Fetal Palate Research Articles

Activin Subunit and Receptor Expression in Normal and Cleft Human Fetal Palate Tissues

Craniofacial malformations, such as cleft palate, present serious complications in the newborn and are often of unknown etiology. Activin BA subunit deletion leads to cleft palate in mice, but the expression of this protein in the human palate has not been explored. Our goal was to determine the spatial and temporal expression of inhibin/activin subunits; the binding protein, follistatin; and activin receptors in the human fetal palate. Residual human fetal palate tissues, with or without cleft, were collected during routine autopsy at Women and Infants Hospital. Inhibin/activin alpha and beta subunits, follistatin, and activin receptor protein and mRNA expression were studied by immunocytochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR) experiments, respectively. Dimeric activin A levels were compared in cleft and normal palate tissue homogenates by immunoassay. Activin BA, follistatin, and activin receptor type IIA proteins were observed in normal and cleft palate tissues throughout pregnancy (gestational weeks 11 to 40). Proteins were predominantly found in developing bone cells, with no significant group differences. Inhibin/activin BA subunit, follistatin, and activin receptor mRNAs were also detected in normal and cleft fetal palate tissues, but inhibin alpha and BB subunit were absent. Inhibin/activin BA subunit expression was consistent with the presence of dimeric activin A, but levels did not differ significantly between cleft and control tissues. Inhibin/activin BA subunit, follistatin, and activin receptor proteins and mRNAs are present in the human fetal palate. These data suggest that activin signalling has the potential to be associated with human palate development.

P40.08: Posterior wall of the fetal palate: is it a reliable sign applicable in routine?

Objectives: To evaluate the assessment methods for fetal lip/palate on 3D ultrasound with various techniques and examination planes. Methods: Stored 3D volumes of fetuses with postnatal confirmation of cleft lip and/or palate were reviewed. The abnormalities at birth were compared with the antenatal 3D ultrasound findings in the following planes obtained on techniques previously described: 1. The axial plane 2. The sagittal plane 3. The coronal plane 4. Oblique axial plane 5. Oblique sagittal plane 6. Targeted reverse face view Results: There were limitations to each technique or examination plane in antenatal assessment of fetal lip/palate on 3D ultrasound. Different parts of the fetal lip and palate could be visualized with each of the above planes. The anterior part of the palate and the fetal lips are better seen in the axial, sagittal and coronal planes. The oblique axial, oblique sagittal planes and the targeted reverse face view allowed better visualization of the hard and the soft palates. The presence of facial cleft led to a distortion of the anatomy. The use of a combination of these planes allowed a more complete assessment of fetal lip and palate from various angles. Conclusions: The use of a combination of planes and techniques provides more information than the use of a single one in evaluation of fetal cleft lip/palate on 3D ultrasound.

P50.10: Fetal lip and palate cleft diagnosed at 13 weeks of gestation by 4D/3D ultrasound : a case report

P50.10: Fetal lip and palate cleft diagnosed at 13 weeks of gestation by 4D/3D ultrasound : a case report

OC180: Comparison between three different methods of visualizing the secondary fetal palate by three‐dimensional ultrasonography

period and to present data on ultrasound–anatomical correlates of the normal human palate between 12 and 24 weeks of gestation. Methods: Thirty fetuses from normal pregnancies underwent examination of the face and palate using 3D ultrasound imaging in both the axial and reverse face views. To establish ultrasound–anatomical correlates, high-resolution images of human embryos and fetuses of comparable gestational age to those examined by ultrasound were obtained from our collection of human embryology. Results: During the embryological period the palatal shelves grew towards the midline and fused during the 8th week (LMP) of gestation. The last component to reach full differentiation was the bilobular (bifid) uvula, which becomes monolobular during the 9th week of gestation. 3D ultrasound images using both maximum and surface rendered modes showed close anatomical correlation with the comparable anatomical specimens, with the exception of the soft palate and uvula. Axial views provided better visualization of the palate up to the 20th week but thereafter coronal sections were superior owing to the avoidance of acoustic shadowing. Discussion: Prenatal 3D ultrasound images from the 12th to the 24th week of gestation correlate well with the landmarks observed in anatomical specimens of comparable gestational ages. However, data from larger series of different ethnic groups are needed to establish reliable patterns of normal growth.

