Extension of orofacial cleft size and gestational bleeding in early pregnancy.

OBJECTIVE: The oronasal cavity in humans develops during embryonic day 30-60. There are three critical periods when this process can be affected, resulting in a specific type of orofacial cleft: cleft lip (CL), cleft palate (CP), or most serious, total cleft lip+palate (CLP). We assessed whether gestational bleeding during early pregnancy might act to produce a non-specific worsening of embryo status resulting in extension of the basic cleft type (CL or CP) into more serious CLP. STUDY DESIGN: In a group of the child patients with orofacial clefts, the cleft spectrum was correlated with first trimester gestational bleeding reported by the mother. Data were also related to the gender of patients, hereditary factors and additional malformations. RESULTS: Among 2524 mothers who gave birth to babies with an orofacial cleft in the Czech Republic during 1983-2009, 253 (10.0%) had gestational bleeding. Among the children with an orofacial cleft, 497 (19.7%) had an orofacial cleft among relatives and 297 (11.8%) exhibited an additional congenital malformation. In comparison with mothers without bleeding, there was significant increase of children with CLP (p < 0.01) at the expense of children with CP, whose number significantly decreased (p < 0.01) in the bleeding mothers. In the group of children with clefts among relatives we did not find any significant change associated with bleeding. The maternal bleeding was more frequent in children with additional malformations, but this difference was not significant (p = 0.112). CONCLUSION: We hypothesize that size/extent and therefore seriousness of orofacial cleft might increase as a consequence of hypoxia resulting from gestational bleeding.

A case of conjoined twin's cephalothoracopagus janiceps disymmetros.

Conjoined twins are rare variants of monozygotic twins, which result from an incomplete late division of the embryonic disk. Here we report the rarest case of conjoined twins - the male cephalothoracopagus janiceps disymmetros - born in prenatal week 30, from the archive of the Department of Teratology of the Institute of Experimental Medicine AS CR in Prague. The crown-rump length of each twin, 21cm, corresponded to prenatal week 22 in a normal gravidity. The head, chest and upper portion of the abdomen of the twins were fused. The anatomical features of these extremely rare conjoined twins and the observed external anomalies as a narrow nose with a single nostril, male hypoplastic genitalia, partially duplicated sella turcica, spina bifida and further abnormalities are described and documented.

The lack of isolated palatal clefts in Czech Gypsies.

Orofacial clefts are usually divided into three basic types: isolated cleft lip (CL), cleft lip and palate (CLP) and isolated cleft palate (CP). The incidence of specific cleft types in a population and their relative numbers show specific differences between ethnic groups and races. However, there are no available data about the incidence and relative numbers of orofacial cleft types (CL, CLP, CP) in the gypsy ethnic group. The aim of this study was to compare relative numbers of specific types of orofacial clefts between the Czech gypsy and non-gypsy populations. We conducted a retrospective epidemiological study using a set of all living patients with orofacial clefts born in the Czech Republic from 1964 until 2002. The cleft patients were subdivided into three groups: 5304 non-gypsy children, both parents of whom were non-gypsies (NN), 98 gypsy children, both parents of whom were gypsies (GG) and 18 children with one parent non-gypsy and one parent gypsy (NG). The relative number of isolated CP was 37.1% in NN children. However, the relative number of CP was significantly reduced to 5.1% (P < 0.01) in the GG group. Conversely, the relative number of CLP was higher (P < 0.01) in the GG group (62.2%) in comparison to the NN group (39.2%). The tendency to decrease in the relative number of CP and increase in the relative number of CLP was also apparent in the NG group, but not so well expressed. We hypothesize that the decrease in CP and increase in CLP and CL in gypsies might be caused by their genetic predis-position to CL. Since the CP originates later than CL during embryonic development, some CP arise in embryos with already existing CL giving rise to CLP. Consequently, the missing isolated CP might be hidden in the group of CLP patients postnatally.

Satellite nucleoli in the megakaryocytic lineage of rats.

The present study was undertaken to provide more information on the incidence of satellite nucleoli in developmental stages of the megakaryocytic lineage. Satellite nucleoli representing solitary silver stained nucleolus organizer regions (AgNORs) present in nuclei in addition to other nucleolar types were observed in all stages of megakaryocytic development. However, the incidence of satellite nucleoli was more frequent in mature megakaryocytes than in less differentiated immature megakaryoblasts and naked megakaryocytic nuclei representing the terminal stages of megakaryocytic development after loss of the cytoplasm transformed to thrombocytes. There is a possibility that the increased incidence of satellite nucleoli in mature megakaryocytes might be due to the loss of AgNORs from active nucleoli characteristic for immature cells. The decreased incidence of satellite nucleoli in naked megakaryocytic nuclei might reflect their disintegration in the terminal stages of the megakaryocytic development.

