Pathology of amniogenesis in the early prenatal period of human development

Morphological and, in a number of cases, cytogenetical investigation has been performed in 420 intact embryonal sacs and in embryos 7-8-week-old, obtained at spontaneous abortions (272) and at tubal pregnancy (148). Among these cases 202 (48.1%) intact empty embryonal sacs, 75 (17.9%) embryos with panorganodysplasia, 25 (6%) embryos with isolated developmental defects and 118 (28%) phenotypically normal embryos have been revealed. Pathology of amniogenesis such as aplasia or hypoplasia of the amniotic cavity is noted in 136 (32.4%) cases. Among 75 embryos with panorganodysplasia anomalies such as hypoplasia of the amniotic cavity in combination with a partial extra-amniotic++ position of the embryos in exocelom (10.7%), aplasia (5.3%) or hypoplasia (17.3%) amniotic peduncle is present in 43 (57.3%) observations. Out of 40 such cases at spontaneous abortions, cytogenetically investigated, in 27 (67.5%) chromosomal disorders (tetraploidy, triploidy, autosomal trisomy and monosomy) are revealed. Aplasia and hypoplasia of the amniotic cavity are considered as pathology of histogenesis at the tissue stage of the early human ontogenesis, that most evidently occurs as a result of asplasia, destruction or anomaly of embryoblast during the first phase of gastrulation on the 7th-11th day of the intrauterine development.     

Direct effects of varying doses of oestradiol on early embryonic development in in vitro culture of rat's two-cell embryos

Hyperstimulation in the rat using pregnant mares' serum gonadotrophin (PMSG) has been known to cause death in pre-implantation embryos, as well as enhancement of oestradiol production. This study examines the effect of oestradiol, in levels that are found in hyperstimulated pregnant rats, on pre-implantation embryonic development. Using a simplified in vitro system, 2-cell embryos retrieved from rats on the 2nd day of pregnancy were cultured in rat two-cell embryo culture medium (R2ECM) containing pharmacological doses of oestradiol for 96 h and scored daily in the morning. Three ngxmL(-1) oestradiol reduced the incidence of >8-cell embryos to morulae on the 5th day and blastocysts on the 6th day of development. Most embryos were retarded at the lower cell stages on the 5th day and degenerated by the 6th day. None of the blastocysts expanded on the last day of culture. Fifteen ngxmL(-1) oestradiol accelerated embryo development on the 3rd day but retarded development on the 4th day, and increased the incidence of degenerated embryos by the 5th and 6th day of development. These results suggest that the elevated oestradiol may constitute a mechanism by which PMSG induces death in pre-implantation rat embryos, possibly via a direct action on the embryos.     

Effect of acute hypoxia on embryonal death and the sex ratio of the progeny

The effect of acute hypoxia on mouse embryogenesis and its influence on the sex ratio in the progeny was studied. On the 7th--10th day of pregnancy mice were subjected to hypoxia at 230 mm HG for 3 hours. They were sacrificed on the 18th day of pregnancy, and gonads of their embryos were taken for investigation. The sex ratio was determined as the ratio of the number of males to that of females. It has been shown that acute hypoxia on the 7th--10th days of pregnancy, resulting in high mortality of adult animals, had no substantial effect on the embryonic development. No selective death of the embryos of either sex was observed both in experimental and control groups.     

Demonstration of dominant lethal mutations in early mouse embryogenesis

Male mice of C57Bl/6Y strain were injected intraperitoneally with 2.5 and 5 mg/kg doses of thioTEPA. Males were mated to tetrahybrid CBWA females during the second week after the treatment. Embryonic mortality was studied by two methods: by standard dominant lethal method on the 15-17th day of pregnancy and cytologically on the 4th day. The rate of fertilization was not affected by thioTEPA. After treatment with 2.5 mg/kg of thioTEPA the frequency of induced dominant lethals was 89.8%; preimplantation losses were 78,5% in treated and 13,8% in control group. The cytological analysis revealed that preimplantation embryonic death is equal to 63,9%. The death of embryos before implantation occurred at 2-20 blastomere stages. After treatment with 5 mg/kg of thioTEPA all embryos died before implantation at 2-16 blastomere stages. It was demonstrated that dominant lethal method gave more complete estimation of dominant lethal frequency, and that cytological analysis is the correct estimation of preimplantation death. Thus the methods used supplement each other.     

