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Parental exposures and risk of nonsyndromic orofacial clefts in offspring: A case-control study in Greece
International Journal of Pediatric Otorhinolaryngology 75 (2011) 695–699
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International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Parental exposures and risk of nonsyndromic orofacial clefts in offspring: A case-control study in Greece§ P. Mirilas a,*, A. Mentessidou a, E. Kontis a, M. Asimakidou a, B.J. Moxham b, A.S. Petropoulos a, E.-N. Emmanouil-Nikolousi c a
2nd Department of Pediatric Surgery, Aristotle University of Thessaloniki Medical School, Papageorgiou General Hospital, Ring Road, Nea Efkarpia, 56429 Thessaloniki, Greece Cardiff School of Biosciences, Cardiff University, Wales, UK c Department of Histology and Embryology, School of Medicine, Aristotle University of Thessaloniki Medical School, Greece b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 December 2010 Received in revised form 15 February 2011 Accepted 16 February 2011 Available online 30 March 2011
Objective: To investigate the association between cleft lip and/or palate and nongenetic factors in Greece. Methods: We designed a case-control study including 35 patients with nonsyndromic cleft lip and/or palate, retrospectively selected, and 35 control patients matched for prefecture of residence, prospectively selected from pediatric population hospitalized for abdominal pain or injury. Parents were interviewed about drug uptake, diseases, habits, non-occupational exposure to pollutants, and occupation. Questions covered the period from one year before until three months after conception. Results: High-risk paternal occupations (mostly farmers) were significantly more frequent in cleft lip and/or palate than in controls (p = 0.039) and increased significantly the cleft lip and/or palate risk in offspring (OR: 3.00; 95% CI: 1.03–8.70). Maternal occupation did not correlate with cleft lip and/or palate. Parental disease, drugs uptake, hazardous habits, maternal folate supplementation and nonoccupational exposure to pollutants did not correlate with cleft lip and/or palate. There was a suggestion of increased risk with maternal passive exposure to tobacco (OR: 1.81; 95% CI: 0.69–4.74) and with residential proximity to industries (OR: 1.70; 95% CI: 0.61–4.74). Conclusions: Paternal high-risk occupations probably exert a teratogenic effect on spermatogenesis or result in maternal contamination, and deserve specific preventive policies. The relation of smoking and residential proximity to industries with occurrence of cleft lip and/or palate deserves further study. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cleft lip and/or palate Parental occupation Smoking Environmental pollutants Drugs Folic acid
1. Introduction Lip and/or palate clefts (CL/P) are congenital malformations with severe medical and psychosocial consequences [1,2]. They may present as cleft lip with or without cleft palate (CL P) or as cleft palate alone (CP). The pathogenesis of nonsyndromic CL/P is complex and both genetic and environmental factors are believed to be involved [3–6]. Identification of the environmental factors that are implicated in the pathogenesis of isolated CL/P is crucial in order to formulate prevention strategies. Many parental exposures have been associated with CL/P, including maternal use of anticonvulsants or retinoids [7,8], paternal and maternal hazardous habits such as smoking [5,7–11] and regular alcohol consumption [8], residential proximity to sites
§ Preliminary results were presented at the International Workshop on Risk Assessment and Human Exposure to Hazardous Materials, Aristotle University of Thessaloniki, 21–24 October 2009. * Corresponding author. Tel.: +30 6945 038083. E-mail address: [email protected] (P. Mirilas).
0165-5876/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijporl.2011.02.018
of increased chemical pollution [12] and first-trimester maternal fever-producing illnesses including influenza [6,13]. In addition, an association between certain occupations and the occurrence of CL/ P has been noted [14–16]. However, there are some discrepancies in findings derived from different countries. For instance, hairdressers and woodworkers were mainly associated with offspring with cleft lip and/or palate in forested areas of Norway [15]. In Italy – where the clothing and shoe industries are very well developed – women involved in leather and shoe manufacturing [16] were most at risk. Furthermore, in the agricultural metropolitan area of Atlanta, USA, farmers and firemen appeared to be at most increased risk of having offspring with CL/P [14]. Similarly, data about the relation between parental socioeconomic status and risk of CL/P are inconclusive [5,17]. Such disparities between countrybased studies determining the most common environmental risk factors for oral clefts may be explained by differences in socioeconomic status and economic policies, occupation frequencies and occupational settings, mentality, level of prevention and information campaigns, habits, hobbies, etc. If cultural, political and socioeconomic differences are the reason for data discrepancies, country-based studies are needed to define risk factors
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associated with CL/P in each country. Only on this basis can effective country-based prevention policies be achieved. In Greece, the prevalence at birth of orofacial clefts was reported as being 0.85 per 1000 newborns. This was determined specifically for the decade 1955–1965 [18]. More recent published reports concerning the prevalence of this anomaly are not available. Therefore, it is not surprising that an etiologic study has not yet been undertaken and consequently the role of hazardous environmental exposures of parents in the development of CL/P has never been investigated. Thus, relevant prevention policies are almost completely lacking in this country. Taking the above into consideration, we designed a case-control study in order to examine the association between both paternal and maternal exposure to a series of environmental factors (including drugs, diseases, chemical and environmental pollutants, smoking, alcohol and occupational hazardous factors) and the occurrence of nonsyndromic CL/P in Greece.
trimester is the critical period for the formation of CL P (5th–12th gestational week) and CP (7th–12th gestational week) [5,9]. Parents’ responses were recorded as dichotomous data (Yes or No). Interviews of both parents of either cases or controls were conducted by the same researchers (AM, MA) using the same questioning strategy. In case a definite ‘yes’ or ‘no’ response could not be elicited, based on the memory of the respondent, it was rather recorded as a missing answer in order to eliminate bias. Due to the wide variety of answers with respect to occupation, paternal and maternal occupations were classified as of high- or low-risk. Classification was based on whether occupational exposure to hazardous materials (including solvents and heavy metals) was likely or not, according to information available in the US National Library of Medicine Haz-Map [12,19]. Thus, for example, farmers/groundsmen who used pesticides were considered of high-risk, while unemployed women/homemakers were classified as of low-risk.