Nucleolar coefficient of granulocyte precursors and granulocytes after visualization of nucleoli by two different methods.

To clarify differences in the incidence and number of nucleoli in the granulopoietic lineage, these nuclear components were studied in human and rabbit granulocyte precursors and granulocytes after visualization by 2 widely employed cytochemical procedures, i.e. a procedure for the demonstration of RNA and the silver reaction for the demonstration of nucleolar silver stainable proteins (SSPs). In early stages of the granulocyte proliferating compartment, substantial differences were not found between specimens in which nucleoli were visualized by both procedures. However, in contrast to specimens stained with the silver reaction, the number of cells without nucleoli was substantially larger in advanced stages of granulocyte development in specimens stained for RNA. The number of nucleoli per cell as expressed by the nucleolar coefficient was generally larger in specimens stained with the silver reaction for nucleolar SSPs. These differences were significant starting with the stage of myelocytes. Moreover, in specimens stained with the silver reaction, most of human mature granulocytes did not contain nucleoli but nucleoli were present in all mature granulocytes of rabbits. Such differences were not observed in specimens stained for RNA in which most granulocytes were without RNA-containing nucleoli. Thus, the evaluation of the presence or absence of nucleoli in specimens depended on the visualization procedure. It is likely that in micronucleoli which are characteristic for terminal differentiation of the granulocytic lineage, RNA-containing structures may be lost or are below the detection limit of the light microscope. In addition, differences in the presence of nucleoli exist apparently between human and rabbit granulocytes in specimens stained for SSPs but not in those stained for RNA.

Significant differences in the incidence of orofacial clefts in fifty-two Czech districts between 1983 and 1997.

Between 1983 and 1997 a total of 2029 children with CL/P (cleft lip, cleft lip and palate or cleft palate), who were born in the Bohemian districts of the Czech Republic and who underwent surgery and treatment at the Clinic of Plastic Surgery in Prague, were analysed. One possibility for decreasing the risk of delivery of a child with CL/P is to decrease or eliminate its prenatal exposure to embryotoxic factors. Detection of the embryotoxic factors acting at the individual level (e.g. elevated temperature, drug consumption, x-ray examination or infection) is easier than the detection of embryotoxic factors operating at the population level (e.g. water contamination, air pollution). When searching for the latter factors, we first have to reveal regional differences in CL/P incidence. The aim of the present paper was to determine significant differences in the mean incidence of newborns with CL/P in Bohemian districts during a 15 year period. The correlation between the incidence of CL/P and the birth rate in the different districts was also examined. The mean incidence of CL/P in all Bohemian districts was 1.86 per 1000 newborns (1.86/1000). Districts were divided into three groups, according to significant differences in the incidence of CL/P using a confidence interval. The lowest mean incidence of CL/P was detected in the Svitavy district (0.72/1000) and Louny (1.05/1000). The highest mean incidence was found in the Beroun district (2.86/1000). Besides Beroun, a high mean incidence of CL/P (more than 1.96/1000) was also found in Klatovy, Melník, Tábor, Kolín, Semily, Ceská Lípa, Pardubice, Teplice, Ceský Krumlov, Sokolov, Chomutov, Praha-západ, Jicín, Rakovník, Kladno, Prachatice, Rokycany, Tachov, Liberec, Pelhrimov. Paradoxically, the districts with a higher or lower birth rate exhibited a lower (1.62/1000) or higher (1.92/1000) incidence of CL/P, respectively. Future studies should elucidate whether the significant regional differences in the incidence of CL/P can be related to differing exposure of pregnant women to harmful environmental embryotoxic factors.

The asymmetric distribution of interphasic silver-stained nucleolus organizer regions in human and rat proerythroblasts.

The distribution of SSPs representing AgNORs was studied in human as well as rat proerythroblasts to provide information on the distribution of these nucleolar components in highly immature and proliferating non-neoplastic cells. The distribution of SSPs was asymmetric and most of the cells contained one nucleolus which possessed a larger number of these nucleolar components than the remaining nucleoli. Such nucleolus might be functionally dominant, since the number of nucleolar SSPs is apparently related to the nucleolar biosynthetic activity. On the other hand, when a proerythroblast possessed only one nucleolus, the number of SSPs in such a cell was very similar to the sum of SSPs in a polynucleolar cell. The asymmetric distribution of SSPs characteristic for most proerythroblasts disappeared in the terminal stages of the erythroblastic development. Cells in such stages, as described previously, were characterized by the presence of a limited number of single SSPs.

