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Rodrigo Soares de Andrade
FATORES AMBIENTAIS ASSOCIADOS À OCORRÊNCIA DE
FISSURAS LABIAIS OU PALATINAS NÃO SINDRÔMICAS
ASSOCIATED ENVIRONMENTAL FACTORS WITH THE
OCCURRENCE OF NON-SYNDROMIC CLEFT LIP OR PALATE
Piracicaba/SP
2019
Rodrigo Soares de Andrade
FATORES AMBIENTAIS ASSOCIADOS À OCORRÊNCIA DE
FISSURAS LABIAIS OU PALATINAS NÃO SINDRÔMICAS
ASSOCIATED ENVIRONMENTAL FACTORS WITH THE
OCCURRENCE OF NON-SYNDROMIC CLEFT LIP OR PALATE
Tese apresentada à Faculdade de Odontologia de Piracicaba da
Universidade Estadual de Campinas como parte dos requisitos
exigidos para a obtenção do título de Doutor em Estomatopatologia na
Área de Patologia.
Thesis presented to the Piracicaba Dental School of the University of
Campinas in partial fulfillment of the requirements for the degree of
Doctor in Pathology in Oral Pathology area.
Orientador: Prof. Dr. Hercílio Martelli Júnior
Este exemplar corresponde à versão final da tese
defendida pelo aluno Rodrigo Soares de Andrade, e
orientado pelo Prof. Dr. Hercílio Martelli Júnior.
Piracicaba/SP
2019
Ficha catalográfica
Universidade Estadual de Campinas Biblioteca da Faculdade de Odontologia de Piracicaba
Marilene Girello - CRB 8/6159
Andrade, Rodrigo Soares de, 1992- An24f AndFatores ambientais associados a ocorrência de fissuras labiais ou palatinas
não sindrômicas / Rodrigo Soares de Andrade. – Piracicaba, SP : [s.n.], 2019.
AndOrientador: Hercílio Martelli Júnior. AndTese (doutorado) – Universidade Estadual de Campinas, Faculdade de
Odontologia de Piracicaba.
And1. Fenda palatina. 2. Cafeína. 3. Poluição por fumaça de tabaco. 4. Ácido
fólico. 5. Anormalidades dentárias. I. Martelli Júnior, Hercílio. II. Universidade
Estadual de Campinas. Faculdade de Odontologia de Piracicaba. III. Título.
Informações para Biblioteca Digital
Título em outro idioma: Associeted enviromental factors with the occurrence of non-
sydromic cleft lip or palate Palavras-chave em inglês: Cleft palate Caffeine Tobacco smoke pollution Folic acid Tooth abnormalities Área de concentração: Patologia Titulação: Doutor em Estomatopatologia Banca examinadora: Hercílio Martelli Júnior [Orientador] Marcelo Sivieri de Araújo Ana Camila Pereira Messetti Renato Assis Machado Alan Roger dos Santos Silva Data de defesa: 06-12-2019 Programa de Pós-Graduação: Estomatopatologia
Identificação e informações acadêmicas do(a) aluno(a) - ORCID do autor: http://orcid.org/0000-0001-6114-0929 - Currículo Lattes do autor: http://lattes.cnpq.br/4936205843300438
UNIVERSIDADE ESTADUAL DE CAMPINAS Faculdade de Odontologia de Piracicaba
A Comissão Julgadora dos trabalhos de Defesa de Tese de Doutorado, em sessão pública
realizada em 06 de dezembro de 2019, considerou o candidato RODRIGO SOARES DE
ANDRADE aprovado.
PROF. DR. HERCÍLIO MARTELLI JÚNIOR
PROF. DR. MARCELO SIVIERI DE ARAÚJO
PROFª. DRª. ANA CAMILA PEREIRA MESSETTI
PROF. DR. RENATO ASSIS MACHADO
PROF. DR. ALAN ROGER DOS SANTOS SILVA
A Ata da defesa, assinada pelos membros da Comissão Examinadora, consta no
SIGA/Sistema de Fluxo de Dissertação/Tese e na Secretaria do Programa da
Dedicatória
Dedico este trabalho e essa vitória a minha família, meu pai Moacir Jose de
Andrade, minha mãe Maria Lucia Soares Andrade, minha irmã Nayara Soares de
Andrade e meu sobrinho Heitor Soares Caixeta, que sem sombra de tudo, são meu maior
tesouro e minha fortaleza!
Agradecimentos Especiais
Agradeço ao meu orientador, Prof. Dr. Hercílio Martelli Júnior, por todos esses anos de
trabalho árduo, por todos os ensinamentos e oportunidades, e por toda paciência por me
formar o profissional que sou hoje. Minha admiração e carinho ultrapassam a relação
orientador/orientado e trilhamos um caminho de uma grande parceria e amizade. O vejo como
um exemplo de ser humano e um como um profissional admirável! Tenho orgulho de poder
ter sido orientado por um profissional tão competente, que no final dessa jornada, podê-lo-ei
chamar de amigo.
Agradecimentos
O presente trabalho foi realizado com apoio das agências Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Código de
Financiamento 001, 88881.188930/2018-01 PDSE; e ao Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq), processo nº 141063/2016-5.
À Universidade Estadual de Campinas (UNICAMP), na pessoa do Magnífico
Reitor, Prof. Dr. Marcelo Knobel e à Faculdade de Odontologia de Piracicaba –
UNICAMP, na pessoa do seu Diretor, Prof. Dr. Francisco Haiter Neto e Diretor Associado,
Prof. Dr. Flávio Henrique Baggio Aguiar.
À Profa. Dra. Karina Gonzales Silvério Ruiz, coordenadora geral dos
programas de Pós-graduação da Faculdade de Odontologia de Piracicaba da Universidade
Estadual de Campinas.
Ao Prof. Dr. Márcio Ajudarte Lopes, coordenador do programa de Pós-
graduação em Estomatopatologia da Faculdade de Odontologia de Piracicaba da Universidade
Estadual de Campinas.
Aos docentes das áreas de Semiologia e Patologia da Faculdade de Odontologia
de Piracicaba – UNICAMP, Prof. Dr. Márcio Ajudarte Lopes, Prof. Dr. Alan Roger dos
Santos Silva, Prof. Dr. Pablo Agustín Vargas, Prof. Dr. Oslei Paes de Almeida, Prof. Dr.
Edgard Graner, Prof. Dr. Ricardo D. Coletta e Prof. Dr. Jacks Jorge Júnior, por todos os
ensinamentos.
A Prof. Dra. Daniella Barbosa Reis Martelli, por todo auxílio e ajuda para elaboração desse
trabalho, por sua disponibilidade e por prontidão em me ensinar.
A Universidade José do Rosário Vellano (UNIFENAS) e seus profissionais, por terem me
recebido e me acolhido tão carinhosamente.
Aos profissionais e pacientes do Centro de anomalias craniofaciais - Pró Sorriso, onde
realizei vários trabalhos, pude evoluir como profissional e como ser humano.O aprendizado e
as oportunidades que tive, são impagáveis.
Aos alunos Ygor, Alison, Juliana, Isadora, Any e Maria Luiza por ter tido o prazer de
orienta-los durante suas iniciações cientificas, e de aprender infinitamente durante todo o
processo.
A Profa. Dra. Adriana Boeri Freiri Tamburini, por todo conhecimento compartilhado, por
toda ajuda, e principalmente por toda a amizade e amparo, serei eternamente grato!
A Dra. Flavia Alves, minha psicóloga e terapeuta, que foi de extrema importância para me
dar luz e conforto em tempos difíceis durante o meu doutorado.
Aos meus amigos Adriano e Matheus, por toda amizade e carinho, me propiciando os
melhores momentos.
Aos meus colegas da FOP/UNICAMP por todo companheirismo durante a Pós-graduação.
Resumo
Esta tese contemplou dois estudos “distintos” envolvendo pacientes com fissuras orais não
sindrômicas. As fissuras de lábio e/ou palato são malformações congênitas que, como
consequência, trazem uma série de alterações como comprometimento da estética, da fala e da
posição dos dentes. De modo geral, são justificadas pela herança multifatorial, coexistindo
fatores genéticos e ambientais, e podem ser associação ou não a síndromes. O primeiro estudo
teve como objetivo avaliar fatores de risco ambientais que tenham influência sobre o
desenvolvimento de fissuras orais não sindrômicas. A suplementação de ácido fólico, fumo
passivo, consumo excessivo de cafeína e de álcool e exposição a produtos químicos como
agrotóxicos foram as variáveis estudadas através da aplicação de um questionário. Com isso,
observou-se que o consumo de cafeína acima de 100 mg diárias se mostrou maior em mães de
filhos com fissuras orais. Esse consumo excessivo acima de 500mg semanais mostrou uma
probabilidade 34 vezes maior do desenvolvimento de fissuras nos filhos dessas mães. O fumo
passivo acima de 15 minutos diários também se mostrou como fator de risco importante e
mais frequentemente encontrado para mães de filhos com fissuras orais não sindrômicas. A
suplementação com ácido fólico e sulfato ferroso, juntamente com seus complexos
multivitamínicos mostraram fator protetor para evitar o desenvolvimento de fissuras orais não
sindrômicas, sendo mais consumido pelas mães de filhos não fissurados. Já o segundo estudo
teve como objetivo analisar anomalias dentárias em pacientes com fissuras labiais isoladas.