2. Methods
2.1. Statistics [20]
Our case-control study included 35 patients operated on for nonsyndromic CL/P and 35 residence-matched control patients. Medical records of all children treated for CL/P in our Pediatric Surgery Department between 2004 and 2009 were retrospectively reviewed. A total of 44 charts were analyzed. The exclusion criterion was the presence of an associated anomaly or syndrome, which would interfere with the evaluation of the disease. We did not manage to reach the families of 5 cases with CL/P. Four other cases were excluded from the study because of associated anomalies or syndromes (e.g., Pierre Robin syndrome). Thus, a total of 35 children with isolated CL/P were included in the study. Both parents of these children were contacted by phone and after informed consent was obtained, they undertook a specially designed structured interview. After the survey of patients with CL/P deformities was completed, we prospectively included during 2009 a control group of 35 children hospitalized for abdominal pain or injury in our department. Controls were excluded from the study if they had a history of known congenital anomalies or recognizable malformations. Inclusion criterion for this group was that controls should be matched with patients for prefecture of residence before and during gestation. Our department is one of four departments of Pediatric Surgery covering an area of 16 prefectures (urban and rural) with a total population of 2.4 millions. Considering the possible variation in the amount of exposure to environmental and/or chemical pollutants between different prefectures, matching of cases and controls for prefecture of residence was undertaken to avoid any bias in results. Initially, families of 52 children were asked to participate in the control group. Three children were excluded from the study because they had previously been operated upon for congenital disorders (e.g., cryptorchidism, hypospadias). Fourteen out of 52 children were not included because they failed to match with controls for prefecture of residence. Finally, 35 children free of congenital anomalies and area-matched with cases ended up participating as controls. Parents of controls were required to provide written informed consent and underwent the same structured interview as for the parents of CL/P cases. Study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. Interview formulation was based on the results of previous research on environmental factors associated with CL/P [3–17] and on specific regional and national characteristics, e.g., common hazardous habits. The interview questions were divided into five categories (Tables 1 and 2). The exposure period of interest during pregnancy was limited to the first three months because the first
Differences in variables (maternal, paternal and common parental variables) between cases and controls were tested by Chi-square tests. For estimation of risk of offspring with CL/P, odds ratios and 95% confidence intervals were used. When Chi-square tables contained cells with an expected count of less than five, the Monte–Carlo method was used for the calculation of p value, OR and 95% confidence intervals. When the observed count in one cell of the crosstabulation was zero, the relative risk was calculated for estimation of risk of CL/P in offsprings [21]. Statistical analysis was performed using SPSS 16.0 for Windows.
3. Results Review of the medical charts showed that 15 out of the 35 CL/P cases suffered from cleft lip without cleft palate (CL), 11 from cleft lip and palate (CLP) and 9 children suffered from cleft palate alone (CP). Estimated risks of paternal and maternal factors for offspring with CL/P are presented in Tables 1 and 2, respectively. As can be seen in Table 1, paternal profession of high risk had a significant correlation with occurrence of CL/P in offspring (Pearson chi square, p = 0.039), and a three times increased risk for the anomaly (odds ratio = 3). From the list of frequencies of high- and low-risk paternal occupations observed in this study (Table 3), it is evident that professions of high risk were more frequent in the CL/P group. In contrast, observed frequencies of low- and high-risk occupations were comparable between mothers of CL/P and mothers of controls (Table 4); thus, no association was detected between maternal occupation risk and occurrence of CL/P (Table 2). As can be seen in Tables 1 and 2, there is a tendency for residential proximity to industries to be associated with CL/P. Although this association is characterized by low level of statistical significance, we should note that the odds ratio value for residential proximity to industrial areas is greater than one by 0.7. These findings suggest that the risk of offspring with CL/P is increased by 70% when the family lives within 5 km of an industry. In addition, there was a suggestion that maternal passive exposure to tobacco before and during pregnancy was more frequent in the CL/P group than in the controls (Table 2) and tended to increase 1.58 and 1.81 times, respectively, the risk of nonexposed women for offspring with CL/P (Table 2). The association between second-hand smoke and CL/P occurrence, however, did not achieve statistical significance. Similarly, odds ratio and 95% confidence intervals suggested an association between folate
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Table 1 Frequency and risk (odds ratios, 95% confidence intervals) of paternal risk factors of offspring with orofacial clefts. Father Frequency (n) CL/P cases (n = 35) Factors Category 1. Diseases and drugs Preparations/face creams containing Vit A Antirheumatic drug therapy Corticosteroids uptake Antidepressants Medication for bone disease Thyroid disease/medication Other disease/drugs Category 2. Habits Smoking before and during pregnancy (>1 cigarette/day) Smoking before but not during pregnancy (>1 cigarette/day) Regular alcohol consumption (>2 glasses per week) Drugs abuse Category 3. Exposure to environmental pollutants Residence proximity to factories (5 km) Residence proximity to electrical power industry (5 km) Residence proximity to high voltage electric cables (5 km) Exposure to radiation (e.g., work in area where X-rays/radiopharmaceuticals are used, residence near cell phone antenna) Exposure to pesticides (e.g., usage of pesticides in own crops, work in or residence near farm where pesticides are used) Category 4. Exposure to chemical contaminants Lead (e.g., living in a house painted with lead-base paints) Cadmium (e.g., coming in contact with vehicle batteries) Vanadium (e.g., coming in contact with vehicle batteries) Category 5. Demographic data Occupation of high risk Parents were blood relatives
Risk for CL/P Controls (n = 35)
OR (95% CI)
1 0 0 0 0 1 0
0 0 0 1 0 0 0
1.02 (0.97–1.09)a
22 0 2 0
20 0 1 0
1.26 (0.48–3.30)
13 3 4 0
9 6 5 0
1.70 (0.61–4.74) 0.45 (0.10–1.97) 0.77 (0.19–3.16)
11
7
1.83 (0.61–5.47)
12 4 8
10 3 6
1.20 (0.42–3.37) 1.72 (0.35–8.43) 1.61 (0.49–5.28)
15 2
7 0
3.00 (1.03–8.70)* 1.06 (0.97–1.15)a
0.97 (0.91–1.02)a 1.02 (0.97–1.09)a
1.00 (0.18–5.33)
CL/P: cleft lip and/or palate. OR: odds ratio. CI: confidence intervals. a Relative risk was calculated as a good approximation to odds ratio. * Significant association (p = 0.03).