Effect of bacterial endotoxin on migration of gonadotropin-releasing, hormone producing neurons in rat embryogenesis

The effect of bacterial lipopolysaccharide endotoxin (LPS), immune system activator, on differentiation and migration of gonadotropin-releasing, hormone producing neurons in rat embryogenesis has been studied. Intraperitoneal introduction of LPS (18 jg/kg) to pregnant rats on the 12th day of pregnancy led to 50% decrease in total number of GRH-neurons in the forebrain of 17-day-old embryos and 17% decrease in 19-day-old embryos. At the same time, the number of GRH-neurons in the nasal area of the head of 17- and 19-day-old embryos increased by 40 and 50%, respectively, whereas it increased by 20% in olfactory bulbs of 17-day-old embryos and did not changed in olfactory bulbs of 19-day-old embryos. Neither the total number of neurons nor their distribution patterns were affected by the introduction of LPS into pregnant rats on the 15th day of pregnancy. Singular localization of GRH-neurons in embryo forebrain was observed after LPS administration, whereas the neurons were located by groups of 3-4 cells in rostral areas. Therefore, at the early stages of pregnancy, LPS was shown to suppress initial stages of differentiation and migration of GRH producing neurons. The effects observed in our study may be mediated by LPS-induced, proinflammatory cytokines.     

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.     

Teratogenic effects of chlorambucil on in vivo and in vitro organogenesis in mice.

Pregnant ICR/DUB mice were each given a single oral injection of chlorambucil (14.2 or 20 mg/kg) on the 10th, 11th, 12th, or 13th day of gestation (plug day = 1st day). Fetuses examined on the 18th day were decreased in weight and had tail, cranial, and limb defects. They type and frequency of malformations differed according to the dosage and day of treatment. Limb defects resulted from treatment on the 11th or 12th days of gestation and tail defects from treatment on all days. Control limb buds from 12th day embryos cultured for 6 days in serum-supplemented BGJ medium containing 0.5-2 mug/ml chlorambucil were retarded in development and had cartilage abnormalities. The extent of the deformities was dose related. Limb buds were also taken from embryos 24 h after in vivo exposure to teratogenic doses of chlorambucil and cultured in control medium. After 6 days in culture these limbs also had growth impairment and cartilage abnormalities. The defects in limbs exposed in vitro were similar to those in limbs exposed in vivo.     

Effect of bacterial endotoxin on migration of gonadotropin-releasing hormone-producing neurons in rat embryogenesis

The effect of bacterial lipopolysaccharide endotoxin (LPS), immune system activator, on differentiation and migration of gonadotropin-releasing hormone-producing neurons in rat embryogenesis has been studied. Intraperitoneal injection with LPS (18 μg/kg) into pregnant rats on the 12th day of pregnancy led to 50% decrease in total number of GnRH-neurons in the forebrain of 17-day-old embryos and 17% decrease in 19-day-old embryos. At the same time, the number of GnRH-neurons in the nasal area of the head of 17- and 19-day-old embryos was increased by 40 and 50%, respectively, whereas it increased by 20% in olfactory bulbs of 17-day-old embryos and was not changed in olfactory bulbs of 19-day-old embryos. Neither the total number of neurons nor their distribution patterns were affected by LPS injection into pregnant rats on the 15th day of pregnancy. Singular localization of GnRH-neurons in embryo forebrain was observed after LPS administration, whereas the neurons were located by groups of 3–4 cells in rostral areas. Therefore, at the early stages of pregnancy, LPS was shown to suppress initial stages of differentiation and migration of GnRH-producing neurons. The effects observed in our study may be mediated by LPS-induced proinflammatory cytokines.     

Effect of ethanol on the cerebellar cortex of the chick embryo

The effect of ethanol on the cerebellar cortex of chick embryos was studied in semi-thin sections of material prepared for electron microscopy. The embryos were injected with ethanol on the 3rd or 6th day of incubation and observed until days 13, 15, 17 and 21 of development. A decrease was seen in the number of germinal cells generated, together with defects in neuronal migration and the existence of a lower quantity of cells due to a generalised process of cell death. At the same time, a progressive neuronal degeneration was observed until the 15th day of incubation, the tissue recovering progressively on days 17 and 21. On the other hand, the embryos treated with ethanol on the 3rd day were less affected than those injected on the 6th day.     