Realizou-se uma comparação entre a quantidade e tipo de anomalia dentária em pacientes
com fissuras labiais que envolviam ou não para o rebordo alveolar. Observou-se que a
agenesia dentária foi à anomalia mais encontrada, seguida de dentes supranumerários e
microdontia. Pacientes com a fissura labial com o envolvimento de rebordo apresentaram
mais anomalias dentárias quando comparados a pacientes com fissura labial sem o
envolvimento do rebordo alveolar. Em síntese, concluímos que o consumo de cafeína acima
de 100 mg diários e o fumo passivo além de 15 minutos ao dia aumentaram o risco do
desenvolvimento de fissuras orais não sindrômicas não sindrômicas. A suplementação com
ácido fólico, complexos multivitamínicos e alimentos ricos em ferro, utilizados durante a
realização do pré-natal, atuam como fatores protetores para o não desenvolvimento de fissuras
orais não sindrômicas não sindrômicas. Pacientes com fissuras labiais com o envolvimento de
rebordo tem mais anomalias dentárias do que os pacientes com fissuras labiais sem o
envolvimento de rebordo e agenesia dentária e supranumerários são as anomalias dentárias
mais comuns nos pacientes com fissuras labiais isoladas.
Palavras–chave: fissuras orais, cafeína, fumo passivo, ácido fólico, anomalias dentarias,
agenesia dental.
Abstract
This thesis contemplated two "distinct" studies involving patients with nonsyndromic oral
clefts. Clefts of the lip and/or palate are congenital malformations that, as a consequence,
bring about a series of alterations such as impairment of aesthetics, speech and tooth position.
In general, they are justified by multifactorial inheritance, coexisting genetic and
environmental factors, and may or may not be associated with syndromes. The first study
aimed to evaluate environmental risk factors that influence the development of nonsyndromic
oral clefts. Folic acid supplementation, secondhand smoking, excessive consumption of
caffeine and alcohol, and exposure to chemicals such as pesticides were the variables studied
by applying a questionnaire. Thus, it was observed that the consumption of caffeine above
100 mg daily was higher in mothers of children with oral clefts. This excessive consumption
above 500 mg per week showed a 34 times greater probability of the development of cracks in
the children of these mothers. Secondhand smoke above 15 minutes daily was also shown to
be an important risk factor and more frequently found for mothers of children with
nonsyndromic oral clefts. Supplementation with folic acid and ferrous sulfate, along with its
multivitamin complexes showed a protective factor to avoid the development of
nonsyndromic oral clefs, being more consumed by mothers of non-cleft children. The second
study aimed to analyze dental anomalies in patients with isolated cleft lips. A comparison was
made between the amount and type of dental anomaly in patients with cleft lip involving or
not the alveolar ridge. It was observed that tooth agenesis was the most commonly found
anomaly, followed by supernumerary teeth and microdontia. Patients with cleft lip involving
the ridge presented more dental anomalies when compared to patients with cleft lip without
the involvement of the alveolar ridge. In summary, we concluded that the consumption of
caffeine above 100 mg daily and secondhand smoking beyond 15 minutes a day increased the
risk of developing nonsyndromic oral clefts. Folic acid supplementation, multivitamin
complexes and iron-rich foods used during prenatal care act as protective factors for the
development of nonsyndromic oral clefts. Patients with cleft lip with lip involvement have
more dental anomalies than patients with cleft lip without lip involvement and tooth agenesis
and supernumerary are the most common dental anomalies in patients with cleft lip alone.
Keywords: oral clefts, caffeine, secondhand smoking, folic acid, dental anomalies, dental
agenesis.
Sumário
1 Introdução ................................................................................................... 14
2 Artigos
2.1 Artigo: Maternal consumption of caffeine and second-hand tobacco smoke as risk
factor for the development of oral clefts .................................................................... 17
2.2– Artigo: Dental anomalies in Brazilian patients with isolated cleft lip with or without
alveolar ridge involvement .................................................................................... 37
3 Discussão ...................................................................................................... 51
4 Conclusão ..................................................................................................... 55
Referências .................................................................................................. 56
Apêndice 1-Ficha cadastral para participantes do projeto. .................................... 62
Anexos
Anexo 1– Parecer Comitê de ética ..................................................................... 65
Anexo 2– Comprovante de Submissão ................................................................ 80
Anexo 3– Verificação de originalidade e prevenção de plágio ................................. 82
14
1 Introdução
1.1 Capítulo 1
As fissuras orais não sindrômicas são as anomalias craniofaciais mais prevalentes na
população. A prevalência destas condições em todo o mundo é de aproximadamente 1
em cada 500 nativivos e varia entre diferentes países, etnias e raças (Rahimov et al.,
2012). As fissuras orais não sindrômicas podem afetar a vida de uma pessoa e de sua
família, tendo uma influência importante na qualidade de vida dos indivíduos.
A etiologia das fissuras orais não sindrômicas envolve fatores ambientais,
genéticos e epigenéticos (Lima et al., 2015, Kumari et al., 2018; Machado et al., 2018).
Consanguinidade, deficiência nutricional materna durante a gravidez, tabagismo e
alcoolismo são considerados como alguns desses fatores de risco (Martelli et al.,
2012).O tabagismo, incluindo a forma passiva (Ayu & Samsudin, 2003; McKinney et
al., 2016), aumenta o risco de fissuras orais não sindrômicas , devido ao efeito
teratogênico dos agentes ativos e radicais livres presentes na fumaça do cigarro
(Martelli et al., 2015; Machado et al., 2016; Machado et al., 2018). Little et al. (2004)
observaram uma probabilidade de2,1 vezes maior de desenvolvimento de fissuras orais
não sindrômicas para mães expostas ao tabagismo passivo.
Existem evidências para associações entre alguns hábitos alimentares da
mãe durante o primeiro trimestre de gravidez, como o consumo de alimentos e bebidas
ricas em cafeína e o desenvolvimento de fissuras orais não sindrômicas (McDonald et
al., 1992; Browne, 2006; Bille et al., 2007; Johansen et al., 2009).A ingestão alimentar
materna e o estado nutricional desempenham um papel significativo no
desenvolvimento das fissuras orais não sindrômicas (Krapels et al., 2006). O baixo
consumo de alimentos ricos em ferro pode estar associado à ocorrência de fissuras orais
não sindrômicas, mas os mecanismos envolvidos nesta associação ainda não são
totalmente compreendidos (McKinney et al., 2016). O uso de multivitamínicos reduz o
risco de fissura na maioria dos estudos (Wilcox et al., 2007; Jia et al., 2011). A
suplementação com ácido fólico por si só mostra diminuir o risco de defeitos do tubo
neural e das fissuras orais não sindrômicas (Rozendaal et al., 2013; De-Regil et al.,
2015; Machado et al., 2016).
Como os fatores de risco podem variar de acordo com diversas variáveis, faz-se
necessário investigar a etiologia das fissuras orais não sindrômicas em diferentes
15
localidades para identificar os fatores de risco que são mais frequentes nos diferentes
países. No Brasil, a prevalência das fissuras orais não sindrômicas varia de 0,36 a 1,54
por 1.000 nativivos (Martelli-Júnior et al., 2007; Rodrigues et al., 2009), sendo
expressiva a prevalência destas anomalias.Por isso, faz-se immportante conhecer e
buscar correlacionar a ocorrência de fissuras orais não sindrômicas com os diversos
fatores de risco ambientais, genéticos e epigenéticos.
1.2 Capítulo 2
O desenvolvimento facial humano ocorre entre a 4ª e 8ª semanas de vida intrauterina e a
união dos processos faciais (maxilares, mandibulares e frontonasais) vão permitir a
correta formação e desenvolvimento da face. A ausência na coalescência desses
processos anatômicos resulta no aparecimento dos vários tipos de fissuras devido à
ruptura da integridade do lábio e/ou do palato (Paranaíba et al., 2013). Este processo é
complexo e ocorre sob o controle de mecanismos genéticos (Koukskoura et al., 2011).
As fissuras orais não sindrômicas são caracterizadas por áreas de
descontinuidades no lábio e/ou palato, representando as anomalias congênitas mais
frequentes na região craniofacial dos seres humanos (Dixon et al., 2011; Marazita et
al.,2012; Haet al., 2015). Para a formação do lábio superior e maxila, as proeminências
maxilares passam por crescimento medial, produzindo fusão de proeminência nasal com
proeminências maxilares (Sarmiento et al., 2017).