Prenatal ultrasound examination of the secondary palate

Prenatal ultrasound examination of the secondary palate

Sonographic assessment of normal fetal palate using three‐dimensional imaging: a new technique

The aim of this study was to describe a novel three-dimensional (3D) ultrasound rendering technique to examine the normal fetal posterior palate and to assess its correspondence with the real fetal anatomy. A prospective longitudinal study was conducted from January to October 2005 and included 100 fetuses in a low-risk population. Fetal ultrasound examinations were performed at 17, 22, 27 and 32 weeks' gestation to determine the normal 3D ultrasound view of the fetal palate at different gestational ages. The ultrasound scans were performed using the strict anterior axial plane of the starting reconstruction volume and the underside 3D view of the fetal palate. The 3D view of the fetal palate was compared with the normal anatomical view of the fetal palate obtained by surgical fetopathological examination of fetuses at the same gestational ages. The sonographic visualization rates of seven defined anatomical landmarks of the fetal palate were computed for each gestational age. The visualization rates across gestational ages were compared by use of the Cochrane Q test. The reliability of detection of each anatomical landmark across gestational ages was determined by Cronbach's Alpha. In all cases a 3D ultrasound view of the fetal maxilla and secondary palate was obtained at each period of gestation and corresponded well to the fetal anatomical specimens. The seven defined anatomical landmarks of the fetal palate were identified in 42-100% of cases. The visualization rates across gestational ages were significantly different in five of these anatomical landmarks. These differences can be explained by different developmental processes of these anatomical structures. The overall reliability of visualization across the gestational ages for the anatomical landmarks was medium to very high (0.73-0.96), except for the landmark interpalatal suture which was low (0.48). This technique of anterior axial 3D view reconstruction of the fetal palate seen by an underside view can provide unique diagnostic information on the integrity of the secondary palate. This innovative, simple and rapid technique may become the reference technique in ultrasound investigation of the fetal palate, and should be of value in diagnosing isolated secondary cleft palate or palatal involvement when cleft lip and alveolus are diagnosed.

Cleft Palate and Keratoconus in a Child Affected by Fetal Alcohol Syndrome: An Accidental Association?

This report describes the case of a 9-year-old girl affected by fetal alcohol syndrome who presented at birth with blepharophimosis and a cleft palate, which was submitted to surgery. She was referred to our hospital for a visual acuity reduction, where a diagnosis of keratoconus was made. This case highlights the rarity of the association between fetal alcohol syndrome and cleft palate and the previously unreported association involving fetal alcohol syndrome-keratoconus and cleft palate-keratoconus.

Amniotic fluid induces rapid epithelialization in the experimentally ruptured fetal mouse palate - implications for fetal wound healing

Cleft of the secondary palate is one of the most common congenital birth defects in humans. The primary cause of cleft palate formation is a failure of fusion of bilateral palatal shelves, but rupture of the once fused palate has also been suggested to take place in utero. The possibility of post-fusion rupture of the palate in humans has hardly been accepted, mainly because in all the cleft palate cases, the cleft palatal edge is always covered with intact epithelium. To verify whether the intrauterine environment of the fetus plays roles in wound healing when the once fused palate is torn apart, we artificially tore apart fetal mouse palates after fusion and cultivated them in culture medium with or without mouse or human amniotic fluid. We thereby found that the wounded palatal edge became completely covered with flattened epithelium after 36 hours in culture with amniotic fluid, but not in culture without amniotic fluid. Using histological and scanning electron microscopic analyses of the healing process, it was revealed that the epithelium covering the wound was almost exclusively derived from the adjacent nasal epithelium, but not from the oral epithelium. Such actions of amniotic fluid on the fetal wound were never simulated by exogenous epidermal growth factor (EGF), albumin, or both. In addition, the rapid epithelialization induced by amniotic fluid was not prevented by either PD168393 (an inhibitor of the EGF receptor-specific tyrosine kinase) or SB431542 (a specific inhibitor of TGFbeta receptor type I/ALK5). The present study provides new insights into the unique biological actions of amniotic fluid in the repair of injured fetal palate.

