Studies on teratological testing using chicken embryos--effects of solvents, injection sites and the age of the embryo.

The purpose of this study was to examine the effects of solvents, injection sites and embryo age when using chicken embryos for teratological testing. The results obtained were as follows: 1) Solvents: distilled water, physiological saline, sesame oil, 25% ethanol, 0.5% carboxymethylcellulose and 0.1% methylcellulose solution were not toxic in Day-4 embryos (eggs incubated for 4 days). 2) With 6-aminonicotinamide, air space injection more effectively induced malformations in chicken embryos. With boric acid, however, yolk sac injection was better. It was shown therefore that the appropriate injection site varied according to the test drug. 3) 6-aminonicotinamide induced characteristic malformations when injected into embryos of various ages ranging from 4 to 13 days of incubation. On the other hand, boric acid was teratogenetic only when injected into Day-3 or Day-4 embryos. It seems, therefore, that the age of the embryo at the time of administration is of critical importance and that the optimum time of administration varies according to the test drug.     

Effects of ethanol on the primitive streak stage mouse embryo

Recent studies of mouse models have suggested that malformations associated with the fetal alcohol syndrome (FAS) are caused by the effects of ethanol on early embryos during gastrulation and neurulation. A study of Xenopus laevis embryos showed that exposure of gastrula stage amphibian embryos to ethanol inhibits migration of the mesodermal cells, causes formation of small neural plates, and subsequently causes hypoplastic craniofacial malformations in tadpoles. We now report effects of ethanol on the primitive streak stage mouse embryos. An ethanol solution (25%) was injected intraperitonealy twice into mice of 6.5-7.0 days of pregnancy at a dose of 0.015 ml/gm of body weight. Histological and morphometric examinations of 7.5-day embryos, 20 hr after the second injection, showed that the epiblast layer was disorganized and shrunk with formation of many blebs. In addition, formation of the mesodermal cell layer was retarded in the ethanol-treated embryos, suggesting that exposure of gastrula stage embryos to ethanol causes similar abnormalities in mouse and Xenopus embryos. These results suggest that the inhibition of the morphogenetic movements during gastrulation may be the primary effect of ethanol in causing major craniofacial malformations of FAS.     

Cardiovascular malformations induced by bromodeoxyuridine in the chick embryo

For the study of morphogenesis and early embryonic development, 5-bromodeoxyuridine (BUdR), a halogenated analogue of thymidine, is incorporated into replicating DNA and serves as a valuable tool. To study the teratogenicity of BUdR on the developing chick cardiovascular system, we topically administered graded doses of BUdR (32.6-325.6 nmol) in ovo during Hamburger-Hamilton stages 15 to 16. We also administered to a parallel group of embryos corresponding nanomole doses of thymidine during identical stages of development. In the thymidine-treated group, survival rates and cardiovascular anomaly rates did not differ statistically from those in the chick Ringer's control group. Both survival rates and cardiovascular anomaly rates in the BudR-treated group were dose-responsive. Among 78 embryos with cardiovascular anomalies induced by BUdR, vascular malformations were found in 96%. These anomalies included interruption of the right fourth aortic arch, absence or hypoplasia of the right and/or left sixth aortic arch, and persistence of the left fourth aortic arch. Interruption of the right fourth aortic arch was always associated with intracardiac anomalies. Intracardiac anomalies were found in 54% of the embryos; these included ventricular septal defect, double outlet right ventricle, and persistent truncus arteriosus. Subclavian artery malformations were noted in 95% of the embryos. Possible mechanisms for BUdR-induced malformations in the cardiovascular system of the chick are discussed.     

The effects of minoxidil on the development of the chick embryo

The effects of minoxidil were studied on chick embryos of 24 and 48 hours of incubation. Minoxidil (3%) was injected into the air sacs of the eggs at doses of 20, 30, 40, and 50 microliters per egg. The controls received 100 microliters of physiological saline. All the embryos, including controls, were examined at day 13. The total number of eggs used in this study was 300. At 24 hours incubation, the percentage of survival ranged from 87 to 21 as the dosages of minoxidil were increased from 20 microliters to 50 microliters per egg (controls = 87%). The survival of the embryos ranged from 79% to 9% after the 48-hour treatment with the similar dosages of minoxidil utilized for the 24-hour group (controls = 83%). A low incidence of gross malformations such as twisted limbs, abnormal beak, short neck and everted viscera were observed; however, the increased incidence was not statistically significant when compared to controls. Body hemorrhage and edema were of high occurrence among the treated embryos. These effects are probably secondary to the known pharmacological effects of minoxidil. The frequency and types of gross malformations did not vary much in the 24 or 48-hour treated groups.     