Para identificar as fissuras labiopalatinas não sindrômicas (FL/PNS), em função
das suas diferentes apresentações clínicas, as mesmas são tradicionalmente divididas em
fissuras de lábio (FLNS), fissuras de lábio e palato (FLPNS) e fissuras palatinas (FPNS)
(Spina et al.,1972). As FL/PNS afetam aproximadamente 1 em cada 500 a 2.500
nativivos, em todo o mundo (Dixon et al., 2011). Martelli et al. (2012) mostraram a
prevalência para cada tipo de fissura, sendo a de maior ocorrência as FLPNS (53,4%),
seguida, respectivamente, das FLNS (26,2%) e das FPNS (20,49%). Também
mostraram que as FPNS ocorrem com maior frequência no sexo feminino, enquanto as
FLPNS e FLNS predominam no masculino. Essas malformações podem levar a
prejuízos na audição, fonação e aparência afetando psicologicamente o indivíduo e sua
família (Fan et al., 2018). Eslami et al. (2013) mostraram que ocorre um aumento na
frequência de ansiedade, baixa autoestima e depressão nos pacientes com fissuras orais
não sindrômicas.
16
Silva et al. (2018) observaram que clinicamente as FLPNS comprometem a
saúde bucal, levando a dentes ausentes ou malformados e podem comprometer a higiene
oral. Küchler et al. (2011) mostraram que a ocorrência de fissuras orais não sindrômicas
e o desenvolvimento dos germes dentários tem uma estreita relação embrionária com
relação à cronologia e posição anatômica, pois os eventos críticos dos dentes, lábio e
palato ocorrem quase que simultaneamente, sugerindo que fissuras orais não
sindrômicas e alterações dentárias tem como determinante, o fator genético. Stahl
(2006) relata que a formação da FLPNS interfere no desenvolvimento dentário, o que
adiciona risco de ocorrência de anomalias dentárias nos indivíduos com esta
malformação congênita. Esses achados podem representar um marcador clínico para
definição de subfenótipos das fissuras (Sá et al., 2016).
Sabe-se que a hipodontia é mais frequente no lado da fissura comparado ao
contralateral, e também à prevalência de hipodontia aumenta com a gravidade da fissura
(Suzuki et al., 2017). As anomalias dentárias podem ser atribuídas à própria fissura ou
ao início da correção cirúrgica dos defeitos, além de estarem relacionadas com a
extensão da fissura (Jamal et al., 2010). Melo Filho et al. (2015) e Cakan et al. (2018)
mostraram que as anomalias dentárias possuem maior frequência em indivíduos
fissurados, podendo ser de número, tamanho e forma. Em pacientes com fissuras orais
não sindrômicas, a anomalia dentária mais frequente é agenesia, seguida por
supranumerário e microdontia (Paranaíba et al., 2013; Sá et al., 2016). Letra et al.
(2007) relataram que o desenvolvimento do germe dentário e a incidência de FLPNS
tem relação embrionária. As anomalias dentárias podem representar marcadores clínicos
adicionais para fissuras orais não sindrômicas , mostrando um fator genético comum
entre as condições (Chu et al., 2016).
A partir da constatação de que anomalias dentárias são mais prevalentes em
pacientes com fissuras orais não sindrômicas , é importante observar se existe ou não
diferença nas anomalias dentárias, seja de número ou forma, e na sua prevalência
quando se compara as fissuras labiais completas (envolvendo lábio e rebordo alveolar) e
fissuras incompletas (que afetem somente o lábio, sem envolvimento do rebordo
alveolar).
17
Artigo 1*
Maternal consumption of caffeine and second-hand tobacco smokers risk factor
for the development of oral clefts
Rodrigo Soares de Andrade1, Daniella Reis Barbosa Martelli2, Alison José Martelli1,
Adriana Boeri Freire Tamburini3, Mário Sérgio Oliveira Swerts3, Verônica Oliveira
Dias2, Hercílio Martelli Júnior1,2
1Department of Oral Diagnosis, School of Dentistry, State University of Campinas,
FOP-UNICAMP, Piracicaba, São Paulo, Brazil.
2School of Dentistry, State University of Montes Claros, Unimontes, Minas Gerais
State, Brazil.
3Center for Rehabilitation of Craniofacial Anomalies, Dentária School, University of
José Rosario Vellano, Alfenas, Minas Gerais, Brazil.
Correspondence: Rodrigo Soares de Andrade,
Oral Pathology, FOP-UNICAMP, Av. Limeira, 901 - Areião, Piracicaba – São Paulo,
Brazil.
CEP: 13414-018 - Phone: +55-19-999665488
E-mail: [email protected]
18
ABSTRACT
Objective: The aim of the present study was to evaluate the possibility of
environmental factors such as caffeine, folic acid, multivitamin complexes, alcohol and
tobacco (secondhand smoking) to act as oral clefts development risk factors.
Methods: This case-control study included 409 mothers, 132 with children presenting
oral clefts (case) and 277 not presenting oral clefts (control). In both groups, children
were 0-2 years-old. A questionnaire was applied to each mother to evaluate their food
consumption and habits during pregnancy’s first trimester.
Results: Eighty-four (63.63%) mothers in the case group reported secondhand smoking
and 5 (3.78%) reported alcohol consumption (p=0.797). Mothers more exposed to
passive smoking were twice as likely to have a child with an oral cleft. Caffeine
consumption was observed in 126 (95.45%) of the mothers in the case group and in 246
(88.80%) in the control group (OR=3.20; 95% CI=1.21-8.43) (p=0.013). Amongst case
group mothers, 107 (84.25%) consumed more than 500 mg of caffeine weekly, leading
to an observed in 34% increase in the frequency of oral cleft in their children. Folic acid
supplementation was observed in 116 (87.87%) cases and 271 (97.83%) were control
(p<0.001). As for ferrous sulfate administration, 114 (86.36%) in the case group and
271 (97.83%) in the control group (p=0.617).
Conclusions: The results suggest a direct relationship between both secondhand
smoking and caffeine consumption to nonsyndromic oral clefts, while folic acid, ferrous
sulfate and multivitamins consumption could be considered protective factors against
nonsyndromic oral clefts occurrence.
Keywords: Caffeine, secondhand tobacco, folic acid, non-syndromic oral cleft, alcohol.
19
Objetivo: Investigar fatores ambientais como cafeína, ácido fólico, complexos
multivitamínicos, álcool e tabaco (tabagismo passivo), que têm sido descritos como
fatores de risco para o desenvolvimento das fissuras orais.
Métodos: Este estudo caso-controle incluiu 409 mães, sendo 132 com crianças com
fissura oral (caso) e 277 com crianças sem fissura oral (controle). Em ambos os grupos,
as crianças tinham idade entre 0 e 2 anos. Um questionário foi aplicado a cada uma das
mães para investigar seus hábitos e consumo de alimentos durante o primeiro trimestre
da gestação.
Resultados: Oitenta e quatro (63,63%) mães do grupo caso relataram tabagismo
passivo e 5 (3,78%) relataram uso de álcool (p=0,797). As mães expostas ao tabagismo
passivo apresentaram duas vezes maior probabilidade de ter um filho com fissura oral.
No grupo caso, 126 mães (95,45%) consumiram cafeína, e no grupo controle 246
(88,80%) (OR=3,20; 95% CI=1,21-8,43) (p=0,013). Entre as mães do grupo caso 107
(84,25%) consumiam semanalmente mais de 500 mg de cafeína, aumentando em 34% a
frequência de fissuras orais nos filhos. A suplementação com ácido fólico foi observada
em 116 (87,87%) casos e 271 (97,83%) do controle (p<0,001). Em relação ao consumo
de sulfato ferroso, 114 (86,36%) no grupo caso aderiram e 271 (97,83%) no controle
(p=0.617).
Conclusões: Os resultados sugerem uma relação direta entre tabagismo passivo,
consumo de cafeína e fissuras orais não sindrômicas. O uso de ácido fólico, sulfato
ferroso e multivitaminas são fatores protetores para a ocorrência de fissuras orais não
sindrômicas.
Palavras-chave: Cafeína, tabagismo passivo, ácido fólico, fissura oral, álcool.
20
INTRODUTION
Nonsyndromic oral clefts are amongst the most prevalent congenital abnormalities in
humans (1) and is a substantial morbidity and mortality source worldwide with
significant social impact (2). In 70% of cases, the cleft lip and/or palate present as non-
syndromic (NSCL/P), that is, without structural malformations in other organs and
without behavioral and cognitive alterations (3,4). Based on epidemiological features
and embryologic timing, NSCL/P is traditionally divided in cleft lip (NSCL), cleft lip
and palate (NSCLP) and cleft palate (NSCP) (5).
The incidence of NSCL/P is approximately 1 in 500-2,500 live births, varying
according to location, ethnicity and socioeconomic status of the population studied (1).
In Brazil, studies on the incidence of NSCL/P are scarce and vary considerably.
According to the Brazilian surveys, the incidence of NSCLP ranges from 0.19 to 1.54
for every 1,000 live births (6,7).