OP02.14: Fetal palate: a new direct sonographic sign of normal, cleft and ogival palate, with 2D and 3D

Objective: Cleft palates are still considered as impossible or very difficult to study in 2D or in 3D, and impossible in routine examination. We described a new approach to identify normal palate and it’s pathology. Since 1 year, a new treatment of 3D acquisition can be applied with this method. Method: This easy 2D approach, has been realised with success on every fetuses seen, aged from 18 to 35 Weeks since 2002 (that is to say more than 7000 fetuses), sometimes completed with 3D view. Using precise guide marks, this approach allows a direct very echogenic visualisation of the osseous palate’s posterior wall, proving it’s integrity. Keeping the same guide lines, 3D thick view often visualise the whole palate. Results: With this approach the visualisation of the posterior wall has always been successful and registered. First we could definite criteria of a normal palate. We could also have cleft lips and we could demonstrate the associated cleft palate and the duplication of the uvula. Further we could diagnose cleft palate without cleft lips, short palate (helped with 3D view), and one suspected ogival palate, confirmed with a sagittal view (3D thick view wasn’t available). All these findings have been confirmed by postnatal examination. There hasn’t be any false negative or false positive cases about the integrity of the palate. We described the guidelines we used in this study with 2D and 3D view, the criteria of normal palate, it’s pitfalls, and the sonographic characteristics of cleft and in ogival forms. Conclusion: This study demonstrate than the palate is possible to visualise, even in routine, with a very good prediction of it’s normality or pathology. This will be a great help for parents avoiding the surprise of this pathology or eliminate the cleft palate’s fear. It will be also useful for the surgeon in case of cleft lip for example to explain the parents the more precisely possible what will occur after birth.

Regional heterogeneity in the developing palate: morphological and molecular evidence for normal and abnormal palatogenesis

Development of the mammalian secondary palate involves the growth, elevation, medial elongation and midline fusion of palatal shelves. Recent morphological and molecular studies on palatogenesis suggest that the developing palate is not a homogeneous organ but each part may behave differently during organogenesis. Especially, some key molecules involved in palate development have been shown to exhibit heterogeneous patterns of expression in the palatal tissue. Therefore it seems necessary to recognize the regional heterogeneity of the developing palate along the dorsoventral and anteroposterior axes when analyzing the mechanisms of normal and abnormal morphogenesis. Based on recent studies, we discuss the issue of the regional heterogeneity in the fetal palate and propose a principle that divides the fetal palate into several regions from the morphological and molecular standpoint.

OC8.02: Sonographic evaluation of the fetal hard palate and maxillary tooth buds

OC8.02: Sonographic evaluation of the fetal hard palate and maxillary tooth buds

Ultrasound antenatal diagnosis of cleft palate by a new technique: the 3D ‘reverse face’ view

To assess the clinical value of a novel three-dimensional (3D) ultrasound technique, the reverse face view (3D RF view), in the antenatal categorization of facial clefting and in particular clefting of the hard palate. Eight cases of suspected orofacial clefting were examined by 3D surface rendering. The fetal lips and alveolar ridge were examined in the frontal plane and the face was then rotated through 180 degrees on the vertical axis to examine the secondary palate by the 3D RF view. In each case described, we were able to visualize the fetal face, lips and palate and make an antenatal diagnosis as to whether the palate was affected. In all cases, the antenatal diagnosis was subsequently confirmed. In one case with a left-sided cleft in the lips and alveolar ridge and an intact hard palate, the correct diagnosis was made but a cleft in the soft palate was missed. Although clefts of the lips and alveolar ridge are readily diagnosed on high-quality antenatal ultrasound, visualization of the fetal palate using existing techniques is unreliable. In the patients described here, the 3D RF technique allowed relatively straightforward assessment of the fetal palate with a high degree of accuracy.