Modulation by ellagic acid of DCA-induced developmental toxicity in the zebrafish (Danio rerio)

The ability of ellagic acid (EA) to modulate dichloroacetic acid (DCA)-induced developmental toxicity and oxidative damage was examined in zebrafish embryos. Embryos were exposed to 20 mM EA administered concomitantly with 32 mM DCA at 4 hours postfertilization (hpf) and 20 h later. Embryos were observed through 144 hpf for developmental malformations, and production of superoxide anion (SA) and nitric oxide (NO) was determined in embryonic homogenates. DCA was shown to produce developmental abnormalities and significant levels of SA and NO in zebrafish embryos. EA exposure alleviated the developmental malformations observed in treated embryos and decreased the levels of SA and NO in those same embryos. Less than 10% of DCA + EA exposed embryos showed developmental malformations compared to 100% of embryos treated with DCA alone. Animals in this group that developed malformations were shown to have fewer defects than those treated with DCA only. Taken together, the results confirm the involvement of oxidative stress in the developmental toxicity of DCA in zebrafish embryos, and suggest possible protection against those effects with the use of antioxidants.     

Teratogenicity of Ni2+ inXenopus laevis,assayed by the FETAX procedure

The teratogenicity of Ni2+ was tested by the FETAX (Frog Embryo Teratogenesis Assay: Xenopus) procedure in the South African frog, Xenopus laevis. In seven assays, beginning at 5 h postfertilization, groups of Xenopus embryos were incubated for 96 h in media that contained Ni2+ (added as NiCl2) at concentrations ranging from 1 x 10(-7) to 3 x 10(-3) mol/L; control groups were incubated in the same medium without added NiCl2. At 101 h postfertilization, surviving embryos were counted, fixed in formalin, and examined by microscopy to determine their developmental stages, malformations, and head-to-tail lengths. In control embryos, survival was greater than or equal to 95% and malformations were less than or equal to 7%. Malformations were found in greater than 95% of embryos exposed to Ni2+ concentrations greater than or equal to 5.6 mumol/L. The most frequent malformations in Ni(2+)-exposed embryos were ocular, skeletal, and intestinal deformities; less common malformations included facial, cardiac, and integumentary deformities. Other abnormalities, not categorized as malformations, included stunted growth, dermal hypopigmentation, and coelomic effusions or hemorrhages. The median embryolethal concentration (LC50) of Ni2+ was 365 (SE +/- 9) mumol/L; the median teratogenic concentration (EC50) was 2.5 (SE +/- 0.1) mumol/L; the Teratogenic Index (TI = LC50/EC50) was 147 (SE +/- 5), indicating that Ni2+ is a potent teratogen for Xenopus laevis. Experiments in which Ni(2+)-exposures were limited to specific 24 h periods showed that Xenopus embryos were most susceptible to Ni(2+)-induced malformations on the second and third days of life, during the most active period of organogenesis.     

Effect of Magnetic Field on Chick Morphogenesis

It is well known that development of organisms, as well as normal functioning of different systems, is to a large extent affected by environmental factors. To study the effect of magnetic field on the chick morphogenesis, fresh fertilized eggs of White Leghorn were incubated to the definitive primitive streak stage [Hamburger-Hamilton (H.H.) stage 4]. The embryos were mounted on a watch glass using News in vitro culturing technique and then exposed to a homogeneous magnetic field of 5000 ± 200 Oe for 1 h. The controls were maintained simultaneously without the magnetic field. The experimental and control embryos were further incubated for 24 h so as to obtain the H.H. stage 12 in the controls. It was observed that experimental embryos were considerably retarded, as evidenced by shortening of the embryonic long axis and by other malformations. In 14 out of the 25 experimental embryos, the brain was poorly developed with open neural tube. In 18 of the 25 experimental embryos, the somites were diffused, and in 12 of the 25 experimental embryos, the heart was developed with slight displacement. All the controls developed normal to H.H. stage 12. Histological observations of the neural structures in the sectioned experimental embryos show increase in the number of dividing cells and their dispersed distribution throughout the neuroepithelium. The malformations recorded are attributed to the environmental stress effect caused by the magnetic field as manifested by its effect on mitotic activity, interkinetic nuclear migration in neuroepithelium and cell locomotion in general.     

