Do prenatal and postnatal hypothyroidism affect the craniofacial structure?: An experimental study
ABSTRACT
Objective: To investigate the effect of experimental prenatal and postnatal hypothyroidism (HT) on the craniofacial structure in rats.
Materials and Methods: Female Wistar albino rats were mated with males for fertilization. Pregnant rats were divided into three groups. Group 1 (methimazole [MMI]-induced prenatal hypothyroidism group) mother rats were given MMI water during and after pregnancy. Group 2 (MMI-induced postnatal hypothyroidism group) mother rats were given MMI water after pregnancy. After the breast-feeding period, group 1 and 2 rat pups received the same water as their lactating mothers drank. Group 3 (control group) pregnant rats and rat pups were given normal tap water. When the rat pups were 90 days of age, lateral cephalometric and posteroanterior films were taken under anesthesia.
Results: Posteroanterior radiographs revealed that palatal, cranial, bizygomatic arch, and bigonial width measurements were significantly shorter in prenatal HT and postnatal HT groups compared to the control group (P < .001). Intragroup comparisons in lateral cephalometric radiographs showed that, nearly all of the comparisons were statistically significant (P < .05), with the exception of the Co-Gn, E-Pg/S-Gn measurements between the prenatal and postnatal HT groups.
Conclusions: Sagittal and transverse measurements showed that untreated HT has detrimental effects on the growth of the maxilla and mandible.
INTRODUCTION
Hypothyroidism (HT) is defined as a metabolic condition that is caused by a deficiency in thyroid hormone (TH) production and gland function.1,2 THs (triiodothyronine-T3 and thyroxine-T4) are very important regulators of the growth, bone maturation, energy metabolism, and turnover of cells.2,3
Thyroid diseases are common, and their effects on craniofacial, dental, and oral structures are defined in the literature. Severe HT causes subnormal growth of the maxilla and mandible and a decrease in the facial dimensions. HT has detrimental effects on tooth development and eruption, which leads to prolonged retention of deciduous teeth and impaction of permanent teeth, resulting in malocclusion.4–6 Oral effects of HT include enlargement of the tongue, thick lips, dysgeusia, poor periodontal health, and delayed wound healing.2,5,7
HT can occur either as a congenital or as an acquired condition.4,8 In prenatal HT, linear growth at birth is normal, but the development of bone and teeth is retarded. After birth, HT disturbs normal growth, resulting in growth deficiency or complete cessation of growth is also seen at athyroidism. Growth of the craniofacial structures is retarded and brachycephalic facial features develop.9
To the best of our knowledge, no study has been reported that involved investigation of craniofacial measures in cases of HT. Here we aim to evaluate the effect of experimental prenatal and postnatal HT on the craniofacial structure in rats.
MATERIALS AND METHODS
Experimental Design
In this prospective, randomized study, 6-month female Wistar albino rats weighing 250–300 g were mated with males for fertilization. Vaginal smears were performed for the determination of pregnancy. When semen was detected in the vaginal smears, rats were thought to be pregnant and were followed. Pregnant rats were divided into three groups, as follows:
Group 1 (methimazole [MMI]-induced prenatal hypothyroidism group): The pregnant rats were fed ad libitum with MMI water during and after pregnancy. MMI (SC-205747A, Santa Cruz Biotechnology Inc, Dallas, Tex; 0.025% wt/vol) was given daily in drinking water starting from pregnancy. Rat pups were fed with breast milk from their lactating mothers. After weaning (19–22 days), pups drank the same MMI water that their lactating mothers drank until day 90. Group 2 (MMI-induced postnatal hypothyroidism group): The pregnant rats were fed ad libitum with water during pregnancy. The mother rats were given the same dose of MMI daily in drinking water from birth. Offspring were fed with breast milk from their mothers. After weaning (19–22 days), pups drank the same MMI water that their lactating mothers drank until day 90. Group 3 (control group): The pregnant rats were fed ad libitum with normal tap water (without MMI). Rat pups were fed with breast milk from their lactating mothers. After weaning and until they were 90 days of age, they were fed ad libitum with regular water, as were their mothers. This protocol and dosage of MMI administration is typically used for the production of HT rats.10–12
The rats were restricted separately in plastic cages under artificial lighting from fluorescence lamps, with a 12-hour light:12-hour dark photoperiod. The room temperature was set at 25°C. When the rat pups were 90 days of age, 10 female and 10 male rats from each group (total = 60 rats) were used. Permission was obtained from the University of Adnan Menderes, Local Ethics Committee for Animal Experiments (reference No, 64583101/2014/036). The experiments were carried out in the Department of Experimental Animals at the Research and Development Center.