The etiology of NSCL/P is attributed to genetic, epigenetic and environmental
factors interplay but the exact interactions are poorly known (1,8). This lack of
knowledge is probably a reflection of the wide diversity of molecular mechanisms
related to facial embryogenesis involving multiple genes and the influence of
environmental factors (9-12). Amongst the major environmental risk factors for the
development of NSCL/P are medication use, smoking, maternal diet and vitamin
supplementation, particularly during pregnancy’s first trimester (13-15).
The aim of the present study was to evaluate the possibility of environmental
factors such as caffeine, folic acid, multivitamin complexes, alcohol and tobacco
(secondhand smoking) to act as oral clefts development risk factors
21
MATERIALS AND METHODS
This case-control study was conducted in the Center for Rehabilitation of Craniofacial
Anomalies, State of Minas Gerais, Brazil, where 132 mothers were recruited. They were
being followed as well as their children to be included in the case group, with children
up to 24 months of age with NSCL/P and 277 mothers to the control group of children
without craniofacial randomly obtained from the Pediatric Dentistry Clinic at the
University. Patients were recruited from February 2018 to February 2019. The sample
was chosen for convenience, that is, all patients during the chronological period of the
study were included.
Case group included mothers with up to 24 months-old children diagnosed with
NSCL/P without gender distinction, while control group included children without any
skull alterations or syndrome. Each control will be paired with a case of gender and age
equal within 3 months. All children with NSCL/P were evaluated by a multidisciplinary
team of specialists to exclude the involvement of other alterations or syndromes.
The sample of the studied population was selected during one year of follow-up,
based on our primary exposure of interest, which is secondhand smoking,
supplementation with folic acid, ferrous sulfate and multivitamin complexes, in addition
to caffeine and alcohol consumption during the first trimester of pregnancy. As
reference values, the consumption of the studied variables was taken into consideration.
The mothers answered a demographic questionnaire with characteristics, habits and
exposures during pregnancy. The questionnaire included questions about consumption
of caffeine-containing beverages (coffee, soft drinks with caffeine, energetic beverages
with caffeine, and dietary supplements of physical performance) during the first 3
months of pregnancy. For each drink, there was 1 question with 5 response categories:
none, number of 100ml cups per week on average (without specifying cup size),
22
consumption up to 500 mg weekly, from 500 mg to 1000 mg weekly and above 1400
mg weekly (13-15). Questionnaire application method was standardized for mothers
from both groups about their food and nutritional supplementation, as well as their
habits during pregnancy, focusing on the first trimester of the gestational period.
The risk of delivering offspring with an NSCL/P was estimated by odds ratio
with a 95% confidence interval in non-conditional logistic regression models. All
beverages were summarized and analyzed in the same way as follows. "Cups per week"
was calculated from the reported number of cups consumed per day, per week (divided
or multiplied by 7). Women whom reported consuming less than 1 cup per week were
categorized as zero consumption. The variable “concentration in milligram per week”
was analyzed as a continuously variable. Finally, categories were created: 0-100mg per
week (reference category), 100mg at 100mg to the level above 1400mg per week (13-
15). Trends between categories were evaluated, with zero as the reference. An estimate
of caffeine from all sources was computed from data on coffee, soft drinks with
caffeine, energetic beverages with caffeine, and dietary supplements of physical
performance.
The caffeine content was estimated as 100mg per cup of coffee, 40mg per cup of
soft drinks with caffeine, 80 mg per cup of dietary supplements of physical
performance, 70mg per thermogenic capsule, drink based on the values of the
international health authorities (http://www.matportalen.no/Emner/Gravide). The risk of
NSCL/P was assessed by increasing the daily caffeine intake by 100mg (continuous
variable) and categories >100mg, increasing every 100 mg up to a proportion greater
than 1000 mg of caffeine (13).
Adjustments were made to potential confounders (factors associated with
NSCL/P in other studies, most of which were also associated in our study), namely, A
23
(quartiles), dietary folate (factors associated with NSCL/P, most of which were also
associated in our study), folic acid supplementation (yes or no), use of vitamin
supplements (yes or no), alcohol consumption in early pregnancy (yes or no), smoking
(passive only, yes or no> 15 min per day). Evaluations of possible interactions with
coffee intake were performed comparing folic acid supplementation and smoking.
When evaluating the effects of coffee or tea separately, we adjusted to the categorized
as cups per week
A logistic regression test was performed to calculate gross and adjusted OR,
corresponding 95% CI and p values between exposures of interest and NSCL/P.
Because of the number of patients, all variations of NSCL/P were combined for our
analysis. We also performed subgroup analyzes replicating our conditional logistic
regression analysis among those with NSCL/P.
All analyzes were performed using the IBM SPSS Statistics (SPSS®, version
20.0; SPSS Inc., Chicago, Illinois, USA) with a significance level of 5%.To address the
impact of uncontrolled bias, p-value, a new measure, was calculated that assesses how
far the actual risk estimate can be given without conflicts and bias in a study in a
sensitivity analysis.
RESULTS
As for deleterious habits such as secondhand tobacco smoke and alcohol consumption
in the first trimester of pregnancy, 84 (63.63%) mothers in the case group reported
being secondhand tobacco smokers during the first trimester of pregnancy (p<0.001;
OR=6.46 CI=4.09-10.20) and 5 (3.78%) mothers stated the frequent use of alcohol, and
we found a significant difference between groups (OR=0.86; 95% CI=0.30-2.52)
24
(p=0.797). In the control, 59 (21.29%) mothers were secondhand tobacco smokers and
12 (4.33%) consumed alcohol during the first trimester of pregnancy (Table 1).
In the case group, 126 mothers (95.45%) frequently ingested caffeine-containing
products during pregnancy, and 277 mothers in the control group (88.80%) (OR=3.20;
95% CI=1.21-8.43) (p=0.013). Of the 127 mothers in the group who consumed caffeine,
83 (65.3%) consumed more than 1,000 mg per week, and of the 246 mothers in the case
group, 124 (50.40%) consumed more than 1,000 mg per week (Table 2). Of the 132
mothers of children with NSCL/P interviewed, 8 (6.06%) reported consanguinity of
their son because they were married to first-degree relatives (p≥0.065). Of those, 18
(13.63%) reported a history of NSCL/P in the family. When we observed folic acid
supplementation in the first trimester of pregnancy, 116 (87.87%) mothers of NSCL/P
were used correctly and 271 (97.83%) were case group (OR=6.23; 95% CI=2.37-16.32)
(p<0.001).
When questioned about consumption of pre-gestational vitamins such as ferrous
sulfate, 114 (86.36%) mothers of the case group had supplementation and 271 (97.83%)
of the mothers in the control group (OR=7.13; 95% CI=2.75-18.43. About changing
eating habits, 109 (82.57%) mothers in the case group reported having changed their
diet in the first trimester of pregnancy, eating healthier foods, rich in vitamins and iron
while 223 (80.50%) of the control mothers also reported having improved their eating
pattern (OR=0.87; 95% CI=0.50-1.49) (p≥0.61) (Table 1).
DISCUSSION
Some studies have raised the possibility that habits of mothers during the gestational
period are directly related to the increased possibility of developing NSCL/P (15,16). In
this study we analyzed the mothers in prenatal care comparing the consumption of
25
dietary supplements related to the good development of the fetus, such as folic acid and
ferrous sulfate (17). These are mainly related to fusion of maxillary processes and
vision. The results of the present study corroborate Taghavi et al. (2012), who also
observed that mothers of children without NSCL/P do prenatal care correctly and follow
better supplementation diet than mothers of children with NSCL/P (18).
Secondhand tobacco smoke is considered deleterious when the mother inhaled
some nicotine-based agent over 15 minutes daily, a parameter established by the WHO
in 2003 (https://www.who.int/tobacco/research/secondhand_smoke/en/). Although
mechanisms through which tobacco increases craniofacial abnormalities in infants are
not precisely understood, they most likely involve the interaction between smoking and
polymorphism of susceptible genes (19).
Inhaled CO2 suffers dissociations within the mother's body releasing free
radicals that have teratogenic effects, wherein the vasoconstrictive action of nicotine can
cause reduction of utero-placental blood flow. Carbon monoxide binds to hemoglobin
so that less oxygen is available to the placenta (20). In addition, endothelial injury
caused by tobacco increases the rupture of placental neovessels, leading to reduced fetal
blood supply and hypoxia, which probably results in abnormal facial morphogenesis.
Therefore, the sum of exposure to toxins, hypoxia and cellular ischemia results in
craniofacial abnormalities (19). Little et al., (2004) analyzed 24 mothers on the smoking
habits of pregnant women and the occurrence of NSCL/P in their children (20). There
was a statistically significant association between relative maternal smoking and
NSCL/P (relative risk=1.34). This form corroborates the evidence of this study, in
which 84 of the 132 mothers with cleft children reported being exposed to smoking
(63.6%), while 59 of the 277 mothers in the control group were exposed (21.3%),
showing an increased risk of children with cleft palate from smoking mothers (21).