Cleft beak induced by hydrocortisone in the chick is prevented by increased cell division after experimental reduction of amniotic fluid.

Hypoplasia of the medial nasal process has been reported in chick embryos on embryonic day (ED) 5, 24 h after their exposure to hydrocortisone (HC). As a result, the cleft beak occurs in 80-100% of specimens on ED 9. In order to analyze its influence on cell proliferation, HC was injected intra-amniotically into embryos on ED 4, and the mitotic index and number of BrdU-positive cells were evaluated 24 h later, both in the epithelium and mesenchyme of the medial nasal processes, on serial frontal histological sections. Two hours after BrdU administration, there were 50% of labeled mesenchymal cells in the embryos exposed to HC and only 23% in the control group. The mitotic index of mesenchymal cells was significantly lower in the HC group than in the controls. The epithelium showed no significant difference. HC seemed to prevent the mesenchymal cells from entering mitosis. The cleft beak in the embryos exposed to HC on ED 4 was totally eliminated by tearing open the amnion (amniotomia) and allowing fluid to leak out on ED 5. In some of specimens exposed to HC, the mitotic index was investigated at six time intervals from 15 to 120 min after amniotomia. A significant increase in the mitotic index was detected in the mesenchymal cells of the medial nasal processes during the first hour after amniotomia. Such a prompt increase of the mitotic activity may be hypothetically explained by release of the HC from its receptor binding as a consequence of outflow of the amniotic fluid together with the HC pool, and freeing of the mesenchymal cells, blocked in the G2 phase, to enter mitosis. As a result, the hypoplasia of the medial nasal process might be compensated and the development of the cleft beak prevented.

Different embryotoxic effect of vitamin A and B-carotene detected in the chick embryo.

Teratogenicity of vitamin A was firstly detected in experimental animals in 1953. Nearly 30 years later, teratogenicity of vitamin A analogue-isotretinoin was reported in humans. Isotretinoin induces serious birth defects of craniofacial and central nervous system, cardiovascular system and thymic malformations--in about 25% of babies exposed during the first trimester of their prenatal development. The biological interconversion of isotretinoin to vitamin A is known. That is why later epidemiological studies focused on high vitamin A intake in pregnant woman: Women who use daily vitamin A supplements during early pregnancy have approximately a two-fold increased risk of giving birth to a malformed baby. On the basis of these data, replacement of vitamin A has been recommended with its natural precursor B-carotene which is supposed to be more safe for pregnant woman due to its limited absorption from intestine. Aim of the present paper was to test a possible direct embryotoxic effect (i.e. lethality + teratogenicity) of B-carotene in chick embryos and to compare these results with known embryotoxicity of vitamin A in the same experimental model. Single subgerminal or intaamniotic injection of vitamin A or B-carotene within day 2-5 of incubation was used for estimation of the beginning of the embryotoxicity range determining the minimal embryotoxic doses. Vitamin A started to affect development between doses 0.3-0.3 microm [corrected] per embryo. Malformations of head, extremities and heart were detected similarly like in laboratory mammals and in man. B-carotene exhibited such an effect neither after injection of the highest tested doses-100 microm [corrected] per embryo. The results documented the strong difference in embryotoxicity between vitamin A and B-carotene. After theoretical extrapolation of the results achieved in the chick embryo, the minimal embryotoxic doses of vitamin A in mammals were estimated to be between 0.1-1 mg/kg of maternal weight and those of B-carotene to be more than 1000 mg/kg of maternal weight. Human epidemiological studies have proved teratogenicity of vitamin A after daily doses 25,000 i.u.-8.3 mg (0.13 mg/kg)- and reduction of its maximum intake has been recommended to 10,000 i.u. per day (0.05 mg/kg). The results about teratogenicity of vitamin A achieved in the chick embryo are in agreement with such a recommendation. Intake of vitamin A in the food is sufficient for pregnant woman in common Czech population. Therefore, an artificial supplementation of vitamin A brings risk of overdosage. If supplementation by vitamin A is unavoidable during pregnancy, B-carotene should be preferred.

Timing of exchange of the maxillary deciduous and permanent teeth in boys with three types of orofacial clefts.