Inhibins and activins in human fetal abnormalities

To date, the only routine clinical application of inhibin or activin measurement in testing for fetal abnormalities has been the use of inhibin A in prenatal screening for trisomy 21 (Down syndrome). Second trimester maternal serum levels of inhibin A are, on average, two-fold higher in Down syndrome than in unaffected pregnancies. Although the biology of altered second trimester maternal serum analyte levels in Down syndrome pregnancy cannot yet be explained, it seems that fetal products tend to be decreased, while placental products tend to be increased. This pattern holds true for inhibin A because maternal serum levels appear to be derived from placental rather than fetal sources. Therefore, the measurement of inhibins and activins in maternal fluids, although clinically useful and relatively easy to obtain, may not be helpful in studying their role in human fetal development. Studies in transgenic mice indicate a role for activin, follistatin, and activin receptor type IIA in development of the palate and craniofacial region. Cleft palate is a common birth defect and is associated with serious feeding and respiratory complications in newborns. We have begun to investigate the potential role of activin in human craniofacial development by examining the spatial and temporal expression of inhibin/activin subunits, follistatin and the activin receptors in the fetal palate. Palate tissues were collected at autopsy from fetuses ranging in gestational age from 9 to 42 weeks, and 8 week embryonic tissues were also examined. Tissues were either stored in paraffin for immunocytochemistry or were frozen for RT–PCR examination of the expression of inhibin/activin proteins or mRNAs, respectively. To date, βA subunit, follistatin, and activin receptor, but not α and βB subunit, mRNAs are present in palate tissues and inhibin/activin βA immunoreactivity has been consistently observed in developing bone. Expression of the activin A subunit and its receptors in the human fetal palate are consistent with a developmental role. Studies are ongoing to determine whether altered activin biosynthesis is associated with cleft palate. Future studies of fetal tissues may help to elucidate other roles for the TGF-β family in human development.

Organ culture of the fetal mouse palate for screening the developmental toxicity of chemicals: a validation study.

Using in vitro organ culture of the fetal mouse palate in a chemically defined serumless medium, the toxicity of 24 chemical compounds was investigated. Explanted palates of day-12.5 mouse fetuses were exposed for 72 h in vitro to various concentrations of each chemical, and the fusion rate and growth parameters were compared between the experimental group and respective controls. The average rate of palate fusion was 84% in vehicle controls. For compounds that are teratogenic in experimental animals in vivo, the fusion rates of palatal shelves decreased as the concentration of the test chemicals increased, showing a dose-dependent relationship. Palate fusion was inhibited by 11 of the 15 in vivo teratogens, and the predictability of in vivo developmental toxicity in this culture system was 73%. Cyclophosphamide itself did not inhibit the growth and fusion of explanted palates, but supplementation of hepatic S-9 fraction and cofactors for a monooxygenase system converted it to a toxic substance, as was shown in other in vitro systems. The 50% inhibitory concentration (IC50) value calculated based on the fusion rate was also found to be a useful parameter for evaluating the developmental toxicity of drugs. The teratogenic risk in the human fetus could be assessed by comparing the minimal toxic concentrations of the test compound on cultured palates with the maximal plasma level in pregnant women under therapeutic conditions and with the plasma concentrations when its minimal teratogenic dose is given to pregnant mice. This organ culture system of the fetal palate should be useful for screening the developmental toxicity of drugs and other environmental agents, and its value should increase when it is used in combination with other battery test systems.

Reduction of the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in mice using an antiulcer drug, geranylgeranylacetone.

The protective effect of geranylgeranylacetone (GGA), an antiulcer drug, against the acute toxicity and teratogenicity produced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was examined in C57BL/6J mice. When mice were co-treated, GGA reduced the loss of body weight gain and lethality produced by TCDD but hepatomegaly and thymic atrophy were not improved. Additionally, no protective effect of GGA was observed in the formation of cleft palate and hydronephrosis in mouse fetuses caused by maternal exposure to TCDD. To clarify the reducing mechanism by GGA, the Hsp70.1 mRNA levels in liver and intestine were analyzed. However, it was difficult to explain the effect of GGA from the induction of Hsp70.1. GGA had also no effect on the induction of hepatic ethoxyresorufin O-deethylase activity by TCDD. These data suggest that GGA exhibits a protective effect against some forms of dioxin toxicity by a mechanism without involving inhibition of arylhydrocarbon receptor activation.

P182: Nomogram of fetal hard palate length, width, and area throughout gestation

P182: Nomogram of fetal hard palate length, width, and area throughout gestation

Embryonic arrhythmia by inhibition of HERG channels: a common hypoxia-related teratogenic mechanism for antiepileptic drugs?