26
Although here is no exact known mechanism through which coffee ingestion can
increase the risk of NSCL/P (14), it seems to involve effects on homocysteine.
Evidences that hyperhomocysteinemia of the mother may be linked to the increased risk
of clefts are also corroborated (16). Coffee ingestion increases homocysteine plasma
concentration (21) as well as secondhand tobacco smoke (22) raising the risk of
spontaneous abortions, chromosomal anomalies, multiple congenital aberrations (23)
and mainly low birth weight (24). Supplements of folic acid and ferrous sulfate are
usually associated with lower risk of NSCL/P (25), and such supplements also reduce
plasma homocysteine (21), which justifies the supplementation of these nutrients during
the pre-gestational period. In the present study, the association between high coffee
intake and NSCL/P was higher in mothers of the case group and also on the use of folic
acid and ferrous sulfate supplements that were lower than in the control (87.00%) and
case (97.00%) groups respectively.
Similarly, mothers that consumed coffee in large quantities (>500mg/week) are
more pre-exposed to chemicals per session than those who do not have NSCL/P.
However, alcohol consumption was not statistically significant between groups. Even if
adjusted for possible confounding factors, the possibility of residual bias by known
factors cannot be ruled out. Although we have no control over the diet, there was no
evidence of a worse diet with iron-rich foods.
Considering that the fusion of the maxillary processes occurs relatively late in
the first trimester (26), it is possible that the reported coffee intake is higher than the
actual consumption in the crucial phase of embryonic development, as over time coffee
intake decreases due to nausea and vomiting. A meta-analysis of three studies on
maternal coffee consumption and NSCL/P found a slight increase in the risk of oral
clefts (27), corroborating our study, which showed that caffeine consumption increases
27
the likelihood of oral clefts development. All three studies used 0 cups of 100ml as a
reference category, as recommended by our methodology. In the study by Kurppa et al.
(1983), drinking more than 4 cups of coffee by day was not associated with higher risk
of NSCL/P (OR=1.0; 95% CI= 0.6-1.6) (28), while in the study by McDonald et al.
(1992), more than 3 cups of coffee per day resulted in an adjusted odds ratio of 1.4
(95%; CI=0.7-2.7) (29), in this study it was observed that caffeine consumption by
mothers of the case group was higher, and mothers of children with NSCL/P consumed
more caffeine. In the study by Billie et al (2007), with 134 mothers, caffeine
consumption was associated with an increased risk of NSCL/P (30), which was
corroborated by the results of this study.
Concentration parameters of caffeine in different sources, portion sizes and
methods of preparation for coffee should be considered to estimate caffeine ingestion.
However, the methodology (13-15) has been extensively validated on the basis of a
subpopulation, using various biomarkers as reference measures. The correspondence
between food diaries was particularly high for coffee and cola-based soda, which are the
main caffeine sources in our population (31,32).
High consumption of caffeine in the first trimester of pregnancy by the mother
increased the risk of nonsyndromic oral cleft in the child, as a unsatisfactory prenatal
leads to ineffective nutritional supplementation, thus becoming an important risk factor.
Harmful habits such as alcohol consumption and especially the inhalation of active
tobacco agents, even if passively, may harm the fetus.
We can observe limitations in this study, some of them may not have been
standardized, or mistakenly answered by mothers of both groups, but to minimize these
biases the questionnaire was applied in a reserved place, giving more privacy and
28
confounding factors were applied to statistical tests. Due to the rarity of finding children
with oral cleft of up to 24 months, the number of research participants was also limited.
In summary, our results suggest a relationship between both secondhand
smoking and caffeine consumption to nonsyndromic oral clefts, while folic acid, ferrous
sulfate and multivitamins consumption could be considered protective factors against
nonsyndromic oral clefts occurrence.
29
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L.B.; Cnattingius, S. E_ect of ca_eine exposure during pregnancy on birth
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34
Table 1 - Values referring to the variables evaluated in this study, which may have a direct relationship with the development of nonsyndromic oral clefts.
Case
n = 132 (%)
Control
n= 277 (%)
p value OD ratio (95%)
Gender
Female
Male
57 (43.18)
75 (56.82)
129(46.57)
148(53.42)
0.520
Reference
1.147 (0.755-1.741)
Consanguinity
No
Yes
127 (96.22)
5 (3.78)
274 (98.92)
3 (1.08)
0.065
R e fe ren c e
3.596(0.846-15.279)
NSCL/P history No
Yes
114 (86.36)
18 (13.64)
270 (97.47)
7(2,52)
<0.001
Reference
1.188(1.097-1.286)
Folic acid Yes
No
116 (87.88)
16 (12.12)
271 (97.83)
6 (2.16)
<0.001 R efe ren c e
6.230 (2.378-16.322)
Nutritional
supplementation
(iron)
Yes
No
109 (82.58)
23 (17.42)
223 (80.50)
54 (19.50)
0.617 Reference
0.871 (0.508-1.494)
Vitamin
supplementation
Yes No
114 (86.37) 18 (13.63)
271 (97.83) 6 (2.17)
<0.001 R efe ren c e 7.132 (2.759-18.431)
Second-hand
tobacco smoke > 15
min
No
Yes
48 (36.37)
84 (63.63)
218 (78.70)
59 (21.30)
<0.001 Reference
6.466 (4.096-10.208)
Alcohol
consumption
No
Yes
127 (96.22)
5 (3.78)
265 (95.66)
12 (4.34)
0.797 Reference
0.869 (0.300-2.521)
35
Exposure to
chemicals
No
Yes
127 (96.22)
5 (3.78)
270 (97.47)
7 (2.53)
0.480
Reference
1.419 (0.743-4.877)
Caffeine
consumption
No
Yes
5 (3.78)
127 (96.22)
31 (11.19)
246 (88.81)
0.013
Reference
3.201 (1215-8.431)
Medicine
Consumption
No
Yes
47 (35.61)
85 (64.39)
187 (67.50)
90 (32.50)
<0.001
Reference
3.758 (2.430-5.812)
36
Table 2 - Reference values of caffeine consumption of the case and control groups
Case
n= 127
(96.2%)
Control
n= 246
(88.8%)
p value OD ratio (95%)
>100<= 500mg 20 (15.74%) 44 (17.88%) 0.070 2.727(0.922-8.066)
>500 mg 107 (84.25%) 203 (82.52%) 0.021 3.163 (1.193-8.387)
Reference: (0 <=100mg)
37
Artigo 2*
Dental anomalies in Brazilian patients with non-syndromic isolated cleft lip with or
without alveolar ridge involvement
Rodrigo Soares de Andrade1 , Alison José Martelli1 ,Adriana Freire Boeri Tamburini2 ,
Daniella Reis B. Martelli3 , Letízia Monteiro de Barros2 , Hercílio Martelli Júnior2, 3
1Department of Oral Diagnosis, School of Dentistry, State University of Campinas, FOP-
UNICAMP, Piracicaba, São Paulo, Brazil.
2Center for Rehabilitation of Craniofacial Anomalies, Dental School, University of José
Rosario Vellano, Alfenas, Minas Gerais, Brazil.
3Dental School, State University of Montes Claros, Unimontes, Minas Gerais State, Brazil.
Correspondence: Rodrigo Soares de Andrade, Oral Pathology, FOP-UNICAMP.
Avenue Limeira, 901 - Areião, Piracicaba – São Paulo, Zip code: 13414-018
Piracicaba – São Paulo – Brazil.
Phone: +55-19-999665488, E-mail: [email protected]
38
Abstract
Background: Some studies have demonstrated a high prevalence of dental anomalies in
patients with nonsyndromic cleft lip and/or palate (NSCL/P). Few studies have evaluated
these anomalies in patients’ cleft lip with or without alveolus (NSCL±A).
Objective: The aim of the current study was to investigate the prevalence of dental anomalies
in a group of Brazilian patients with NSCL±A isolated.
Methods: Of the 1,528 patients with oral clefts treated at the Centre for Rehabilitation of
Craniofacial Anomalies, 154 (10.07%) subjects with NSCL±A were included in this study.
Panoramic and periapical radiographs were performed and dental anomalies of size, number
and shape were considered.
Results: Of the 154 patients, 87 (56.50%) were male and 67 (43.50%) female. Ninety
(58.40%) patients presented NSCL+A and 64 (41.60%) without NSCL-A. Of these 70
subjects with dental anomalies, 44 (62.85%) were male and 26 (37.14%) female (p=0.146)
and, 98 anomalies were observed, being 66 (67.34%) in the NSCL+A group and 32 (32.65%)
in the NSCL-A. Dental agenesis was the most prevalent anomaly, representing 56.12%
(n=55). Supernumerary teeth representing 29.59% (n=29) of all anomalies and microdontia
representing 12.24% (n=12).