Timing of exchange of the deciduous and permanent maxillary teeth was investigated using dental plaster casts of 163 boys with total unilateral cleft (UCLP), 82 boys with bilateral cleft (BCLP), and 97 boys with isolated cleft palate (CP). All patients were treated at the Prague Plastic Surgery Clinic. The results were compared with a control group of 294 schoolboys. To evaluate the course of eruption, the proportion of each erupted teeth in each year of age was employed. In boys with UCLP, eruption of the permanent maxillary lateral incisors and the permanent maxillary second molar was retarded on the cleft side. On the non-affected side, no delay of eruption was observed, but earlier eruption was found in the permanent maxillary canine and in the permanent maxillary first and second premolars. In boys with BCLP, the highest retardation of eruption was found in the permanent maxillary lateral incisor and in the permanent maxillary first molar. The permanent maxillary canine and both permanent maxillary premolars erupted earlier than in the control group. In boys with CP, only the permanent maxillary central incisors erupted earlier. The maxillary deciduous canines and the second molars were both lost early. We conclude that the developmental disturbances of the maxillary jaw and teeth in patients with orofacial clefts are also associated with alteration of timing of dental exchange.

Teratogenic and lethal effects of long-term hyperthermia and hypothermia in the chick embryo.

The teratogenic effect of maternal hyperthermia is well known in laboratory animals and is presumed to exist also in humans. The aim of our study was to describe the embryotoxic effect of long-term higher and lower incubation temperatures on the chick embryo. Chick embryos were incubated within days 1 to 9 at 12 different incubation temperatures ranging from 31 to 42 degrees C. On the basis of our results, we estimated that there are three upper and lower critical thresholds of the incubation temperature: the first thresholds are 31 and 42 degrees C, at which all embryos died; the second thresholds are 32 and 41 degrees C, at which all living embryos were malformed; the third thresholds are 33 and 40 degrees C, at which some of the living embryos were without structural malformations, but their weight was shifted down and up with lower and higher temperature, respectively. The incubation temperature of 37 to 38 degrees C was optimal. Typical malformations detected on day 9 of incubation were microphthalmia, gastroschisis, caudal regression syndrome, and hyperlordosis, all of which occurred in dead embryos several times more frequently than in living embryos. CNS malformations were only sporadically present on day 9, as most of specimens bearing CNS defects died during the first days of incubation.

Sex differences in the incidence of orofacial clefts and the question of primary prevention in families with genetic risk.

Systematic registration of all children with orofacial clefts in Bohemia (Czech Republic) started at the Clinic of Plastic Surgery, Prague in 1964. A sample of 181 affected children with positive family histories (i.e. one of the parents had some type of orofacial cleft) was selected for the present study. The aim of this study was to follow the relation not only between the type of cleft in the child and in its parent, but also between the sex of the child and of the affected parent. Among children of mothers with cleft lip 68% were boys and only 32% were girls with cleft lip or cleft lip and palate. If the mother had cleft lip and palate, the same cleft type was found in 64% of boys and only 15% of girls. If the mother had cleft palate, the same cleft type was found in 37% of boys and 51% of girls. Very similar results were found for affected fathers and their children, with only one exception: among children of fathers with cleft lip and palate, the percentage of boys and girls with CLP was 43% and 40%, respectively. We can conclude that the cleft type in a child depends not only upon the cleft type present in the mother or father, but also upon the sex of the child. There was higher risk to have the orofacial cleft in sons of mothers with CL or CLP or fathers with CL and daughters of mothers or fathers with CP. The combination of the preconception choice of the sex of the baby with the ultrasonography method for the prenatal screening of malformations could decrease the risk delivering a child with an orofacial cleft in families with a genetic predisposition.

Asymmetric mitoses revealed by AgNORs in developing rat blood cells.

Immature cells of erythroid as well as granulocytic series were studied in rat hemopoietic bone marrows to provide a basic information on the asymmetric distribution of AgNORs in anaphases and telophases of these cells. The results clearly demonstrated that such asymmetric mitotic divisions with respect to the distribution of AgNORs to future daughter nuclei represent a regular phenomenon in the course of erythroid as well as granulocytic maturation and thus are not limited to neoplastic cells in which this phenomenon was described previously.

Nucleoli and argyrophil nucleolus organizer regions (AgNORs) of cells of the megakaryocytic line in the rat.

The main maturation stages of Norway rat megakaryocytic series, megakaryoblasts and mature megakaryocytes, stained by silver for demonstration of argyrophil nucleolus organizer regions (AgNORs) were investigated to provide basic information on the number of nucleoli and interphasic AgNORs in these cells. The results showed that megakaryoblasts as well as mature megakaryocytes possess numerous nucleoli; their number and also the number of AgNORs is significantly higher in less mature than in more mature cells. The number of AgNORs in megakaryocytes of the Norway rat and man are virtually the same, although the numbers of nucleolar organizers per haploid chromosome set differ markedly. This fact leads to the conclusion that the number of interphasic AgNORs depends on the function and metabolic state of the cell rather than on the number of nucleolar organizers.