Threatened abortion, hormone therapy and malformed embryos

Causal relations between maternal genital bleeding, supportive hormone therapy and external malformations of the embryos were investigated with special reference to the critical period of organogenesis. This was done using morphological and obstetrical data obtained by Nishimura and his associates from 667 undamaged embryos derived from induced abortions whose mothers had genital bleeding in early pregnancy. In addition, data from 90 embryos with polydactyly and 38 with limb reductions in the Nishimura collection were used for case history studies. Evidence was presented to demonstrate that, for major malformations such as CNS anomalies, cleft lip, polydactyly and limb reductions, maternal genital bleeding was not a cause but a consequence of the conception of an abnormal embryo. No indication was revealed that exogenous female hormones currently used in Japan for preventing miscarraiges could produce major malformations recognizable at the embryonic stage, including limb reductions, nor salvage the severely malformed embryos. This does not however mean to exclude the possible relationship of progestogens/estrogens intake during early pregnancy with an increased incidence at birth of certain internal and/or external malformations. It was suggested that most, if not all, of the minor anomalies observed at certain embryonic stages are kinds of normal variants without any functional impairment of embryonic development.     

Effects of pulsing electromagnetic fields on the prenatal and postnatal development in mice and rats: in vivo and in vitro studies

Electromagnetic fields (EMF) might have various biological effects on the developing embryo. We studied the effects of pulsing electromagnetic fields (PEMF) on the in vitro development of preimplantation mouse embryos and of early somite rat embryos as well as on the in vivo development of rat embryos. We used PEMF at frequencies of 1, 20, 50, 70, and 100 Hz with a tension of 0.6 V/m. The embryos were exposed to PEMF throughout the experimental period. PEMF at frequencies of 20 and 50 Hz were embryotoxic, inhibiting over 50% of blastocysts from hatching and further development, all within 72 h of culture. PEMF at frequencies of 50 and 70 Hz induced 22% and 30% incidence of malformations in 10.5 day old rat embryos after 48 h in culture. The main malformations were absence of telencephalic, optic, and otic vesicles and of forelimb buds. In addition, retarded growth and development manifested by fewer somites, reduction in crown-rump length, and retarded closure of the neural tube were found in many embryos. No significant pathological changes were found by TEM in PEMF-exposed embryos. Disappearance of microvilli and collapse of apical parts of endodermal cells were observed by SEM in many yolk sacs of embryos exposed to 50 and 70 Hz PEMF. A slightly reduced litter average, a reduction or increase of weight, and a delay in eye opening was observed among offspring of pregnant rats exposed throughout pregnancy to PEMF at frequencies of 20, 50, and 100 Hz. No malformations were observed among these offspring. The mechanism of PEMF-induced embryotoxicity and teratogeneity is unknown, as is the mechanism of the "protective effects" of the mother on the rat embryos exposed to PEMF in vivo.     

Heat-shock induced tolerance to the embryotoxic effects of hyperthermia and cadmium in mouse embryos in vitro

Mammalian embryos growing in vitro are harmed by short elevations in the culture temperature. However, a relatively mild hyperthermic exposure can induce thermotolerance, a transient state of resistance to the effects of a subsequent heat exposure. The present study examines the induction of tolerance to heat and cross-tolerance to another teratogen, cadmium, in day 8 CD-1 mouse embryos in vitro. The ability of a mild heat pretreatment (5 min at 43 degrees C) to partially protect embryos against an embryotoxic heat exposure (20 min at 43 degrees C) was demonstrated. The frequency of death was reduced from 43% to 20%, abnormal branchial arches from 44% to 13.2%, and retarded turning from 22% to 5% in pretreated embryos. Other malformations, such as small forebrains and microphthalmia, were not affected, and the rate of exencephaly was significantly increased. The same heat pretreatment (5 min at 43 degrees C) was also found to reduce the damaging effects of a subsequent exposure to 1.75 microM cadmium. In the absence of pretreatment, cadmium caused 55% embryo deaths and 87% malformations, but prior heat exposure caused significant reductions in these frequencies to 29% and 55%. The total morphological score was higher in the pretreated group, as were the measurements of the yolk sac diameter, crown-rump length, and head length. Thus, embryos that have developed resistance to hyperthermia are also partially protected against the harmful effects of a second teratogen, cadmium. The response of the embryo to elevated temperatures may be involved in the development of tolerance to a variety of stresses.     