There is evidence that drug-induced embryonic arrhythmia initiates phenytoin (PHT) teratogenicity. The arrhythmia, which links to the potential of PHT to inhibit a specific potassium channel (Ikr), may result in episodes of embryonic ischemia and generation of reactive oxygen species (ROS) at reperfusion. This study sought to determine whether the proposed mechanism might be relevant for the teratogenic antiepileptic drug trimethadione (TMO). Effects on embryonic heart rhythm during various stages of organogenesis were examined in CD-1 mice after maternal administration (125-1,000 mg/kg) of dimethadione (DMO), the pharmacologically active metabolite of TMO. Palatal development was examined after administration of a teratogenic dose of DMO and after simultaneous treatment with DMO and a ROS-capturing agent (alpha-phenyl-N-tert-butyl-nitrone; PBN). The Ikr blocking potentials of TMO and DMO were investigated in HERG-transfected cells by using voltage patch-clamping tests. DMO caused stage-specific (gestation days 9-13 only) and dose-dependent embryonic bradycardia and arrhythmia at clinically relevant maternal plasma concentrations (3-11 mM). Hemorrhage in the nasopharyngeal part of the embryonic palate (within 24 h) preceded cleft palate in fetuses at term. Simultaneous treatment with PBN significantly reduced the incidence of DMO-induced cleft palate, from 40 to 13%. Voltage patch-clamping studies showed that particularly DMO (70% inhibition), but also TMO, had Ikr blocking potential at clinically relevant concentrations. TMO teratogenicity, in the same way as previously shown for PHT, was associated with Ikr-mediated episodes of embryonic cardiac arrhythmia and hypoxia/reoxygenation damage.

615 Prospective comparison of three-dimensional versus two-dimensional ultrasound in the diagnosis of fetal cleft lip and palate

615 Prospective comparison of three-dimensional versus two-dimensional ultrasound in the diagnosis of fetal cleft lip and palate

Tissue uptake of EGF receptor antisense oligonucleotides in organ culture of fetal mouse palates and their effects on in vitro palatogenesis

To investigate the incorporation of oligonucleotides (ODNs) into the tissues of cultured fetal mouse palates and their effects on in vitro palatogenesis, we cultured day-12.5 fetal mouse palates in a chemically defined serumless medium supplemented with either antisense or sense ODNs to epidermal growth factor receptor (EGF-r). The EGF-r ODNs were found to be incorporated into the palatal tissue and remained detectable for at least 72 hr. Immunohistochemical and immunoblot analyses revealed that the treatment with 5μM EGF-r antisense ODN suppressed the production of EGF-r protein. No pathological change was observed in the explanted palates when they were treated with 5 μM EGF-r antisense or sense ODNs, but the treatment with 10 or 20 μM ODN caused pyknotic changes in the palatal epithelium, probably due to the ODN toxicity. The present results indicate that under optimal conditions, antisense ODNs to EGF-r can be incorporated into fetal organs cultured in vitro and specifically inhibit the production of EGF-r protein. Since the suppression of the production of EGF-r protein did not prevent the palate fusion, EGF and/or EGF-r alone may not play a critical role in palatogenesis, as suggested by previous studies. The antisense ODN technique could be of potential use for analyzing the roles of specific molecules in normal and abnormal morphogenesis.

Induction of hepatic CYP2B1/2 by a teratogenic compound cis-1-[-4-(p-menthane-8-yloxy)phenyl]piperadine (YM9429) in rats.

A teratogenic compound cis-1-[-4-(p-menthane-8-yloxy)phenyl]piperadine (YM9429) selectively induces skeletal malformations characterized by cleft palate in rat fetuses. In the present study, we investigated the effect of YM9429 on hepatic cytochrome P-450s and their activities in rats. Oral administrations of YM9429 at a dose of 250, 500 or 750 mg/kg daily for 3 days induced cytochrome P-450 contents in a dose-dependent manner. Concomitant induction of enzyme activities of benzphetamine N-demethylase, erythromycin N-demethylase and, to a lesser extent, aminopyrine N-demethylase was observed. Immunoblot analysis revealed that YM9429 up-regulated hepatic levels of CYP2B1/2 and CYP3A1/2 proteins. A single dose of YM9429 at 250 mg/kg induced CYP2B1/2 protein levels significantly. These results suggest that YM9429 is a strong inducer of cytochrome P-450 with characteristics resembling those of phenobarbital.