinary or gastrointestinal infections during periconceptional and gestational periods observed in this study is consistent with the findings of The NS et al., but they observed an association between respiratory infections during the periconceptional period and the occurrence of BA 9 .This is the first report from Brazil of environmental risk factors for BA.The low incidence of this disease made our work challenging, but the high morbidity/ mortality rate associated with BA makes it crucial to further investigate infectious and non-infectious risk factors associated with its etiology.In conclusion, this study showed that date of conception during winter decreased the risk of BA, while smoking during pregnancy seemed to increase it.

Table 1 :
The investigation was conducted during the first weeks after birth or at different points during outpatient follow-up.Mothers of infants, children and adolescents (up to 18 years old) Mothers who agreed to participate had adequate memory skills and lived in Rio Grande do Sul during pregnancy and at birth.They were informed about objectives and methods of the study, signed an informed consent form, and then answered a standard questionnaire about environmental risk factors of BA during periconceptional and gestational (second and third trimesters) periods.Investigated risk factors comprised 45 items, including consumption of cigarettes, alcohol, illicit drugs, teas, and erva-mate (Ilex paraguaiensis St. Hill), and use of medications, condoms and skin creams.Additional topics were parents' age, pregnancy planning, season at conception and at birth, and diagnosis of infections.To control a possible bias related to mothers' long-term memory, children of both groups were matched by age at questionnaire application.The study was approved by the Research Ethics Committee of HCPA.Characterization of samples according to studied variables.