Lectin teratogenesis. II: Demonstration of increased binding of concanavalin A to limb buds of rabbit embryos during the teratogenically sensitive period

The plant lectin concanavalin A (con A) causes malformations of rabbit embryos when 160 micrograms (in 40 microliter) are injected into the exocoelom on gestational days 12-15 but does not cause malformations on days 10-11. The purpose of this study was to investigate the mechanism for increased susceptibility of day 12-15 embryos to con A teratogenicity. Light microscopy of day 11 embryos 15-20 hr after treatment with con A revealed no observable difference from controls. Day 13 embryos at similar times exhibited limb buds with large areas that were denuded of ectoderm. Concurrent addition of alpha-methyl-D-mannoside (alpha MM), a specific inhibitor of con A, to the injection solution of day 13 embryos resulted in limb buds that appeared normal. The regions of con A binding to day 11 and day 13 embryos were visualized through epifluorescent microscopy of untreated embryos stained with fluorescein-labelled con A. Day 11 embryos exhibited moderate fluorescence on the surface of limb buds and the pericardial region. Day 13 embryos exhibited strong fluorescence of limb bud surfaces; the pericardial region remained moderately fluorescent. Addition of alpha MM to the incubation medium resulted in no fluorescence above background. Visualization of con A receptors was accomplished by ultrastructural analysis of forelimb buds stained with ferritin-labelled con A. Ferritin label was observed only on the surfaces of the ectoderm and was sparse over all regions of day 11 limb buds. In contrast, ferritin label was moderately heavy in all regions of the day 13 limb buds. No labelling occurred when the ferritin-labelled con A was preincubated with alpha MM. These observations indicate that the number of exposed con A receptors on limb buds of teratogenically sensitive embryos (day 13) is increased, compared with the number of exposed receptors on limb buds of younger, insensitive (day 11) embryos. The increased number of exposed con A receptors on limb buds during the teratogenically sensitive period provides not only increased binding of the lectin to sensitive embryos but also a potential mechanism for the anomalous attachment of distal regions of the limb buds to the body wall.     

The spontaneous occurrence of aortic arch and cardiac malformations in the white Leghorn chick embryo (Gallus domesticus)

Spontaneous aortic arch and cardiac malformations occur in White Leghorn chick embryos at a relatively high rate. Although this breed of Gallus domesticus is widely used for biomedical and biological research, no previous study has recorded the incidence of these defects. We found aortic arch malformations in 7.1% (14 of 196) and ventricular septal defects in 11.7% (23 of 196) of living embryos. Defects occurred alone or as a combined pattern. Our findings suggest that the cardiovascular defects in the chick embryo documented in past studies may, in some cases, have been part of normal spontaneous occurrence, rather than the major result of experimental manipulation.     

Elevated transforming growth factor beta2 enhances apoptosis and contributes to abnormal outflow tract and aortic sac development in retinoic X receptor alpha knockout embryos

Septation of the single tubular embryonic outflow tract into two outlet segments in the heart requires the precise integration of proliferation, differentiation and apoptosis during remodeling. Lack of proper coordination between these processes would result in a variety of congenital cardiac defects such as those seen in the retinoid X receptor alpha knockout (Rxra(-/-)) mouse. Rxra(-/-) embryos exhibit lethality between embryonic day (E) 13.5 and 15.5 and harbor a variety of conotruncal and aortic sac defects making it an excellent system to investigate the molecular and morphogenic causes of these cardiac malformations. At E12.5, before the embryonic lethality, we found no qualitative difference between wild type and Rxra(-/-) proliferation (BrdU incorporation) in outflow tract cushion tissue but a significant increase in apoptosis as assessed by both TUNEL labeling in paraffin sections and caspase activity in trypsin-dispersed hearts. Additionally, E12.5 embryos demonstrated elevated levels of transforming growth factor beta2 (TGFbeta2) protein in multiple cell lineages in the heart. Using a whole-mouse-embryo culture system, wild-type E11.5 embryos treated with TGFbeta2 protein for 24 hours displayed enhanced apoptosis in both the sinistroventralconal cushion and dextrodorsalconal cushion in a manner analogous to that observed in the Rxra(-/-). TGFbeta2 protein treatment also led to malformations in both the outflow tract and aortic sac. Importantly, Rxra(-/-) embryos that were heterozygous for a null mutation in the Tgfb2 allele exhibited a partial restoration of the elevated apoptosis and of the malformations. This was evident at both E12.5 and E13.5. The data suggests that elevated levels of TGFbeta2 can (1) contribute to abnormal outflow tract morphogenesis by enhancing apoptosis in the endocardial cushions and (2) promote aortic sac malformations by interfering with the normal development of the aorticopulmonary septum.     