Responses of mitochondrial biogenesis and function to maternal diabetes in rat embryo during the placentation period

Mitochondria are cellular organelles that have been reported to be altered in diabetes, being closely related to its associated complications. Moreover, mitochondrial biogenesis and function are essential for proper embryo development throughout the placentation period, occurring during organogenesis, when a great rate of congenital malformations have been associated with diabetic pregnancy. Thus, the aim of the current work was to investigate the effect of the diabetic environment on mitochondrial function and biogenesis during the placentation period. For this purpose, we studied the oxidative phosphorylation system (OXPHOS) enzymatic activities as well as the expression of genes involved in the coordinated regulation of both mitochondrial and nuclear genome (PGC-1alpha, NRF-1, NRF-2alpha, mtSSB, and TFAM) and mitochondrial function (COX-IV, COX-I, and beta-ATPase) in rat embryos from control and streptozotocin-induced diabetic mothers. Our results reflected that diabetic pregnancy retarded and altered embryo growth. The embryos from diabetic mothers showing normal morphology presented a reduced content of proteins regulated through the PGC-1alpha mitochondriogenic pathway on gestational day 12. This fact was accompanied by several responses that entailed the activation of OXPHOS activities on the same day and the recovery of the content of the studied proteins to control levels on day 13. As a result, the mitochondria of these embryos would reach a situation close to control on day 13 that could allow them to follow the normal mitochondriogenic schedule throughout a gestational period in which the mitochondrial differentiation process is critical. Nevertheless, malformed embryos from diabetic mothers seemed to show a lower adaptation capability, which could exacerbate their maldevelopment.     

Interaction of beta-carboline with chick embryo spontaneous motility.

The effect of beta-carboline (beta-CCE) on spontaneous motility and its development was studied in chick embryos between the 11th and 19th day of incubation. 1. Acutely administered beta-CCE (7.5 mg/kg e.w.) already induced significant activation of motility in 11-day-old embryos. From the 17th day of incubation activation acquired a paroxysmal character. 2. In spinal embryos (decapitated on the second day of incubation) there was no such activating effect, demonstrating that it is associated with supraspinal components of the CNS. 3. In chronic administration from the fourth day of incubation (1.55 +/- 0.24 mg/kg e.w./24h), beta-CCE led to reduced development of spontaneous motility. The effect was concentrated in the period between the fourth and eighth day of incubation. The chronic administration of beta-CCE augmented the activating effect of metrazol and weakened GABA-inhibition of spontaneous motility. 4. On the basis of their findings, the authors express the hypothesis that the benzodiazepine beta-CCE-sensitive component of the complex GABA receptor evidently already functions from the beginning of the second half of incubation of chick embryos.     

Effect of bacterial lipopolysaccharide pyrogenal on the development of the Syrian hamster embryo

Pyrogenal is injected to female hamsters (Mesocricetus auratus) at various time of pregnancy twice, with a 24 hour's interval: at first 100 or 50, and then--50 or 25 minimal pyrogenic doses. The embryos are examined on the 15th day of the intrauterine development by means of main teratologic methods. Pyrogenal injected on the 9th--10th day of the embryonal development produces hydrocephaly, micrognathia, retardation in development, hemorrhagic syndrome. Pyrogenal injected during some other time of pregnancy produces hemolysis of erythrocytes. This is proved by presence of hemosiderin in the foetal liver.     

Limb reduction defects in the first generation and deafness in the second generation of intrauterine exposed fetuses to diethylstilbestrol

Maternal treatment with diethylstilbestrol (DES) during pregnancy can produce vaginal adenocarcinoma and other abnormalities of the vagina in her daughters when they reach adolescence or adulthood, miscarriages and absence of full term infants. Concerning malformations in newborns whose mothers were treated with DES, clitoromegaly and malformations of the uterus were reported in females and genital lesions in males. However, the frequencies of major congenital anomalies were not greater than expected. We report three cases of limb reduction defects (LRD) in the first generation of children whose mothers were treated with DES during pregnancy, and two children (one male, one female) with deafness in the second generation after intrauterine exposure to DES. The LRD were not associated with other congenital anomalies. The malformed children with LRD were born between 1965 and 1973. The deafness was also isolated. The two mothers who have no hearing problems and who are healthy were exposed in utero to DES in 1963 and 1965, respectively. Their children were born in 1989 and 1994, respectively. In conclusion, the association of LRD and hearing loss with intrauterine exposure to DES could be coincidental. However, some hypothesis may explain these associations. Congenital hearing loss in the second generation may suggest a transgenerational effect.     