uptake and occurrence of orofacial clefts, although this association was statistically non significant (Table 2). Pre- and post-conceptional parental exposure to medication or diseases was not correlated with the occurrence of CL/P (Tables 1 and 2). Likewise, we did not detect any association between occurrence of CL/P and paternal or maternal active smoking – either before or during pregnancy – or parental exposure to any other environmental or chemical factor (Tables 1 and 2).
4. Discussion Our findings indicate that paternal occupation with an increased likelihood of exposure to hazardous materials (classified as high-risk occupation) is correlated with an increased risk of offspring with isolated CL/P. In our sample population, high-risk paternal occupations in the CL/P group included farmers, chemical factory laborers, heavy equipment mechanics, carpenters and cabinetmakers, vehicle mechanics and plumbers, with nearly 50% of the fathers being farmers. Thus, the most common high-risk paternal occupation was farming. These results are in line with the study of Schnitzer et al. [14] conducted in a population from Atlanta, USA, in which farmering was the main occupation correlated with the occurrence of CL/P. Results of other studies on the relation between oral clefts and paternal exposure to pesticides and paternal agricultural work are inconclusive [22–24]. It is plausible that occupational exposures to hazardous agents (e.g., pesticides, toxic solvents) may induce a genotoxic mutagenic effect on spermatozoa [15]. Some paternal behaviors may also
result in maternal exposure (e.g., contaminated clothing brought home) [15]. It is conceivable that the formulation of prevention policies against occupational exposure of future fathers to toxic agents is necessary. For instance, in Greece, where a great part of the population works in agriculture (especially in rural areas), rigorous information campaigns and elimination measures against the use of toxic pesticides may contribute to the prevention of CL/P. Similar country-based studies could determine occupational settings of high-risk and associated hazardous materials in each country and provide data so that proper country-based prevention policies can be designed. Maternal occupational exposure to toxic agents seems a priori to have a more direct effect on CL/P, and was previously shown to be associated with an increased risk of CL/P in offspring [15,16,22,25]. Experimental data support this: an increased incidence of oral clefts in offspring was noticed when pregnant mice were exposed to the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) [26]. However, no correlation was found here between CL/P and maternal exposure to such toxic agents (e.g., agricultural work). This could be attributed to the fact that the majority of mothers included in our study were concerned with the upkeep of their household. The second more frequent maternal profession was office employees, also of low-risk. Further study is needed to detect those occupations in our country, which may have an increased risk of CL/P and to establish, accordingly, preventive measures. We did not detect any significant difference between children with CL/P and controls with respect to maternal folic acid
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Table 2 Frequency and risk (odds ratios, 95% confidence intervals) of maternal risk factors of offspring with orofacial clefts. Mother Frequency (n)
Factors Category 1. Diseases and drugs Preventive folate supplementation Preparations/face creams containing Vit A Antirheumatic drug therapy Corticosteroids uptake Venous disease in pregnancy/venoactive drugs Anticoagulants Antidepressants Medication for bone disease Thyroid disease/medication Other disease/drugs Category 2. Habits Smoking before and during pregnancy (>1 cigarette/day) Smoking before but not during pregnancy (>1 cigarette/day) Second-hand smoking before and during pregnancy Second-hand smoking before but not during pregnancy Regular alcohol consumption (>2 glasses per week) Drugs abuse Category 3. Exposure to environmental pollutants Residence proximity to factories (5 km) Residence proximity to electrical power industry (5 km) Residence proximity to high voltage electric cables (5 km) Exposure to radiation (e.g., work in area where X-rays/radiopharmaceuticals are used, residence near cell phone antenna) Exposure to pesticides (e.g., usage of pesticides in own crops, work in farm where pesticides are used, residence near productive crops) Category 4. Exposure to chemical contaminants Lead (e.g., living in a house painted with lead-base paints) Cadmium (e.g., coming in contact with vehicle batteries) Vanadium (e.g., coming in contact with vehicle batteries) Category 5. Demographic data Occupation of high risk Parents were blood relatives
Risk for CL/P
CL/P cases (n = 35)
Controls (n = 35)
OR (95% CI)
21 4 1 2 2 3 1 0 1 1
15 0 0 3 3 0 1 1 1 1
2.00 1.12 1.02 0.64 0.64 1.09 1.00 0.97 1.00 1.00
(0.77–5.18) (1.00–1.27)a (0.97–1.09)a (0.10–4.12) (0.10–4.12) (0.98–1.21)a (0.06–16.64) (0.91–1.02)a (0.06–16.64) (0.06–16.64)
6 9 16 18 1 0
7 13 11 14 2 0
0.82 0.66 1.81 1.58 1.00
(0.24–2.76) (0.25–1.83) (0.69–4.74) (0.61–4.06) (0.18–5.33)
13 3 4 4
9 6 5 1
1.70 0.45 0.77 2.12
(0.61–4.74) (0.10–1.97) (0.19–3.16) (0.36–12.45)
6
3
2.20 (0.50–9.63)
12 4 8
10 3 6
1.20 (0.42–3.37) 1.72 (0.35–8.43) 1.61 (0.49–5.28)
5 2
6 0
0.80 (0.22–2.93) 1.06 (0.97–1.15)a
CL/P: cleft lip and/or palate. OR: odds ratio. CI: confidence intervals. a Relative risk was calculated as a good approximate of odds ratio.