Exposure of neural crest cells to elevated glucose leads to congenital heart defects, an effect that can be prevented by N-acetylcysteine

BACKGROUND: Diabetes mellitus during pregnancy increases the risk for congenital heart disease in the offspring. The majority of the cardiovascular malformations occur in the outflow tract and pharyngeal arch arteries, where neural crest cells are essential for normal development. We studied the effects of specific exposure of neural crest cells to elevated glucose on heart development. Antioxidants reduce the damaging effect of glucose on neural crest cells in vitro; therefore, we investigated the effect of supplementing N-acetylcysteine in vivo. METHODS: Cardiac neural crest of HH 8-12 chicken embryos was directly exposed by a single injection in the neural tube with 30 mM D-glucose (or 30 mM L-glucose as a control). To examine the effect of a reduction in oxidative stress, we added 2 mM N-acetylcysteine to the injected D-glucose. RESULTS: Exposure of neural crest cells to elevated D-glucose-induced congenital heart malformations in 82% of the embryos. In the embryos injected with L-glucose, only 9% developed a heart malformation. As expected, all malformations were located in the outflow tract and pharyngeal arch arteries. The frequency of heart malformations decreased from 82% to 27% when 2 mM N-acetylcysteine was added to the injected D-glucose. CONCLUSIONS: These data are the first to confirm that the vulnerability of neural crest cells to elevated glucose induces congenital heart malformations. The fact that N-acetylcysteine limits the teratogenicity of glucose implies that its damaging effect is mediated by an increase of oxidative stress in the neural crest cells.     

Effects of secondary bile acids on the intrauterine development in rats

The effects of secondary bile acids (lithocholic--LCA, and deoxycholic--DCA) on the in vivo development of rat embryos and fetuses were studied. Daily intraperitoneal injections of 2 ml of 1 mM LCA and of 5 mM DCA during days 6 till 15 of pregnancy resulted in an increase of resorptions among 20 day-old fetuses to 22.8% and 9.9%, respectively, vs. 6.2% in controls. Similar injections on days 12 to 19 resulted in an increase of resorptions to 10.3% after treatment with LCA and to 36% after treatment with DCA. Percent of retarded embryos was similar for both bile acids: 7.7 and 8.7% after injections on days 6-15 and 12.3-12.5% after injections on days 12-19 of gestation. This was accompanied by a significant increase in the wet weight of the placenta of living embryos. Intraamniotic injections of 2 microliters of 1 mM LCA into 10 day-old embryos resulted in 18.5% resorptions (vs. 7.5% in controls), 9.2% malformations, and 3.1% growth retardations observed on day 12 of pregnancy. The rate of resorptions following this treatment increased on day 20 of pregnancy to 71% vs. 16% in controls. No differences were found in the wet weight of 20 day-old living fetuses or their livers and placentas between experimental and control groups following i.p. or intraamniotic injections. In addition, single intrauterine instillation of 0.2 ml of 1 mM LCA 10-14 days before mating with normal isogeneic males resulted in 9% of malformations among 12 day-old embryos while malformations were absent in the saline-injected controls. The deleterious effects of secondary bile acids to the embryos were accompanied by damage to the visceral yolk sac. These findings may be significant in relation to the complications previously associated with cholestasis of pregnancy in humans.     