Lack of effect of 2.45-GHz microwave radiation on the development of preimplantation embryos of mice

The development of preimplantation embryos after exposure to microwave radiation was studied. Female CD-1 mice were induced to superovulate, mated, and exposed to 2.45-GHz microwave or sham radiation for 3 h at power densities of 9 mW/cm² and 19 mW/cm² on either day 2 or 3 of pregnancy (plug day was considered day 1). Another group of mice was exposed to heat stress by placing the dams in an environmental room at an ambient temperature of 38 °C and relative humidity at 62% for 3 h on day 2 of pregnancy. All groups were euthanized on day 4 of pregnancy and embryos were recovered by flushing excised uterine horns. Embryos were examined for abnormalities and classified by the developmental stages. They were then treated with hypotonic solution and dissociated for counting blastomeres. Heat stress caused stunted development of embryos, but no remarkable effect of microwave radiation could be found on the development of preimplantation embryos.     

Influence of inbreeding on reproductive performance, ejaculate quality and testicular volume in the dog

The influence of fast freezing and thawing on bovine embryos at different stages of development was investigated. A total of 20 day-7 embryos and 37 day-8 embryos were thawed and classified morphologically before being transferred nonsurgically to synchronized recipients. Ninety percent (18 20 ) of the day 7 embryos (late morulae and blastocysts) were classified as transferable and a pregnancy rate of 52.9% (9 17 ) was obtained with these embryos. Seventy eight percent (29 37 ) of the day 8 embryos (expanded blastocysts) were classified as transferable, but only 24.1% (7 29 ) of these embryos resulted in pregnancy. The best pregnancy rate was obtained with the blastocysts of day 7 (5 8 or 62.5%), which compares favorably with that of freshly collected embryos. It is suggested that the low pregnancy rate found in the day 8 embryos is related to ultrastructural damages of the desmosomes and tonofilaments within the trophoblast layer, which results in a disturbance of the normal hatching process.     

Development of the cerebrospinal fluid in chick embryos. Physicochemical properties.

The development of the physicochemical properties of the cerebrospinal fluid (CSF) was studied in chick embryos from the 9th day of incubation up to hatching. Some of these properties were compared with the corresponding blood or blood plasma properties. During the second half of incubation the CSF pressure rose from 13.2 plus or minus 0.18 mm H2O in 9-day-old embryos to 80.7 plus or minus 0.48 mm H2O just prior to hatching. The critical stages of this development were the 13th to 15th and the 19th to 21st day of incubation. In 13- and 15-day-old embryos, CSF pressure fell sharply after the intracerebral injection of ouabain, but in 19-day embryos it was unaffected. Except for the 15th and 19th incubation day, the CSF pH was always lower than the plasma pH. From the 11th day of incubation up to hatching, the CSF pH fell from 7.36 plus or minus 0.002 to 7.2 plus or minus 0.005. On the 11th and 13th day, specific CSF resistance was higher than plasma resistance, whereas from the 17th incubation day it was significantly lower than the plasma value. During the second half of incubation, specific CSF resistance fell from 1.059 times 10(6) to 0.824 times 10(6) omega mm.m(-1). A difference between the D.C. potential of the venous blood and the CSF appeared for the first time in 15-day-old embryos, the CSF being negative in relation to the blood. By the end of the incubation period this potential difference rose to 10.82 times 0.07 mv.     

Maternal panic disorder and congenital abnormalities: a population-based case-control study