supplementation, which has been documented to play a significant role in the prevention of CL/P [4]. The absence of a significant correlation could be attributed to the lack of pregnancy planning in Greek families. The vast majority of Table 3 Frequencies of paternal occupations of high- and low-risk. CL/P group (n = 35) High-risk occupation Farmer Chemical factory worker Heavy equipment mechanic Carpenter/cabinetmaker Automotive mechanic Plumber Total Low-risk occupation Office employee Merchant Stockbreeder Fisherman Construction worker Construction driver Ironsmith
Total
Control group (n = 35) 7 2 2 2 1 1 15
Farmer Chemical factory worker Radiation technician Carpenter Automotive mechanic Electrician Total
13 2
Office employee Low-risk labourer (transportation labourer etc.) Driver (bus, taxi) Manager Medical doctor Priest Pottery maker Teacher Electrical engineer Peddler Total
1 1 1 1 1
20
2 1 1 1 1 1 7 11 7 3 1 1 1 1 1 1 1 28
mothers interviewed in our study did not plan pregnancy and, as a result, started folate uptake during the 2nd or 3rd month of pregnancy, and therefore only as soon as they found out that they were pregnant. Adequate folate levels are reached three months later [5]. Consequently, folate treatment was null or inadequate during the first gestational trimester, which is the crucial period for CL/P development. Our data emphasize the need for information campaigns about the significance of pregnancy planning and prompt onset of folate supplementation (at least 3 months before conception) [27].
Table 4 Frequencies of maternal occupations of high- and low-risk. CL/P group (n = 35) High-risk occupation Farmer Factory worker Shoe manufacturer Total Low-risk occupation Unemployed/housekeeper Office employee Nurse Kindergarten teacher Total
Control group (n = 35) 3 1 1 5 20 8 1 1 30
Farmer Gas station worker
5 1
Total
6
Unemployed/homemaker Office employee Dentist Manager Total
18 9 1 1 29
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Unlike some previous reports, we did not observe a significant correlation between CL/P and smoking habits of parents. There were only some data suggesting that passive exposure of the mother to tobacco may increase the risk of giving birth to offspring with CL/P, but this was characterized by low level of statistical significance. Lack of a significant correlation between smoking and CL/P occurrence could be a statistical bias resulting from the high percentage of smokers in the Greek population, and, hence, the recording of many smokers (first or second hand) in both study and control groups. Consumption of alcohol is also common in Greece; nevertheless, we recorded insignificant numbers of regular drinkers (even with less strict criteria such as the 2 glasses/week). It seems that alcohol is already avoided by stable future parents or pregnant mothers. Similarly, no significant relation was found between parental exposure to diseases, drugs uptake and nonoccupational exposure to heavy metals or other pollutants. In line with the study of Brender et al. [12], we found some evidence suggestive of increased risk for CL/P in offspring when parents resided within 5 km of factories. The strengths of our study include its clinically selected cases and controls – a selection based on medical records and physical examination instead of choosing a registry derived population –, its residence-matched controls to eliminate the effect of environmental pollutants associated with certain areas, and its method of data acquisition via a structured interview conducted by the researchers themselves. Moreover, the study is characterized by a high level of validity as a result of limited selection bias. Although the study groups were hospital-derived and not populationderived, both cases and controls can be considered representative of the population of our region. There was no referral predilection for CL/P cases because our department offers the same services, qualitatively and quantitatively, as the other pediatric surgery departments in our region; they all cover the same 16 prefectures. In addition, controls were selected from the same department as the CL/P cases and were admitted according to the ‘emergency rotation’ system within the four departments. This selection of controls corrects for referral bias and issues relating to internal validity. Finally, the participation rate was high for both cases and controls, decreasing further the probability of a selection bias [28]. However, our study has some limitations. First, our sample size was not big enough to apply a multivariate statistical analysis and examine the combined effects of the factors under investigation. Further study, possibly multicentric, is required to adjust for confounding. Nevertheless, taking into consideration the prevalence of CL/P and the population referring to our center, a worthy number of cases was actually studied. With 0.85 CL/P cases per 1000 neonates [18] and 110,000 births per year [29] approximately 85 CL/P cases are expected every year in Greece. It is estimated that a quarter of these cases, i.e., 21 cases occur per year in Northern Greece, which is served by our department and three other pediatric surgery departments. We had 35 cases from 2004 to 2009, i.e., approximately six cases every year, moderately exceeding the number of cases expected per year in each center of our region. Second, the limited sample size for each subtype of the disease (CL: n = 15, CP: n = 9, CLP: n = 11) did not allow us to examine risks for CL, CP and CLP separately; a much bigger sample size, sufficient for each of them, would be needed. Another issue is that the interviewers could not be blinded to case/control status because review of medical charts and the obtaining of informed consent from the parents were prerequisites for the interview. The lack of a blind method during interviews is mitigated by reaching a consensus on common questioning strategy prior to interviews. Finally, we did not identify specific hazardous exposures in each occupation, except for the farmers who admitted usage of organophosphate pesticides. Further investigation is needed in
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Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Parental exposures and risk of nonsyndromic orofacial clefts in offspring: A case-control study in Greece§ P. Mirilas a,*, A. Mentessidou a, E. Kontis a, M. Asimakidou a, B.J. Moxham b, A.S. Petropoulos a, E.-N. Emmanouil-Nikolousi c a