Accumulation of activated lymphocytes in liver of Listeria factor Ei treated rabbits.

Factor Ei isolated from Listeria monocytogenes caused in rabbits 1 d after intravenous administration activation of peripheral blood lymphocytes in terms of rRNA biosynthesis. Increase of the number of these active lymphocytes was observed not only in peripheral blood, but also in spleen, lung, kidney, and liver. when the number of lymphocytes was related to the amount of the organ tissue cells, the increment appeared significant only in the liver where the number of active lymphocytes exceeded the control value by one order of magnitude. Based upon this observation we concluded that the accumulation of Listeria factor Ei activated lymphocytes occurred in liver. This characteristic is considered an additional immunomodulative property of Listeria factor Ei similar to the Escherichia coli lipopolysaccharide.

Development of nucleolar apparatus in the chick primitive erythroid cells.

The primitive erythroid line cells of chick embryos were studied during embryonic days 2-14 by means of a cytochemical method to investigate the appearance and frequency of the main nucleolar types. The populations of erythroblasts and erythrocytes were classified according to the presence of functionally dominant nucleoli in their nuclei. In the course of primitive erythroid cell differentiation and maturation, compact nucleoli and nucleoli with nucleolonemas (both supposed to be RNA biosynthetically active) were gradually replaced by ring-shaped nucleoli and finally by micronucleoli reflecting the reversible and irreversible inhibition of RNA synthesis, respectively. The occurrence of the main nucleolar types and their values in primitive erythroid cells of the developing chick depend not only on the maturation stage of the blood cells, but also on the developmental stage of the chick embryo. In comparison with the definitive erythroid line of the post-hatching chick and hen, the cells of the chick embryonic primitive erythroid line possess relatively high values of "active" nucleolar types. These are still present in advanced maturation stages, and occur also as definitive erythroid lines of lower vertebrates.

The effect of actinomycin D on the megakaryocytic line in rat.

Four-day administration of actinomycin D to the albino rat in daily doses of 25 micrograms/kg decreased--in relation to all blood cells of the bone marrow--the values of the more mature stages of the megakaryocytic line, i.e. megakaryocytes and naked megakaryocyte nuclei. The number of megakaryoblasts was not influenced.

rRNA synthesis in proerythroblasts and hepatocytes after cyclosporin A administration in chick embryo.

The ribosomal RNA (rRNA) synthesis in the proerythroblasts and hepatocytes of the chick embryo was examined using the nucleolar test during embryonic days 5-10 after intraamniotic administration of single doses of cyclosporin A (CsA). Administration of the 0.75 or 7.5 g of CsA 5th day of embryonic development caused a significant decrease in proerythroblasts and hepatocytes numbers with active rRNA synthesis. Five days after the CsA administration, the percentage of proerythroblasts with biosynthetically active nucleoli already did not differ from the control while the decreased numbers of hepatocytes actively synthetizing rRNA remained statistically significant. Our results indicate, that CsA inhibits the rRNA biosynthesis in embryonic immature cells. Depending on the cell type, the effect of CsA and its dynamics may be apparently different.

The effect of cyclosporin A on the proliferating chick embryo erythroblasts.

The proportion of proliferating erythroblasts, i.e. proerythroblasts, basophilic erythroblasts and polychromatophilic erythroblasts in blood islands of the chick embryo yolk sac, were counted during embryonic days 2-10. From day 2 when high amounts of erythroblasts signalized the onset of embryonic erythropoiesis, the percentage of less mature erythroid cells gradually decreased. Intraamniotic injection of cyclosporin A in doses 1.5 or 15.0 micrograms per embryo on day 5 led to significant changes in the proportion of proliferating erythroblasts in the yolk sac blood islands. We speculate that these changes were caused initially by the release of the more mature cells into the circulation and later by a dose-dependent decrease in the number of stem cells. The estimation of proerythroblast percentage from all proliferating erythroblasts in the yolk sac blood islands may serve as a valuable indication of toxic damage in the late avian embryo.

Maternal hyperthermia and infection as one of possible causes of orofacial clefts.

It has been proposed that the hyperthermia in pregnant women may be associated with birth defects in their offsprings. We analysed the retrospective interview data on probable febrile illness during critical period of orofacial clefts development in 992 mothers of boys and girls with three types of orofacial clefts--cleft lip, unilateral or bilateral cleft lip and palate and cleft palate. A number of mother that gave the positive answer was relatively high and varied from 24% to 33%. There were no significant differences of the incidence of mother febrile illness between three groups of orofacial clefts in boys and girls. These data support presumption on the harmful effect of febrile illness during early pregnancy on craniofacial development.