Conclusion: In both groups of NSCL, complete and incomplete, the most common dental
anomalies were the agenesia followed by supranumerary teeth, followed for microdontia.
NSCL+A was the group with the highest number of dental anomalies.
Keywords: Cleft lip, dental anomalies, oral clefts, taurodontism, oligodontia.
Introduction
39
Nonsyndromic oral clefts are common congenital birth defects in humans
characterized by defective fusions of the facial processes, occurring in 1 in 500-2,500 live
births worldwide.1 In Brazil, considering the geographic variation, the prevalence varies
between 1:650 to 1:2,700 live birth.2,3 About 70% of cases occur as a nonsyndromic form
[nonsyndromic cleft lip and/or cleft palate (NSCL/P)], and the remaining 30% are associated
with Mendelian disorders or chromosomal, teratogenic and sporadic conditions.4
According to the anatomical site, NSCL/P are commonly classified in cleft lip with or
without alveolus (NSCL±A), cleft lip and palate (NSCLP) or cleft palate only (NSCP).5,6 A
complex and heterogeneous etiology with interplay among environmental, genetic and
epigenetics factors has been reported, but the factors associated remain partially defined.7,8
The human face and the upper jaw begin their development in the fourth week of
gestation by mesenchymal migration and fusion of the primitive facial elements: the paired
medial nasal processes and maxillary processes. As the medial nasal processes fuse with each
other, they form the anterior portion of the upper jaw – the pre-maxilla – from which the
dental incisors originate, and also the medial portion of the upper lip (philtrum) and the
primary palate. An isolated soft tissue cleft lip is caused by a disturbance in the
approximation, fusion or mesenchyme penetration through the epithelial membrane
surrounding the medial, and lateral nasal processes and the maxillary process.9,10
The clinical term cleft lip is often used in the literature to describe both isolated soft
tissue cleft lip and cleft of the lip and the alveolar process; both originate in the embryonic
formation of the primary palate. However, the two are significantly different from one another
from a clinical point of view.10
Studies involving different populations show that patients with NSCL/P have a higher
prevalence of dental anomalies in relation to the general population.6,11-14 Although dental
40
anomalies are more prevalent in the NSCL/P, few studies have investigated these alterations
in the isolated NSCL±A.6,10 Thus, the aim of the current study was to investigate the
prevalence of dental anomalies in a group of Brazilian patients with NSCL±A isolated.
Material and methods
This cross-sectional, epidemiological and observational study. All participants were
recruited from the same institution (Centre for Rehabilitation of Craniofacial Anomalies).
Records from 2013 to 2018 to patients with NSCL/P were reviewed; those without complete
dental history, panoramic and periapical radiographs were not included in the final analysis.
Similarly, patients with a history of dental extraction, who had previous orthodontic treatment
or were at a young age (12 years old), due to the inability to accurately identify all anomalies,
with associations or syndromes, with NSCP or NSCLP were excluded as well. Were included
only patients with NSCL±A. Dental anomalies in third molar were also not considered in this
study. Of the 2.484 patients, only 154 with NSCL±A remained in the study, after the
application of the exclusion criteria.
All patients were carefully evaluated by experts of Center for Rehabilitation of
Craniofacial Anomalies. In order to eliminate inter examiner differences, dental anomalies
were classified by a single calibrated examiner. The determination of cleft phenotype was
based on the description in the NSCL-A files and confirmed by examination of the dental
casts and radiographs.11,14
All of the subjects were from Minas Gerais, Brazil, where there is an admixed
population of Europeans (mostly from Portugal and Italy) and Africans, with a small
percentage of native Brazilian Indians.15 All patients presented similar ethnicities and social
culture and were assisted by the Brazilian Public Health System.The information collected
41
was stored in a database and analysed using the statistical program SPSS® version 24.0
(Statistical Package for Social Sciences for Windows, Inc., USA). Comparisons were made by
cross-tabulation and standard chi-square test, with statistical significance set at p≤.05. This
study was approved by the Research Ethics Committee (#2.746.400).
Results
Of the 1,528 patients treated at the Centre for Rehabilitation of Craniofacial
Anomalies, 154 (10.07%) subjects with NSCL±A were included in this study. Of the 154
patients, 87 (56.50%) were male and 67 (43.50%) female. Ninety (58.40%) patients presented
NSCL+A and 64 (41.60%) without NSCL-A. Of the 154 patients with NSCL±A, 84 (54.55%)
had no dental anomalies and 70 (45.45%) (p=0.353) patients presented 98 dental anomalies in
total. Of the 70 patients with dental anomalies, 44 (62.85%) were male and 26 (37.14%)
female (p=0.146).
Among the 70 patients with dental anomalies, 98 changes were observed, being 66
(67.34%) in the NSCL+A group and 32 (32.65%) in the NSCL-A (Table 1 and 2). Dental
agenesis was the most prevalent anomaly, representing 56.12% (n=55) of the 98 observed. In
the NSCL+A group, the agenesis accounted for 56.06% (n=37) and in the NSCL-A occurred
in 56.25% (n=18) of the cases. It is emphasized that of 55 dental agenesis in both groups
studied, 46 (83.63%) affected the lateral incisors.
Supernumerary teeth were the second most common dental anomalies, representing
29.59% (n=29) of all anomalies. In the NSCL+A group, the supernumerary teeth accounted
for 30.30% (n=20) and in the NSCL-A occurred in 39.13% (n=9) of the cases. In both groups,
the highest occurrence of supernumerary teeth was observed in the lateral incisors (n=14), as
well as the dental agenesis.
42
Subsequently, the third most observed dental anomaly was microdontia, representing
12.24% (n=12) of all dental anomalies. In the NSCL+A group, the microdontia accounted for
12.12% (n=8) and in the NSCL-A occurred in 12.50% (n=4) of the cases. In the NSCL+A
group, the occurrence of microdontia was also more observed among the lateral incisors
(n=6). In addition to the described dental anomalies, it was observed the occurrence of one
case of taurodontism (NSCL+A group) and one case (NSCL-A group) of gemination.
Discussion
The NSCL is an anatomical form of the cleft, implies less mesenchymal tissue, allowing
greater incidences of crown malformation of central and lateral incisors, supernumerary
reflected in sufficient space for the dichotomous development of the dental root.16 NSCL is
caused by a disturbance in the mesenchymal fusion of the medial and lateral nasal processes
with the maxillary process.17 The process of tooth formation involves from the dental blade to
the mesenchymal tissue around it (also called the dental papilla), and this mesenchymal tissue
becomes the dentinofibrous complex, which is the main topografic influence of the dental
tissue.18
The isolated soft tissue NSCL without evidence of any type of bone involvement is
a relatively rare phenomenon.19,20 Sekhon et al. (2009) reported a prevalence of 2.1%, equally
distributed among male and female patients21, different from this study, where we found a
higher prevalence in male patients. These findings corroborate the findings of many studies
that have emphasized sexual dimorphism in oral clefts.22-25
The reason for the high prevalence of dental abnormalities is not fully understood, but
the assumption is that etiology is a prenatal outcome that interacts with an altered genotype.26
Dental agenesis is largely genetically determined and transmitted by autosomal dominant
43
inheritance, with incomplete penetration and variable expression.27,28 The agenesis of one or
more permanent teeth is the most common congenital anomaly found in humans, affecting
about 20% of the world population29, which is still present in NSCL patients, as we observed
in this study where agenesis was the most frequent dental anomaly (78.5%).
Oral clefts in humans are usually associated with delayed development of teeth and
abnormalities in tooth number, size and shape, both on the cleft side and on the non-cleft
side.30 Rawashdeh and Bakir (2007) showed a that reduction of tooth size occurred in all types
of permanent teeth and between early and late teeth in patients with cleft compared to
controls31, which in this work we observed the presence of microdontia in 8 patients with
NSCL+A, more frequent in upper lateral incisors, corroborating with others studies29,31,32,
who also found a higher frequency of microdontia in upper lateral incisors.
The results of this study support this assumption, that the oral cleft patient has a
higher prevalence of almost all types of dental anomalies and was evaluated in this study and
found to be more accurate, the group more affected by the NSCL+A compared to the NSCL-
A group, with the normal population32. In this study, dental anomalies were analyzed only in
the permanent dentition, indicating a high percentage of supernumerary teeth about 41.4%.
The reported prevalence of supernumerary teeth in the general population for permanent
dentition ranges from 0.1% to 3.8%33, in patients with NSCL+A, twice the frequency (28.5%)
when compared to patients with NSCL-A (12.8%).