2nd Department of Pediatric Surgery, Aristotle University of Thessaloniki Medical School, Papageorgiou General Hospital, Ring Road, Nea Efkarpia, 56429 Thessaloniki, Greece Cardiff School of Biosciences, Cardiff University, Wales, UK c Department of Histology and Embryology, School of Medicine, Aristotle University of Thessaloniki Medical School, Greece b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 16 December 2010 Received in revised form 15 February 2011 Accepted 16 February 2011 Available online 30 March 2011
Objective: To investigate the association between cleft lip and/or palate and nongenetic factors in Greece. Methods: We designed a case-control study including 35 patients with nonsyndromic cleft lip and/or palate, retrospectively selected, and 35 control patients matched for prefecture of residence, prospectively selected from pediatric population hospitalized for abdominal pain or injury. Parents were interviewed about drug uptake, diseases, habits, non-occupational exposure to pollutants, and occupation. Questions covered the period from one year before until three months after conception. Results: High-risk paternal occupations (mostly farmers) were significantly more frequent in cleft lip and/or palate than in controls (p = 0.039) and increased significantly the cleft lip and/or palate risk in offspring (OR: 3.00; 95% CI: 1.03–8.70). Maternal occupation did not correlate with cleft lip and/or palate. Parental disease, drugs uptake, hazardous habits, maternal folate supplementation and nonoccupational exposure to pollutants did not correlate with cleft lip and/or palate. There was a suggestion of increased risk with maternal passive exposure to tobacco (OR: 1.81; 95% CI: 0.69–4.74) and with residential proximity to industries (OR: 1.70; 95% CI: 0.61–4.74). Conclusions: Paternal high-risk occupations probably exert a teratogenic effect on spermatogenesis or result in maternal contamination, and deserve specific preventive policies. The relation of smoking and residential proximity to industries with occurrence of cleft lip and/or palate deserves further study. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cleft lip and/or palate Parental occupation Smoking Environmental pollutants Drugs Folic acid
1. Introduction Lip and/or palate clefts (CL/P) are congenital malformations with severe medical and psychosocial consequences [1,2]. They may present as cleft lip with or without cleft palate (CL P) or as cleft palate alone (CP). The pathogenesis of nonsyndromic CL/P is complex and both genetic and environmental factors are believed to be involved [3–6]. Identification of the environmental factors that are implicated in the pathogenesis of isolated CL/P is crucial in order to formulate prevention strategies. Many parental exposures have been associated with CL/P, including maternal use of anticonvulsants or retinoids [7,8], paternal and maternal hazardous habits such as smoking [5,7–11] and regular alcohol consumption [8], residential proximity to sites
§ Preliminary results were presented at the International Workshop on Risk Assessment and Human Exposure to Hazardous Materials, Aristotle University of Thessaloniki, 21–24 October 2009. * Corresponding author. Tel.: +30 6945 038083. E-mail address: [email protected] (P. Mirilas).
0165-5876/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijporl.2011.02.018
of increased chemical pollution [12] and first-trimester maternal fever-producing illnesses including influenza [6,13]. In addition, an association between certain occupations and the occurrence of CL/ P has been noted [14–16]. However, there are some discrepancies in findings derived from different countries. For instance, hairdressers and woodworkers were mainly associated with offspring with cleft lip and/or palate in forested areas of Norway [15]. In Italy – where the clothing and shoe industries are very well developed – women involved in leather and shoe manufacturing [16] were most at risk. Furthermore, in the agricultural metropolitan area of Atlanta, USA, farmers and firemen appeared to be at most increased risk of having offspring with CL/P [14]. Similarly, data about the relation between parental socioeconomic status and risk of CL/P are inconclusive [5,17]. Such disparities between countrybased studies determining the most common environmental risk factors for oral clefts may be explained by differences in socioeconomic status and economic policies, occupation frequencies and occupational settings, mentality, level of prevention and information campaigns, habits, hobbies, etc. If cultural, political and socioeconomic differences are the reason for data discrepancies, country-based studies are needed to define risk factors
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associated with CL/P in each country. Only on this basis can effective country-based prevention policies be achieved. In Greece, the prevalence at birth of orofacial clefts was reported as being 0.85 per 1000 newborns. This was determined specifically for the decade 1955–1965 [18]. More recent published reports concerning the prevalence of this anomaly are not available. Therefore, it is not surprising that an etiologic study has not yet been undertaken and consequently the role of hazardous environmental exposures of parents in the development of CL/P has never been investigated. Thus, relevant prevention policies are almost completely lacking in this country. Taking the above into consideration, we designed a case-control study in order to examine the association between both paternal and maternal exposure to a series of environmental factors (including drugs, diseases, chemical and environmental pollutants, smoking, alcohol and occupational hazardous factors) and the occurrence of nonsyndromic CL/P in Greece.
trimester is the critical period for the formation of CL P (5th–12th gestational week) and CP (7th–12th gestational week) [5,9]. Parents’ responses were recorded as dichotomous data (Yes or No). Interviews of both parents of either cases or controls were conducted by the same researchers (AM, MA) using the same questioning strategy. In case a definite ‘yes’ or ‘no’ response could not be elicited, based on the memory of the respondent, it was rather recorded as a missing answer in order to eliminate bias. Due to the wide variety of answers with respect to occupation, paternal and maternal occupations were classified as of high- or low-risk. Classification was based on whether occupational exposure to hazardous materials (including solvents and heavy metals) was likely or not, according to information available in the US National Library of Medicine Haz-Map [12,19]. Thus, for example, farmers/groundsmen who used pesticides were considered of high-risk, while unemployed women/homemakers were classified as of low-risk.