Cephalometric Technique and Data Analysis
Rats were anesthetized with intraperitoneal injections of 100 mg/kg and 20 mg/kg xylazine and ketamine combination, respectively. A 1-mL xylazine + ketamine combination was prepared per kilogram of rat. Lateral and posteroanterior cephalometric radiographs were taken with a Comed X-ray machine (Comed Medical System, Seongnam Si Jungwon-gu GYEONGGI-DO, Korea) at a setting of 2 mAs at 70 Kv.
Dentofacial parameters were measured using a cephalometric analysis program (Romexis 3.2.0.R; Planmeca, Helsinki, Finland). Cephalometric landmarks were marked and digitized by one author. All of the measurements were repeated 2 weeks later to determine the measurement error.
Fifteen cephalometric landmarks were identified (Figure 1), and 21 measurements (17 linear, 3 angular, and one proportional) were performed on lateral radiographs (Table 1). Eight cephalometric landmarks and four linear cephalometric measurements were identified on posteroanterior radiographs (Figure 2; Table 2).
Citation: The Angle Orthodontist 86, 6; 10.2319/080315-521.1
Citation: The Angle Orthodontist 86, 6; 10.2319/080315-521.1
At the end of the radiograph acquisition period, blood samples were collected from the heart under general anesthesia and serum levels of total T4 were analyzed using enzyme-linked immunosorbent assay.
Statistical Analysis
The distributions of the measurements taken on the lateral and posteroanterior radiographs were examined by Shapiro-Wilk’s test. Normal distributed measurements were shown as mean ± standard deviation (mean ± SD), and nonnormal ones were expressed with a median (Interquartile Range).
The interclass correlation coefficient (ICC) was calculated to evaluate the intrarater agreement. Since all ICCs were greater than 0.900 and significant, the first measurements were analyzed.
Total T4 serum levels of control, prenatal, and postnatal HT groups were compared by Kruskal-Wallis analysis of variance (ANOVA) and one-way ANOVA for males and females, respectively. The sample sizes in each combination of gender and groups were near 10, and the lateral and posteroanterior radiographic measurements did not satisfy the homogeneity of the variances. Therefore, two-way ANOVA could not be applied. The analyses were carried out for gender and group categories separately. In each gender category, the groups were compared by one-way ANOVA. For each group category, the genders were compared by an independent-sample t-test. All p values obtained by these ANOVA and t-tests were adjusted by the Bonferroni-Holm correction method. Groups were compared generally by Kruskal-Wallis ANOVA with respect to the Go-Mn measurements and by one-way ANOVA with respect to all other measurements. F-statistics or Welch’s statistics were provided and Tukey honestly significantly different or Games Howell post hoc tests were applied depending on the Levene test results. χ2 statistics were written, and the Mann-Whitney U-test with Bonferroni correction was applied as a result of the Kruskal-Wallis ANOVA. A P values of less than .05 were accepted as statistically significant.
All analysis and calculations were performed in IBM SPSS 21.0 (IBM Corp, Released 2012, IBM SPSS Statistics for Windows [Version 21.0], Armonk, N.Y.).
RESULTS
The rats did not show any signs of systemic disease during the study period. The body weight of rats on the last day of the study period is given at Table 3. Total T4 hormone levels showed that experimental intervention (administration of MMI) effectively produced the expected condition of HT in the prenatal and postnatal HT groups (Table 4).
Significant agreement was found between the two sets of measurements on the lateral and posteroanterior radiographs (P < .001), indicating excellent intrarater reliability (all ICC > 0.900).
When we evaluated the male lateral cephalometric radiographs for control prenatal and postnatal HT groups, S-Ba, Co-Gn, and E-Pg/S-Gn measurements did not show significant differences between the prenatal and postnatal HT groups (P > .05) (Table 5). There were not significant differences in Snlu, SN-PgGn, and MI-II measurements between the control and postnatal HT groups (P > .05) (Table 5). Comparisons of female lateral cephalometric radiograph measurements showed that S-Ba, Go-Mn, E-Pg/S-Gn, SNIu, SN-PgGn, and Ml-II measurements were not statistically different between the prenatal and postnatal HT groups (P > .05) (Table 5). There were significant differences between groups with respect to the other measurements except these ones (P < .05) (Table 5).
Intragroup comparisons in lateral cephalometric radiographs showed that N-A, S-Ba, Po-A, Go-Mn, Go-Pg S-Gn, E-Pg, and Mu-Iu measurements for the control group; S-Ba, Co-Gn, and E-Pg/S-Gn measurements for the prenatal HT group; and Po-N, Go-Mn, Go-Pg, Co-Gn, E-Pg, Co-Pg, Co-Iu, SnPg, SN-PgGn, Co-II, and Mu-Iu measurements for the postnatal HT group were statistically significant between the sexes (P < .05) (Table 5).