The knowledge of dental anomalies, such a dental agenesis or supernumerary teeth,
is fundamental for oral treatment planning, since such anomalies can lead to edentulous
spaces in the maxillary arch that must be closed by orthodontic movements, prostheses or
implants.30 Microdontia was observed in 12.12% (NSCL+A) and 12.50% (NSCL-A) in this
sample. In this sample of microdontia was observed mainly in the upper lateral incisors. It has
44
been reported that taurodontism is associated with certain syndromes and disorders of dental
development33. The results of the present study showed that the general prevalence of
taurodontism was 1.42% for individuals with NSCL and it was found only in patients with
NSCL+A. However, when we compare this prevalence with normal and healthy individuals
(0.1%)31 it seems that this result is in agreement with Burzynski & Escobar (2003), Aizenbud
et al. (2011) and Mangione et al. (2018) that found that taurodontism occurred more
frequently in individuals with oral clefts.9,17,28
Regarding the differences in the prevalence of dental anomalies between NSCL+A
and NSCL-A, dental agenesis, supernumerary and microdontia, were significantly higher in
individuals with NSCL+A. This is probably due to multiple anatomical, genetic and
environmental factors, mesenchymal deficiency, and the direct effect of cleft on the
primordial tissues.19,25,28,29,32
It can be concluded from the results of this study that the frequency of certain dental
anomalies is strongly related to the phenomena of NSCL is a correlated to have the
involvement of the component tissues of the alveolar ridge. Sample size was limited due to
the rarity of isolated NSCL and the limited number of patients. We also observed as a
limitation of the study that the sample was established geographically in the same region.
More evidence to establish the exact nature of this relationship with each dental anomaly is
necessary.
Acknowledgments
The Minas Gerais State Research Foundation – Fapemig, Brazil and National Council for
Scientific and Technological Development – CNPq, Brazil
45
Conflict of interest
None declared.
Ethical approval
Approval for this retrospective study was granted by the research ethics committee of the Jose
do Rosário Vellano University and the Piracicaba School of Dentistry/University of Campinas
(Registration number: 3.574.351). All clinical data were anonymized, and all potential patient
identifiers were removed after the return of the database surveys.
Patient consent
Not required.
ORCID
Rodrigo Soares de Andrade http://orcid.org/ 0000-0001-6114-0929
Alison José Martelli http://orcid.org/0000-0002-5361-9240,
Adriana Freire Boeri Tamburini http://orcid.org/0000-0002-0153-3875
Daniella Reis B. Martelli http://orcid.org/0000-0002-3979-7497
Letízia Monteiro de Barros http://orcid.org/0000-0002-5181-6224
Hercílio Martelli Júnior http://orcid.org/0000-0001-9691-2802
46
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49
Table 1- Dental anomalies in patients nonsyndromic cleft lip with alveolus (NSCL+A).
Maxillary Arch
Agenesis Supernumerary Taurodontism Microdontia Gemination Fusion Concrescence
Tooth R L R L R L R L R L R L R L
Central incisor 0 0 4 1 0 0 1 1 0 0 0 0 0 0
Lateral incisor 19 10 5 3 0 0 5 1 0 0 0 0 0 0
Canine 0 1 1 2 0 0 0 0 0 0 0 0 0 0
1st premolar 1 2 0 2 0 0 0 0 0 0 0 0 0 0
2nd premolar 0 1 0 1 0 0 0 0 0 0 0 0 0 0
1st molar 0 0 0 0 0 1 0 0 0 0 0 0 0 0
2nd molar 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Mandibular Arch
Central incisor 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Lateral incisor 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Canine 1 0 1 0 0 0 0 0 0 0 0 0 0 0
1st premolar 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2nd premolar 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1st molar 1 0 0 0 0 0 0 0 0 0 0 0 0 0
2nd molar 1 0 0 0 0 0 0 0 0 0 0 0 0 0
*Right (R) and Left (L)
50
Table 2 - Dental anomalies in patients nonsyndromic cleft lip without alveolus (NSCL-A).
Maxillary Arch
Agenesis Supernumerary Taurodontism Microdontia Gemination Fusion Concrescence
Tooth R L R L R L R L R L R L R L
Central incisor 1 0 0 0 0 0 0 0 0 0 0 0 0 0
Lateral incisor 7 10 4 2 0 0 0 1 0 1 0 0 0 0
Canine 0 0 0 3 0 0 0 1 0 0 0 0 0 0
1st premolar 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2nd premolar 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1st molar 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2nd molar 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Mandibular Arch
Central incisor 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Lateral incisor 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Canine 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1st premolar 0 0 0 0 0 0 1 1 0 0 0 0 0 0
2nd premolar 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1st molar 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2nd molar 0 0 0 0 0 0 0 0 0 0 0 0 0 0
*Right (R) and Left (L).
51
3 Discussão
Devido à abordagem de dois assuntos “distintos” envolvendo o tema das fissuras orais
não sindrômicas não sindrômicas, a presente tese foi dividida em dois capítulos. O Capítulo
1 contemplou a avaliação de fatores de riscos durante o primeiro trimestre de gravidez que
possam ter relação direta com o desenvolvimento das fissuras orais não sindrômicas. A
importância do primeiro capítulo consiste no fato de devido à ocorrência, e a necessidade de
um tratamento multidisciplinar integrado, as FLPNS são consideradas um problema de saúde
pública pela OMS.
Observou-se a associação entre tabagismo passivo materno e fissuras orais não
sindrômicas, e um risco duas vezes maior de uma mãe que inala os agentes ativos da fumaça
de cigarros durante o primeiro trimestre de gravidez de ter o filho com fissura oral,
comparado a mães controles. Estes resultados corroboram com os encontrados por Martelli et
al. (2015) e Machado et al (2018).
O uso de ácido fólico ou multivitaminas em gestantes durante o primeiro trimestre
reduz substancialmente o risco de fissuras orais não sindrômicas (Jia et al.,2011). O ácido
fólico é um nutriente essencial para o desenvolvimento do feto através do metabolismo de um
carbono, que desempenha função essencial na síntese de DNA e RNA. A deficiência de folato
pode alterar a proliferação celular e a diferenciação na embriogênese (Krapels et al., 2006;
Wu et al.,2012). A presente investigação encontrou efeito preventivo da suplementação de
ácido fólico em mães durante o primeiro trimestre de gravidez, dados que corroboram os de
Krapels et al. (2006) e Wu et al. (2012).
Sabe-seque a cafeína tem efeitos teratogênicos nos seres humanos, porém essa via não
é plenamente compreendida. A cafeína pode aumentar o nível de homocisteína no plasma
(Grubben et al., 2000), que tem demonstrado estar associada a um aumento do risco de fissura
oral (Dien et al., 2018). A homocisteína é um produto intermediário na biossíntese dos
aminoácidos cisteína e metionina. A homocisteína pode seguir duas vias principais: a
remetilação para formar metionina, ou a trans-sulfuração para formar cistetionina. A cafeína é
uma metilxantina, que pode atuar como antagonista da vitamina B6 e perturbar a via de trans-
52
sulfuração dependente da vitamina B6, resultando no aumento do nível de homocisteína
plasmática (Grubben et al., 2000).
Portanto, a ingestão de bebidas com cafeína pode ser um fator de risco adicional para
hiper-homocisteinemia, predispondo à fissura oral (Johansen et al., 2009). Este trabalho
revelou que as gestantes que bebiam e ingeriam alimentos ricos em cafeína tiveram um risco
34% maior de ter um bebê com fissura oral do que o grupo controle. No entanto, os resultados
desse estudo sobre o consumo de cafeína e fumo passivo devem ser interpretados com cautela.
Embora a associação observada apresentasse uma prevalência considerável, o intervalo de
confiança foi amplo, devido ao tamanho limitado da amostra.
Com relação ao segundo capítulo desta Tese, buscou-se avaliar a ocorrência de
anomalias dentárias em pacientes com FLNS, avaliando separadamente estas fissuras com e
sem o envolvimento de rebordo, através de radiografias panorâmicas. A FLNS sem evidência
de qualquer tipo de envolvimento ósseo é uma condição relativamente incomum (Lehtonen et
al.,2015). Sekhon et al. (2009) reportaram uma prevalência de 2,1%, igualmente distribuída
entre homens e mulheres, diferente dos resultados encontrados nesse trabalho, onde se
observou uma prevalência maior em homens (66%) do que em mulheres (44%). Esses
achados concordam com estudos que enfatizaram o dimorfismo sexual em fissuras orais não
sindrômicas (Martelli-Junior et al., 2007; Martelli et al., 2012; Machado et al., 2018).
A FLNS é causada por um distúrbio na fusão mesenquimal dos processos nasal medial
e lateral com o processo maxilar (Machado et al., 2018). O processo de formação do dente
envolve desde a lâmina dentária até o tecido mesenquimal ao seu redor (também chamada de
papila dentária), e esse tecido mesenquimal torna-se o complexo dentinofibroso, que é a
principal influência patológica do tecido dentário (Rawashdeh & Bakir, 2007).
Paranaiba et al., (2013) mostraram um predomínio do lado esquerdo para a presença
de fissuras orais não sindrômicas. Os dados desse estudo ratificam essa suposição para a
população estudada, onde 39 pacientes apresentavam fissura esquerda (56%), 23 direitas
(33%) e 8 bilateral (11%), tanto para FLNS com e sem acometimento do rebordo alveolar.