2. Methods
2.1. Statistics [20]
Our case-control study included 35 patients operated on for nonsyndromic CL/P and 35 residence-matched control patients. Medical records of all children treated for CL/P in our Pediatric Surgery Department between 2004 and 2009 were retrospectively reviewed. A total of 44 charts were analyzed. The exclusion criterion was the presence of an associated anomaly or syndrome, which would interfere with the evaluation of the disease. We did not manage to reach the families of 5 cases with CL/P. Four other cases were excluded from the study because of associated anomalies or syndromes (e.g., Pierre Robin syndrome). Thus, a total of 35 children with isolated CL/P were included in the study. Both parents of these children were contacted by phone and after informed consent was obtained, they undertook a specially designed structured interview. After the survey of patients with CL/P deformities was completed, we prospectively included during 2009 a control group of 35 children hospitalized for abdominal pain or injury in our department. Controls were excluded from the study if they had a history of known congenital anomalies or recognizable malformations. Inclusion criterion for this group was that controls should be matched with patients for prefecture of residence before and during gestation. Our department is one of four departments of Pediatric Surgery covering an area of 16 prefectures (urban and rural) with a total population of 2.4 millions. Considering the possible variation in the amount of exposure to environmental and/or chemical pollutants between different prefectures, matching of cases and controls for prefecture of residence was undertaken to avoid any bias in results. Initially, families of 52 children were asked to participate in the control group. Three children were excluded from the study because they had previously been operated upon for congenital disorders (e.g., cryptorchidism, hypospadias). Fourteen out of 52 children were not included because they failed to match with controls for prefecture of residence. Finally, 35 children free of congenital anomalies and area-matched with cases ended up participating as controls. Parents of controls were required to provide written informed consent and underwent the same structured interview as for the parents of CL/P cases. Study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. Interview formulation was based on the results of previous research on environmental factors associated with CL/P [3–17] and on specific regional and national characteristics, e.g., common hazardous habits. The interview questions were divided into five categories (Tables 1 and 2). The exposure period of interest during pregnancy was limited to the first three months because the first
Differences in variables (maternal, paternal and common parental variables) between cases and controls were tested by Chi-square tests. For estimation of risk of offspring with CL/P, odds ratios and 95% confidence intervals were used. When Chi-square tables contained cells with an expected count of less than five, the Monte–Carlo method was used for the calculation of p value, OR and 95% confidence intervals. When the observed count in one cell of the crosstabulation was zero, the relative risk was calculated for estimation of risk of CL/P in offsprings [21]. Statistical analysis was performed using SPSS 16.0 for Windows.
3. Results Review of the medical charts showed that 15 out of the 35 CL/P cases suffered from cleft lip without cleft palate (CL), 11 from cleft lip and palate (CLP) and 9 children suffered from cleft palate alone (CP). Estimated risks of paternal and maternal factors for offspring with CL/P are presented in Tables 1 and 2, respectively. As can be seen in Table 1, paternal profession of high risk had a significant correlation with occurrence of CL/P in offspring (Pearson chi square, p = 0.039), and a three times increased risk for the anomaly (odds ratio = 3). From the list of frequencies of high- and low-risk paternal occupations observed in this study (Table 3), it is evident that professions of high risk were more frequent in the CL/P group. In contrast, observed frequencies of low- and high-risk occupations were comparable between mothers of CL/P and mothers of controls (Table 4); thus, no association was detected between maternal occupation risk and occurrence of CL/P (Table 2). As can be seen in Tables 1 and 2, there is a tendency for residential proximity to industries to be associated with CL/P. Although this association is characterized by low level of statistical significance, we should note that the odds ratio value for residential proximity to industrial areas is greater than one by 0.7. These findings suggest that the risk of offspring with CL/P is increased by 70% when the family lives within 5 km of an industry. In addition, there was a suggestion that maternal passive exposure to tobacco before and during pregnancy was more frequent in the CL/P group than in the controls (Table 2) and tended to increase 1.58 and 1.81 times, respectively, the risk of nonexposed women for offspring with CL/P (Table 2). The association between second-hand smoke and CL/P occurrence, however, did not achieve statistical significance. Similarly, odds ratio and 95% confidence intervals suggested an association between folate
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Table 1 Frequency and risk (odds ratios, 95% confidence intervals) of paternal risk factors of offspring with orofacial clefts. Father Frequency (n) CL/P cases (n = 35) Factors Category 1. Diseases and drugs Preparations/face creams containing Vit A Antirheumatic drug therapy Corticosteroids uptake Antidepressants Medication for bone disease Thyroid disease/medication Other disease/drugs Category 2. Habits Smoking before and during pregnancy (>1 cigarette/day) Smoking before but not during pregnancy (>1 cigarette/day) Regular alcohol consumption (>2 glasses per week) Drugs abuse Category 3. Exposure to environmental pollutants Residence proximity to factories (5 km) Residence proximity to electrical power industry (5 km) Residence proximity to high voltage electric cables (5 km) Exposure to radiation (e.g., work in area where X-rays/radiopharmaceuticals are used, residence near cell phone antenna) Exposure to pesticides (e.g., usage of pesticides in own crops, work in or residence near farm where pesticides are used) Category 4. Exposure to chemical contaminants Lead (e.g., living in a house painted with lead-base paints) Cadmium (e.g., coming in contact with vehicle batteries) Vanadium (e.g., coming in contact with vehicle batteries) Category 5. Demographic data Occupation of high risk Parents were blood relatives
Risk for CL/P Controls (n = 35)
OR (95% CI)
1 0 0 0 0 1 0
0 0 0 1 0 0 0
1.02 (0.97–1.09)a
22 0 2 0
20 0 1 0
1.26 (0.48–3.30)
13 3 4 0
9 6 5 0
1.70 (0.61–4.74) 0.45 (0.10–1.97) 0.77 (0.19–3.16)
11
7
1.83 (0.61–5.47)
12 4 8
10 3 6
1.20 (0.42–3.37) 1.72 (0.35–8.43) 1.61 (0.49–5.28)
15 2
7 0
3.00 (1.03–8.70)* 1.06 (0.97–1.15)a
0.97 (0.91–1.02)a 1.02 (0.97–1.09)a
1.00 (0.18–5.33)
CL/P: cleft lip and/or palate. OR: odds ratio. CI: confidence intervals. a Relative risk was calculated as a good approximation to odds ratio. * Significant association (p = 0.03).
