Maternal Folic Acid Deficiency Is Associated to Developing Nasal and Palate Malformations in Mice

Abstract

Craniofacial development requires extremely fine-tuned developmental coordination of multiple specialized tissues. It has been evidenced that a folate deficiency (vitamin B₉), or its synthetic form, folic acid (FA), in maternal diet could trigger multiple craniofacial malformations as oral clefts, tongue, or mandible abnormalities. In this study, a folic acid-deficient (FAD) diet was administered to eight-week-old C57/BL/6J female mouse for 2-16 weeks. The head symmetry, palate and nasal region were studied in 24 control and 260 experimental fetuses. Our results showed a significant reduction in the mean number of fetuses per litter according to maternal weeks on FAD diet (p < 0.01). Fetuses were affected by cleft palate (3.8%) as well as other severe congenital abnormalities, for the first time related to maternal FAD diet, as head asymmetries (4.6%), high arched palate (3.5%), nasal septum malformed (7.3%), nasopharynx duct shape (15%), and cilia and epithelium abnormalities (11.2% and 5.8%). Dysmorphologies of the nasal region were the most frequent, appearing at just four weeks following a maternal FAD diet. This is the first time that nasal region development is experimentally related to this vitamin deficiency. In conclusion, our report offers novel discoveries about the importance of maternal folate intake on midface craniofacial development of the embryos. Moreover, the longer the deficit lasts, the more serious the consequent effects appear to be.

Keywords: congenital abnormalities; maternal folic acid-deficient diet; nasal region; palate.

Figure 1

Head and nasopalatine region measurements. (a) Lateral view of a control head E17-fetus. The yellow rectangle crossing the eye indicates the block of sections selected for measurements. (b) Coronal section showing the landmarks for measurements: The nasal meatus (arrows), the eyes (E) and the developing molars (arrows heads) (En: encephalon; P: palate; T: tongue; asterisk: nasal septum; double asterisk: nasopharynx). (c) Description of head measurements on the same black and white image: width (red line), height from palate to top of the head (yellow line) and area of the half head (discontinuous black line). (d) Magnification of the nasopalatine region (T: tongue; P: palate; M: developing molar; double asterisk: nasopharynx; asterisk: nasal septum; En: encephalon). (e) Description of nasopalatine region measurements on the same black and white image: thick of the palate (red line); area of the half of the palate (green line); area of the bone tissue inside the palate area (orange line), area of the nasopharynx (pink line), area (yellow line) and height (blue line) of the nasal septum. (f) Magnification of the right half palate: bone tissue (arrowhead) and the osteogenic condensation (asterisk). (g) Magnification of the rectangle marked in (f): bone tissue (arrowhead) and the osteogenic condensation (asterisk). Scale bars: (a–c) 1 mm; (d–e) 500 µm; (f) 100 µm; (g) 50 µm.

Figure 2

Mean number of fetuses per litter according to weeks on maternal folic acid deficient (FAD) diet. ** p ≤ 0.01 (Spearman’s rho test).

Figure 3

Head and palatine malformations. Control fetus head: lateral view (a), frontal view (b) and coronal section (c). Arrowhead: ear. Arrow: eye. Asterisk: mandible. Severely malformed fetus: lateral view (d), frontal view (e,f) coronal section. The eye, ear and mandible were not developed, the palate is not recognized. Malformed fetus head: lateral view (g), frontal view (h) and coronal section (i). This case showed asymmetrical head and high arched palate (HAP), lack of mandible and tongue. Arrowhead: ear. Arrow: eye. Magnification of nasopalatine region of control fetus (j): the palate (P) showed a form of truncated pyramid, the nasopharynx was like lips and they were two nasal meatus on each side of the nasal septum. Arrow: nasopharynx. Arrowheads: nasal meatus. Asterisk: nasal septum. Magnification of a malformed fetus (k) with HAP palate, aglossia and head asymmetry because the meatus dysmorphology. M: developing molar. T: tongue; Scale bar: (a–i) 1 mm; (j–k) 500 µm.

Figure 4

Nasopalatin region. (a) Coronal section of a control fetus: the nasopharyngeal duct (N) was placed between palate (P).and the nasal septum (NS), which was below the encephalon (En). Arrowhead marked the area of interest. (b) Magnification the control respiratory epithelium with the columnar and ciliated cells. (c) Malformed fetus with the tongue (T) placed between palatal shelves (PS). (d) Magnification of the rectangle showing the transition epithelium (TE) among respiratory (RE) to medial edge epithelium (MEE). Arrows: cilia. (e) Measurement of nasopharynx area (yellow line) in cleft palate. The lower limit was an imaginary line (discontinuous black line) drawn on the transitional epithelium in clef palate fetuses. (f) Anomalous nasopharyngeal cilia. (g) Arrows pointed lack of cilia in some cells. (h) The cells were not columnar, and cilia seemed to be atrophied. (i) Aberrant shape of the nasal septum (NS) and nasopharyngeal duct (N). The palate (P) was normal. En: Encephalon. (j) Magnification of altered nasal septum showing how the area was measured (yellow line) the height of the septum (blue line). Scale bars: (a,c,i) 500 µm; (b,d,f–h) 10 µm; (e,j) 100 µm.

Figure 5

Correlations between head measurements with weeks on maternal FAD diet. (a) Head width; (b) head height; (c) right half head area; (d) left half head area and (e) total head area. ** p ≤ 0.01; Spearman’s Rho.

Figure 6

Correlations between palate measurements with weeks on maternal FAD diet. (a) Palate thickness; (b) right half palate area; (c) left half palate area; (d) total palate area and (e) palate bone area. * p ≤ 0.05; ** p ≤ 0.01.

Figure 7

Correlations between palate measurements and total palate area. (a) Palate thickness and (b) palate bone area. ** p ≤ 0.01; Spearman’s Rho.

Figure 8

Correlations between palate measurements and total head area. (a) Total palate area and (b) palate bone area. ** p ≤ 0.01; Spearman’s Rho.

Figure 9

Correlations between nasal measurements with weeks on maternal FAD diet. (a) Nasopharynx duct area, (b) nasal septum area and (c) nasal septum height. * p ≤ 0.05; Spearman’s Rho.

Figure 10

Correlations between nasal measurements and total head area. (a) Nasopharynx duct area and (b) nasal septum area. ** p ≤ 0.01; Spearman’s Rho.