Galieet al. (2009) revelaram porcentagens semelhantes em seu estudo sobre crianças romanas
afetadas por FLNS sem envolvimento de rebordo.
As razões para o desenvolvimento das anomalias dentárias não são totalmente
compreendidas, mas a teoria mais aceita é que sua etiologia seja um resultado de interações
53
genéticas (Melo-Filho et al., 2014). Dentre essas anomalias dentárias, a agenesia é a mais
comum, acometendo cerca de 20% da população mundial, e geralmente é geneticamente
determinada e transmitida por herança autossômica dominante, com penetrancia incompleta e
expressividade variável (Machado et al., 2016, Machado et al., 2018).
Fissuras orais não sindrômicas em humanos são geralmente associadas ao atraso no
desenvolvimento dos dentes e anomalias dentárias de número, tamanho e forma, tanto no lado
da fissura, quanto no lado não fissurado (Paranaíba et al., 2013). Os resultados do presente
estudo sustentam essa hipótese de que o paciente fissurado tem maior prevalência de
anomalias dentárias ea população acometida pela FLNS com o envolvimento de rebordo foi
mais afetada em relação ao paciente com FLNS sem o envolvimento de rebordo.
Neste estudo as anomalias dentárias analisadas foram apenas em dentição
permanente, onde foi observado um percentual de 41,4% de dentes supranumerários nos
pacientes estudados. A prevalência de dentes supranumerários relatada na população geral
varia de 0,1% a 3,8% (Lehtonen et al., 2015). Nesse estudo, pacientes com FLNS com o
envolvimento de rebordo apresentaram maior quantidade de dentes supranumerários (28,5%),
quase duas vezes maiores do que os pacientes com FLNS sem o envolvimento de rebordo
(12,8%).
O conhecimento das anomalias dentárias, como a agenesia ou dentes
supranumerários, é fundamental para o planejamento do tratamento e reabilitação dos
pacientes fissurados, pois tais anomalias podem levar a espaços desdentados na arcada que
devem ser reabilitados (Melo-Filho et al. 2015). A microdontia é encontrada mais
especificamente nos incisivos laterais superiores, e foi observada em 11,42% em pacientes
com FLNS com o envolvimento de rebordo e 5,7% em pacientes comFLNS sem o
envolvimento de rebordo, de nossa amostra.
Tem sido relatado que o taurodontismo está associado a certas síndromes e distúrbios
de desenvolvimento dentário (Sá et al., 2016). Os resultados do presente estudo mostraram
que a prevalência geral de taurodontismo foi de 1,42% sendo encontrada apenas em pacientes
com FLNS com o envolvimento de rebordo. Entretanto, quando comparamos essa prevalência
com indivíduos clinicamente saudáveis (0,1%) (Rawashdeh & Bakir, 2007), observa-se que
os resultados desse trabalho corroboram com o estudo de Burzynski & Escobar et al.
54
(2013)que constataram que o taurodontismo ocorreu mais frequentemente em indivíduos com
FLNS, quando comparado aos outros tipos de fissuras orais não sindrômicas .
Nesse estudo, indivíduos com FLNS com o envolvimento de rebordo, em
números totais, tiveram mais anomalias dentárias. Isto se deve, provavelmente, à
multiplicidade anatômica, ambientais, deficiência mesenquimal e efeito direto da fissura nos
tecidos primordiais, que são mais pronunciados quando o paciente possui fissuras bilaterais
que afetem diretamente o rebordo alveolar (Mertelli et al., 2012). Mais evidências para
estabelecer a natureza exata dessa relação com cada anomalia dentária individualmente, são
necessárias.
55
4 Conclusão
De acordo com os objetivos propostos e dos resultados obtidos pode-se concluir que:
1. O consumo de cafeína acima de 100 mg diários e o fumo passivo além de 15 minutos
ao dia aumentaram o risco do desenvolvimento de fissuras orais não sindrômicas não
sindrômicas.
2. A suplementação com ácido fólico, complexos multivitamínicos e alimentos ricos em
ferro, utilizados durante a realização do pré-natal, atuam como fatores protetores para
o não desenvolvimento de fissuras orais não sindrômicas não sindrômicas.
3. Pacientes com fissuras labiais com o envolvimento de rebordo tem mais anomalias
dentárias do que os pacientes com fissuras labiais sem o envolvimento de rebordo.
4. Agenesia dentária e supranumerários são as anomalias dentárias mais comuns nos
pacientes com fissuras labiais isoladas.
56
* De acordo com as normas da UNICAMP/FOP, baseadas na padronização do International Committee of Medical Journal Editors - Vancouver Group. Abreviatura dos periódicos em conformidade com o PubMed.
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Apêndice
APÊNDICE 1 – Ficha cadastral para participantes do projeto.
Prezado Senhor (a), com o objetivo de avaliar a possível associação entre o desenvolvimento de
FLP/NS e fatores habituais e nutricionais das mães durante a gestação, a Universidade Estadual de
Campinas está realizando a pesquisa intitulada “Análise e estimativa de fatores nutricionais e
habituais em pacientes com fissuras lábio-palatinas não sindrômicas em uma população
brasileira”. O senhor (a) pode participar respondendo a este questionário que dura cerca de 10
minutos? SIM__ ou NÃO___
1. Iniciais do nome: ____________ Prontuário:
2. Gênero: 1. Masculino ; 2. Feminino . Data:
3. Data Nascimento: _____/_______ /________ ( ) anos
4. Nacionalidade:
5. Naturalidade:
6. Cor da pele: 1. leucoderma ; 2. melanoderma ; 3. feoderma 4.
7. Cosanguidade na família: 1. Positiva ; 2. Negativa
8. Gemeos : 1. Sim ; 2.Não
9. Tem histórico de Fissura labial e/ou
palatina não-sindrômica (FL/PNS) na
família?
1. Sim ; 2. Não ; 55. Não sabe informar .
10.Se tem histórico positivo para FL/PNS, especifique o grau de parentesco e o tipo de
FL/ FL/PNS:
10.1.Grau de parentesco: 1.pai ; 2.mãe ; 3.irmão ; 4.filho
99. Não se aplica ; 55. Não sabe informar .
10.2. Tipo de FL/PNS : 1.Fissura labial, unilateral ; 2.Fissura labial, bilateral
3.Fissura labiopalatina, unilateral ; 4.Fissura labiopalatina, bilateral ;
5.Fissura palatina ; 99. Não se aplica ; 55.Não sabe informar .
63
11 - Uso de Ácido Fólico durante a gestação?
1. Sim ; 2. Não ; 55. Não sabe informar .
12- Fez uso de vitaminas durante a gestação?
1. Sim ; 2. Não ; 55. Não sabe informar .
13. Foi fumante passiva durante a gestação (acima de 15 min diários) ?
1. Sim ; 2. Não ; 55. Não sabe informar .
14. Fez consumo de Álcool durante a gestação?
1. Sim ; 2. Não ; 55. Não sabe informar .
15 – Teve exposição a algum produto químico durante a gravidez? (agrotóxicos, produtos
industriais de limpeza pesada, etc)
1. Sim ; 2. Não ; 55. Não sabe informar .
16 – Fez uso de Cafeína durante a gravidez ( café, Coca-Cola, termogênicos, energéticos) ?
1. Sim ; 2. Não ; 55. Não sabe informar .
16.1 Se a resposta da questão anterior for sim, qual a quantidade de cafeína foi ingerida
semanalmente? (Cada copinho de café e copo de Coca-Cola tem aproximadamente 40 mg de
cafeína. O Termogênico depende da dosagem ingerida, e o energético aproximadamente 70 mg
de cafeína a cada 100 ml.)
1. 0 a 100 mg ; 2.100 mg a 200 mg
3.300mg a 400 mg ; 4. 400 mg a 500 mg ;
5. 500mg a 600 mg ; 99. Não se aplica ; 55.Não sabe informar .
17. Fez uso de algum medicamente durante a gestação?
1. Sim ; 2. Não ; 55. Não sabe informar .
17.1 Se a resposta da questão anterior for sim, qual a classe do medicamente ingerido?
1. Analgésico ; 2.Antibióticos
3. Anti inflamatórios ; 4. Antidepressivos ;
5. Anti hipertenssivos ; 6. Hipoglicemiantes orais
99. Não se aplica ; 55.Não sabe informar .
64
18. Ingeriu alimentos ricos em ferro durante a gestação (feijão, fígado, etc)?
1. Sim ; 2. Não ; 55. Não sabe informar .
65
Anexo 1- PARECER DO COMITÊ DE ÉTICA
CEP – Capítulo 1
66
67
68
69
70
71
72
CEP – Capítulo 2
73
74
75
76
77
78
79
80
Anexo 2- COMPROVANTE DE SUBMISSÃO
81
82
Anexo 3- VERIFICAÇÃO DE ORIGINALIDADE E PREVENÇÃO
DE PLÁGIO