uptake and occurrence of orofacial clefts, although this association was statistically non significant (Table 2). Pre- and post-conceptional parental exposure to medication or diseases was not correlated with the occurrence of CL/P (Tables 1 and 2). Likewise, we did not detect any association between occurrence of CL/P and paternal or maternal active smoking – either before or during pregnancy – or parental exposure to any other environmental or chemical factor (Tables 1 and 2).
4. Discussion Our findings indicate that paternal occupation with an increased likelihood of exposure to hazardous materials (classified as high-risk occupation) is correlated with an increased risk of offspring with isolated CL/P. In our sample population, high-risk paternal occupations in the CL/P group included farmers, chemical factory laborers, heavy equipment mechanics, carpenters and cabinetmakers, vehicle mechanics and plumbers, with nearly 50% of the fathers being farmers. Thus, the most common high-risk paternal occupation was farming. These results are in line with the study of Schnitzer et al. [14] conducted in a population from Atlanta, USA, in which farmering was the main occupation correlated with the occurrence of CL/P. Results of other studies on the relation between oral clefts and paternal exposure to pesticides and paternal agricultural work are inconclusive [22–24]. It is plausible that occupational exposures to hazardous agents (e.g., pesticides, toxic solvents) may induce a genotoxic mutagenic effect on spermatozoa [15]. Some paternal behaviors may also
result in maternal exposure (e.g., contaminated clothing brought home) [15]. It is conceivable that the formulation of prevention policies against occupational exposure of future fathers to toxic agents is necessary. For instance, in Greece, where a great part of the population works in agriculture (especially in rural areas), rigorous information campaigns and elimination measures against the use of toxic pesticides may contribute to the prevention of CL/P. Similar country-based studies could determine occupational settings of high-risk and associated hazardous materials in each country and provide data so that proper country-based prevention policies can be designed. Maternal occupational exposure to toxic agents seems a priori to have a more direct effect on CL/P, and was previously shown to be associated with an increased risk of CL/P in offspring [15,16,22,25]. Experimental data support this: an increased incidence of oral clefts in offspring was noticed when pregnant mice were exposed to the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) [26]. However, no correlation was found here between CL/P and maternal exposure to such toxic agents (e.g., agricultural work). This could be attributed to the fact that the majority of mothers included in our study were concerned with the upkeep of their household. The second more frequent maternal profession was office employees, also of low-risk. Further study is needed to detect those occupations in our country, which may have an increased risk of CL/P and to establish, accordingly, preventive measures. We did not detect any significant difference between children with CL/P and controls with respect to maternal folic acid
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Table 2 Frequency and risk (odds ratios, 95% confidence intervals) of maternal risk factors of offspring with orofacial clefts. Mother Frequency (n)
Factors Category 1. Diseases and drugs Preventive folate supplementation Preparations/face creams containing Vit A Antirheumatic drug therapy Corticosteroids uptake Venous disease in pregnancy/venoactive drugs Anticoagulants Antidepressants Medication for bone disease Thyroid disease/medication Other disease/drugs Category 2. Habits Smoking before and during pregnancy (>1 cigarette/day) Smoking before but not during pregnancy (>1 cigarette/day) Second-hand smoking before and during pregnancy Second-hand smoking before but not during pregnancy Regular alcohol consumption (>2 glasses per week) Drugs abuse Category 3. Exposure to environmental pollutants Residence proximity to factories (5 km) Residence proximity to electrical power industry (5 km) Residence proximity to high voltage electric cables (5 km) Exposure to radiation (e.g., work in area where X-rays/radiopharmaceuticals are used, residence near cell phone antenna) Exposure to pesticides (e.g., usage of pesticides in own crops, work in farm where pesticides are used, residence near productive crops) Category 4. Exposure to chemical contaminants Lead (e.g., living in a house painted with lead-base paints) Cadmium (e.g., coming in contact with vehicle batteries) Vanadium (e.g., coming in contact with vehicle batteries) Category 5. Demographic data Occupation of high risk Parents were blood relatives
Risk for CL/P
CL/P cases (n = 35)
Controls (n = 35)
OR (95% CI)
21 4 1 2 2 3 1 0 1 1
15 0 0 3 3 0 1 1 1 1
2.00 1.12 1.02 0.64 0.64 1.09 1.00 0.97 1.00 1.00
(0.77–5.18) (1.00–1.27)a (0.97–1.09)a (0.10–4.12) (0.10–4.12) (0.98–1.21)a (0.06–16.64) (0.91–1.02)a (0.06–16.64) (0.06–16.64)
6 9 16 18 1 0
7 13 11 14 2 0
0.82 0.66 1.81 1.58 1.00
(0.24–2.76) (0.25–1.83) (0.69–4.74) (0.61–4.06) (0.18–5.33)
13 3 4 4
9 6 5 1
1.70 0.45 0.77 2.12
(0.61–4.74) (0.10–1.97) (0.19–3.16) (0.36–12.45)
6
3
2.20 (0.50–9.63)
12 4 8
10 3 6
1.20 (0.42–3.37) 1.72 (0.35–8.43) 1.61 (0.49–5.28)
5 2
6 0
0.80 (0.22–2.93) 1.06 (0.97–1.15)a
CL/P: cleft lip and/or palate. OR: odds ratio. CI: confidence intervals. a Relative risk was calculated as a good approximate of odds ratio.