When the sex differences were ignored, nearly all of the comparisons were statistically significant (P < .001), except for the Co-Gn, E-Pg/S-Gn measurements between the prenatal and postnatal HT groups (P > .05) (Table 6).
Comparisons of posteroanterior cephalometric radiograph measurements for control, prenatal, and postnatal HT groups showed that all of the group comparisons were statistically different from each other (P < .001) (Table 7). Go1-Go2, C1-C2, P1-P2, and Z1-Z2 measurements changed as follows: control group> postnatal HT group > prenatal HT group.
Intragroup comparisons in posteroanterior cephalometric radiographs showed that there were statistically significant differences between the sexes for Z1-Z2 measurements in the control group (padj = 0.045); P1-P2 measurements in the prenatal group (padj = 0.009); and C1-C2 measurements in the postnatal group (padj = 0.018) (Table 7).
DISCUSSION
Thyroid disorders during development cause major and minor birth defects. Congenital HT is known to lead to deficiencies in growth, bone maturation, and motor development.3 In this study, we aimed to investigate the effect of prenatal and postnatal HT on the craniofacial structures in rat models. To the best of our knowledge, there is no published study that investigates the effect of HT on craniofacial structures; therefore, we were unable to compare the results of this study with the results of others.
HT is caused by iron deficiency, radiation of the thyroid gland, surgery, and excessive antithyroid drugs.2,8 MMI is an antithyroid agent and has been widely used in studies. MMI exposure during the development process blocks TH synthesis and leads to growth retardation.10 In this study, we suppressed the TH synthesis using MMI.
Untreated HT has detrimental effects on the growth and development of the maxilla and mandibula.13 Loevy et al.13 discussed the dental evaluation of a patient with untreated congenital HT in their case report. Their cephalometric radiographs indicate an overwhelming reduction in the dimensions of the craniofacial complex, particularly evident in the patient’s posteroanterior and lateral cephalometric tracings. The findings are similar to those reported by Bedi and Brook4 in a patient with juvenile HT. In this study, significant decreases were found in the sagittal position of the maxilla and mandible effective maxillary and mandibular length, anterior and posterior cranial base length, nasal bone length, occipital bone height, and palatal length between the HT groups and the control group.
Spiegel et al.14 reported the occurrence of a small facial height with a tendency to open-bite malocclusion in HT patients. We have found significant increases in anterior/posterior facial height ratio and mandibular plane angle and reduction in anterior and posterior facial height in each HT group compared with the control group.
Many linear and angular cephalometric measurements were higher in male than in female lateral and anteroposterior radiographs, as was the case in human studies. These results were in accordance with those of other cephalometric human studies.15–18
THs are important for the synthesis of insulin-like growth factor–1 (IGF-1), which promotes bone growth and differentiation in a variety of tissues.9,19,20 THs enhance cartilage growth through IGF-1 and by directly accelerating the differentiation of chondrocytes. HT patients show low serum levels of IGF-I and reduced IGF bioactivity.9,20
Backeljauw et al.21 reported that children with severe primary IGF-1 deficiency may have underdeveloped facial bones (small facial dimensions and a retrognathic maxilla and mandible). It seems that growth retardation in HT groups is related to a deficiency of serum IGF-1 levels, and craniofacial growth is under the endocrine regulation.
Neonatal screening for congenital HT after birth is important to avoid mentally handicapping conditions and growth disorders. HT in the newborn period is often overlooked, and delayed diagnosis leads to the most severe outcome of HT in developing countries.13 Many factors, such as lack of awareness about HT among the health care practitioners and the inaccessibility and higher cost of the laboratory examinations, play a role in the limited diagnosis and management of HT.5 This is especially true for the acquired HT clinical manifestations depend on the age of onset and the length of time before the disease is effectively treated. Once the HT is diagnosed, the retardation of skeletal maturity improves quickly after adequate treatment.4 Generally, the dental development is less responsive to treatments than is skeletal maturity (eg, the height of the patient). Fortunately, craniofacial growth of HT patients does not show a modification in growth. Craniofacial structures are generally affected by the retardation of growth velocity, which in turn could be treated by an early orthopedic treatment in early childhood.
In this study, we used two-dimensional imaging to assess cephalometric parameters in the rats. This imaging method limits the amount of information to be gained from analysis, since the three-dimensional situation is reduced to a two-dimensional projection. Therefore, future studies should evaluate the relationship using three-dimensional computerized tomography.
CONCLUSIONS
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Palatal, cranial, bizygomatic arch, and bigonial width measurements were significantly shorter in the prenatal HT and postnatal HT groups compared to the control group.
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Cephalometric radiographs indicated that statistically significant differences were present between the three groups for almost all of the linear and angular measurements.
Location of cephalometric landmarks on lateral radiographs.
Location of cephalometric landmarks on posteroanterior radiographs.
Contributor Notes