BACKGROUND: Maternal panic disorder in pregnancy is the most common manifestation of anxiety disorders in Hungary. The association between panic disorder during pregnancy and structural birth defects, i.e., congenital abnormalities, was studied. METHODS: The prevalence of maternal panic disorder in cases with different congenital abnormalities was compared to that of matched controls in the population-based Hungarian Case-Control Surveillance System of Congenital Abnormalities. RESULTS: Of 22,843 cases with congenital abnormalities, 210 (0.9%) had mothers with panic disorder during pregnancy compared to 187 (0.5%) of 38,151 controls (adjusted prevalence odds ratio [POR] 1.6; 95% CI, 1.3-2.0). Specific groups of congenital abnormalities were also assessed versus controls. Cases with isolated cleft lip with or without cleft palate (CL/P) (adjusted POR, 3.4; 95% CI, 1.3-9.0) and multiple congenital abnormalities (adjusted POR, 3.0; 95% CI, 1.2-7.2) were more likely to have had mothers with panic disorder during the study pregnancy. Notably, among mothers with panic disorders, the associations were found only in offspring of untreated mothers. CONCLUSIONS: A higher rate of isolated CL/P and multiple congenital abnormalities may be caused by the direct biological effect of panic disorder or by the interaction of maternal panic disorder and lifestyle factors. Antipanic drug treatment seems to have a protective effect for isolated CL/P and multiple congenital abnormalities. Birth Defects Research (Part A), 2006.     

Developmental changes in prolyl hydroxylase activity and protein in chick embryo.

Changes in prolyl hydroxylase activity and immunoreactive protein were studied in various chick embryo tissues during the embryonic development. Both the enzyme activity and the amoung of immunoreactive protein increased till the 16th day of development and declined thereafter in all tissues studied. Comparison of the enzyme activity to the content of the total immuno-reactive protein indicated that there are distinct differences in the degree of enzyme activity between different chick embryo tissues, and in the same tissue between different stages of embryonic development. The highest relative enzyme activities were found in cartilage and skin, in which about 60% of the enzyme was active on the 16th day of development and only 20-30% was active on the 20th day of development; the lowest values were observed in spleen and large vessels, in which below 10% of the enzyme protein was in the active form on the 20th day of development Gel filtration studies demonstrated that in cartilage of 16-day-old chick embryos about 60% of the total immunoreactive enzyme in the tissue was present in the form of active prolylhydroxylase tetramer, whereas on the 20th day of development only 30% of the enzyme protein in cartilage was in the tetramer form. By contrast, in large vessels of the 16-day-old chick embryos, essentially all the enzyme was in the form of prolyl hydroxylase monomers.     

The acute and the chronic effects of imipramine on the spontaneous motility in chick embryos

The effect of imipramine on the spontaneous motility and development of chick embryos was studied from the 4th to the 19th day of incubation. On acute administration (a single dose of 12.5 of 25 mg/kg egg weight), imipramine already induced significant depression of spontaneous motility in 11-day embryos--an effect which increased significantly after the 15th day of incubation. The similar effect of imipramine in spinal embryos testifies to its direct action on the spinal cord and draws attention to certain details of the role of supraspinal structures of the CNS in the acute effect of imipramine. The chronic administration of imipramine showed that it had an almost 100% lethal effect from 4th to the 7th day of incubation. Between the 8th and the 10th day it caused longlasting depression of spontaneous motility. When it was administered between the 11th and 16th day of incubation, no significant effect on the development of spontaneous motor activity was found in chick embryos.     

Epidemiologic analysis of prenatal exposure to cough medicines containing dextromethorphan: no evidence of human teratogenicity

BACKGROUND: In a recent experimental study on chick embryos, Andaloro et al. (1998, Pediatr. Res. 43:1-7) observed a relationship between neural crest/neural tube defects (NTDs) and prenatal exposure to dextromethorphan. These authors made an extrapolation of their results to the human embryo, indicating that this drug could produce NTDs in humans. Rosenquist (1999, Teratology 60:58-60) based on the results of the epidemiologic study performed by Ferencz et al. (1997, Perspect. Pediatr. Cardiol. 5:50-162), concluded that dextromethorphan could cause congenital heart defects in humans. Because this drug is an over-the-counter drug, the suggestion of those authors has led to great controversy and public concern about the possible teratogenic effect of this drug on the human embryo. METHODS: We present the results of a case-control study by using logistic regression analyses on the effect of prenatal exposure to drugs containing dextromethorphan only, or in combination with other drugs, on human development. We mostly analyzed dextromethorphan. The study was designed in part as hypothesis confirmation for NTDs and congenital heart defects and, in part, as hypotheses generation by testing the association with many other congenital defects. RESULTS: The results do not show a relation between the occurrence of NTDs and heart defects or other defects with exposure to drugs containing dextromethorphan. CONCLUSIONS: The usual use of dextromethorphan in cough medications during pregnancy does not increase the risk for congenital defects.