Microbubble-enhanced sonoporation: efficient gene transduction technique for chick embryos

The gene transduction technique is a useful method to study gene functions that underlie vertebrate embryogenesis. In this study, a new gene transduction technique is reported using microbubble-enhanced sonoporation (hereafter referred to as sonoporation) to achieve ectopic and transient gene expression for several embryonic organs including embryonic chick limb bud mesenchymes. The technique proposed in this study has the advantages of 1) relatively simple gene transduction procedures, and 2) efficient exogenous gene transduction and expression with lower damages to embryos. Green fluorescent protein (GFP) or LacZ was misexpressed in limb bud mesenchymes by sonoporation, with the introduced expression transiently detected in the injected sites. Most of the transduced chick embryos survived without showing significant embryonic abnormalities or cell death after sonoporation. To demonstrate its efficacy for assessing the effect of transient gene transduction, the Shh (sonic hedgehog) was transduced into the developing chick limb bud. The transduced limb bud displayed limb malformations including partial digit duplication. Advantages and possible future applications in relation to this method are discussed.     

Chick embryos exposed to trichloroethylene in an ex ovo culture model show selective defects in early endocardial cushion tissue formation

BACKGROUND: Formation of the primitive heart is a critical step for establishing a competent circulatory system necessary for continued morphogenesis, and as such has significant potential as a target for environmental insult. The goal of this study was to identify the initial cellular events that precede more superficially observable abnormalities resulting from exposing early chick embryos to trichloroethylene (TCE). METHODS: A whole embryo culture method was used to assess the susceptibility of endocardial epithelial-mesenchymal transformation in the early chick heart to TCE. This method has the benefits of maintaining the anatomical relationships of developing tissues and organs, instantaneously exposing precisely staged embryos to quantifiable levels of TCE in a protein-free medium, and the ability to directly monitor developmental morphology. RESULTS: A minority of embryos (Hamburger and Hamilton [HH] stage 13-14) exposed to TCE (10-80 ppm) were not viable after 24 hr in culture and exhibited a variety of gross malformations in a dose-dependent fashion. However, the majority of treated embryos remained viable and developed into HH stage 17 embryos that were superficially indistinguishable from vehicle-treated controls. Further analysis of the hearts of these superficially normal embryos by whole-mount confocal microscopy revealed selective reduction in the number of atrioventricular canal mesenchymal cells. Additionally, those mesenchymal cells that did develop migrated abnormally as long thin cords of adherent cells. CONCLUSIONS: The regional selectivity of these effects in the chick heart suggests a critical window of susceptibility to TCE in the epithelial-mesenchymal transformation of atrioventricular canal endocardium.     

D-(-)-beta-hydroxybutyrate-induced effects on mouse embryos in vitro

The effects of the "physiologically occurring" D-isomer of beta-hydroxybutyrate (D-BOHB) were evaluated in neurulating mouse embryos by using the technique of whole embryo culture. Following a 24-hour culture period D-BOHB induced malformations and growth retardation in a concentration-dependent manner. At a 48 mM concentration essentially all embryos exhibited neural tube defects, and decreased rates of glucose metabolism by the pentose phosphate pathway (PPP) and Krebs cycle were observed when compared to controls. The relationship between an inhibition of the PPP and induction of malformations by DL-BOHB has been reported, and thereby suggests a similar mechanism may be operating for the D-isomer. In contrast, the effect of the D-isomer on the Krebs cycle may result from a replacement of glucose intermediates by those generated from metabolism of D-BOHB. Concentrations as high as 20 mM D-BOHB have been reported in the serum of uncontrolled diabetic patients, and since ketones rapidly equilibrate across extraembryonic membranes, embryos in vivo may be exposed to concentrations equivalent to those which induced malformations in vitro. However, the incidence of malformations induced by D-BOHB was less than that reported for the DL-racemic mixture at equivalent concentrations, thereby suggesting that the L-isomer is also teratogenic. Therefore, until the presence and concentration of L-BOHB in the serum of diabetics are documented, the assessment of the impact of maternal ketosis on the embryo remains incomplete.     

Teratogenicity of antibacterial agents

The aim of our study was to study the possible correlation between use of antibacterial drugs in pregnancy and occurrence of congenital malformations. Among 6099 investigated pregnant women, 392 (6.43%) used antibacterial drugs. The most frequently used antibacterials belonged to category B (75.77%), while 14.54% antibiotics belonged to category D and 1.02% to category X. The most often used antibiotics were cephalexin (22.19%), amoxicillin (20.66%) and ampicillin (14.29%). In 14 embryos exposed to effects of beta-lactams in utero, malformations were detected. The results of this study show possible teratogenic potential even with those antibacterials which are considered safe, but as those are usually minor malformations, they often pass undetected. Because of that and because of frequent use of antibacterials during pregnancy, detailed examinations concerning their safety should be made.