supplementation, which has been documented to play a significant role in the prevention of CL/P [4]. The absence of a significant correlation could be attributed to the lack of pregnancy planning in Greek families. The vast majority of Table 3 Frequencies of paternal occupations of high- and low-risk. CL/P group (n = 35) High-risk occupation Farmer Chemical factory worker Heavy equipment mechanic Carpenter/cabinetmaker Automotive mechanic Plumber Total Low-risk occupation Office employee Merchant Stockbreeder Fisherman Construction worker Construction driver Ironsmith
Total
Control group (n = 35) 7 2 2 2 1 1 15
Farmer Chemical factory worker Radiation technician Carpenter Automotive mechanic Electrician Total
13 2
Office employee Low-risk labourer (transportation labourer etc.) Driver (bus, taxi) Manager Medical doctor Priest Pottery maker Teacher Electrical engineer Peddler Total
1 1 1 1 1
20
2 1 1 1 1 1 7 11 7 3 1 1 1 1 1 1 1 28
mothers interviewed in our study did not plan pregnancy and, as a result, started folate uptake during the 2nd or 3rd month of pregnancy, and therefore only as soon as they found out that they were pregnant. Adequate folate levels are reached three months later [5]. Consequently, folate treatment was null or inadequate during the first gestational trimester, which is the crucial period for CL/P development. Our data emphasize the need for information campaigns about the significance of pregnancy planning and prompt onset of folate supplementation (at least 3 months before conception) [27].
Table 4 Frequencies of maternal occupations of high- and low-risk. CL/P group (n = 35) High-risk occupation Farmer Factory worker Shoe manufacturer Total Low-risk occupation Unemployed/housekeeper Office employee Nurse Kindergarten teacher Total
Control group (n = 35) 3 1 1 5 20 8 1 1 30
Farmer Gas station worker
5 1
Total
6
Unemployed/homemaker Office employee Dentist Manager Total
18 9 1 1 29
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Unlike some previous reports, we did not observe a significant correlation between CL/P and smoking habits of parents. There were only some data suggesting that passive exposure of the mother to tobacco may increase the risk of giving birth to offspring with CL/P, but this was characterized by low level of statistical significance. Lack of a significant correlation between smoking and CL/P occurrence could be a statistical bias resulting from the high percentage of smokers in the Greek population, and, hence, the recording of many smokers (first or second hand) in both study and control groups. Consumption of alcohol is also common in Greece; nevertheless, we recorded insignificant numbers of regular drinkers (even with less strict criteria such as the 2 glasses/week). It seems that alcohol is already avoided by stable future parents or pregnant mothers. Similarly, no significant relation was found between parental exposure to diseases, drugs uptake and nonoccupational exposure to heavy metals or other pollutants. In line with the study of Brender et al. [12], we found some evidence suggestive of increased risk for CL/P in offspring when parents resided within 5 km of factories. The strengths of our study include its clinically selected cases and controls – a selection based on medical records and physical examination instead of choosing a registry derived population –, its residence-matched controls to eliminate the effect of environmental pollutants associated with certain areas, and its method of data acquisition via a structured interview conducted by the researchers themselves. Moreover, the study is characterized by a high level of validity as a result of limited selection bias. Although the study groups were hospital-derived and not populationderived, both cases and controls can be considered representative of the population of our region. There was no referral predilection for CL/P cases because our department offers the same services, qualitatively and quantitatively, as the other pediatric surgery departments in our region; they all cover the same 16 prefectures. In addition, controls were selected from the same department as the CL/P cases and were admitted according to the ‘emergency rotation’ system within the four departments. This selection of controls corrects for referral bias and issues relating to internal validity. Finally, the participation rate was high for both cases and controls, decreasing further the probability of a selection bias [28]. However, our study has some limitations. First, our sample size was not big enough to apply a multivariate statistical analysis and examine the combined effects of the factors under investigation. Further study, possibly multicentric, is required to adjust for confounding. Nevertheless, taking into consideration the prevalence of CL/P and the population referring to our center, a worthy number of cases was actually studied. With 0.85 CL/P cases per 1000 neonates [18] and 110,000 births per year [29] approximately 85 CL/P cases are expected every year in Greece. It is estimated that a quarter of these cases, i.e., 21 cases occur per year in Northern Greece, which is served by our department and three other pediatric surgery departments. We had 35 cases from 2004 to 2009, i.e., approximately six cases every year, moderately exceeding the number of cases expected per year in each center of our region. Second, the limited sample size for each subtype of the disease (CL: n = 15, CP: n = 9, CLP: n = 11) did not allow us to examine risks for CL, CP and CLP separately; a much bigger sample size, sufficient for each of them, would be needed. Another issue is that the interviewers could not be blinded to case/control status because review of medical charts and the obtaining of informed consent from the parents were prerequisites for the interview. The lack of a blind method during interviews is mitigated by reaching a consensus on common questioning strategy prior to interviews. Finally, we did not identify specific hazardous exposures in each occupation, except for the farmers who admitted usage of organophosphate pesticides. Further investigation is needed in
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