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Attempts to gain a better understanding of the relationship between the epidermal ridge patterns (dermatoglyphics) and flexion creases on the volar aspects of human hands and feet and specific medical disorders led to a search for a suitable animal model, allowing studies of the fetal development of the pertinent structures. A common experimental animal, the rat (Rattus norvegicus), was found to be an excellent candidate, owing to the strong resemblance of the volar pads and flexion creases on its palmar and plantar surfaces to those of human subjects. A hereditary preaxial polydactyly mouse (Pdn) provides an opportunity to study the effects of this malformation on the surrounding morphological structures and, specifically, on the volar pads, i.e., the sites over which the dermatoglyphic patterns develop. The hands and feet of the wild-type (+/+) mice show no anomalies, and their major pad and flexion crease configurations correspond to those of normal rats. The heterozygous (Pdn/+) mice, in spite of having a thumb/big toe with a duplicated distal phalanx on their hands/feet, did not display any alterations in palmar/plantar pads. The homozygous (Pdn/Pdn) mice have a protrusion in the thenar area and one to three supernumerary digits on the preaxial portion of both the hands and feet. The effect of these anomalies was found to be limited to the pad and flexion crease configurations in the preaxial areas; the postaxial sites were not affected. The original number of pads on the thenar/first interdigital areas of Pdn/Pdn mice was apparently identical to that of the +/+ and Pdn/+mice. The preaxial protrusion, however, affected the number, size, and location of the pads observed in the newborn mice, resulting in varying pad configurations, such as fused and scattered pads or a pad cluster formed by gathering the neighboring pads. These pad modifications were induced by the preaxial plantar/palmar protrusion only and were not affected by the presence of supernumerary preaxial digits. In view of the similarities in the morphology and fetal development of human and mouse distal limbs, the present study is relevant to human subjects, particularly to the understanding of the significance of dermatoglyphic variations in individuals with specific medical disorders. Future studies of naturally occurring or experimentally induced limb malformations in mice or rats should provide valuable insights into the development of human hands and feet and into factors contributing to their congenital anomalies.

Studies on the mouse strain curly tail, a mutant for neural tube defects, have indicated that axial curvature is an important factor in neural tube closure. Previously reported results from experimental interventions in both mouse and chick embryos indicated that curvature along the craniocaudal axis and closure of the posterior neuropore (PNP) are inversely related, a correlation that is also proposed for the rabbit embryo. It was hypothesized that this relationship is a sign of a more general mechanism. Therefore, in the present report the number of species in which axial curvature is described along the craniocaudal axis was extended to include the rat and human. Next, the closure rate of the neural tube as well as the curvature of the PNP region was determined morphometrically for embryos of the following species: chick, rabbit, mouse, rat and human. Although the relationship between neural tube closure and axial curvature appeared specific for each species in the comparative analysis, a general association of increased rate of closure with a decreased curvature emerged. It is concluded that axial curvature is an important factor in neurulation.

Longitudinal bone growth is determined by the process of endochondral ossification in the cartilaginous growth plate, which is located at both ends of vertebrae and long bones and involves many systemic hormones and local regulators. Natriuretic peptides organize a family of three structurally related peptides: atrial natriuretic peptide, brain natriuretic peptide (BNP), and C-type natriuretic peptide. Atrial natriuretic peptide and BNP are cardiac hormones that are produced predominantly by the atrium and ventricle, respectively. C-type natriuretic peptide occurs in a wide variety of tissues, where it acts as a local regulator. These peptides can influence body fluid homeostasis and blood pressure control through the activation of two guanylyl cyclase (GC)-coupled natriuretic peptide receptor subtypes—GC-A and GC-B. We report here marked skeletal overgrowth in transgenic mice that overexpress BNP. Transgenic mice with elevated plasma BNP concentrations exhibited deformed bony skeletons characterized by kyphosis, elongated limbs and paws, and crooked tails. Bone abnormalities resulted from a high turnover of endochondral ossification accompanied by overgrowth of the growth plate. Studies using an in vitro organ culture of embryonic mouse tibias revealed that BNP increases the height of cartilaginous primordium directly, thereby stimulating the total longitudinal bone growth. The present study demonstrates that natriuretic peptides can affect the process of endochondral ossification.

Expression of 105 k-Da heat shock protein (HSP105) during mouse embryo development was investigated. Although HSP105 was detected scarcely in mouse embryos at gestational day (GD) 8, its level of HSP105 increased markedly in the embryos from GD 9 to 11, and then decreased gradually by GD 14. In contrast, 70 k-Da heat shock cognate protein (HSC70) was detected at constantly high levels in embryo from GD 8 to 17 (before birth). The transient increase in level of HSP105 was observed in most embryonic tissues. Since the levels of HSP105 mRNA was detected at constantly high levels in the embryos between GD 8 and 12, the increased expression of HSP105 at GD 9 to 11 seemed to be regulated at posttranscriptional level. Immunohistochemically, HSP105 as well as HSC70 were localized in the cytoplasm and the nuclei of cells in various mouse embryonic tissues. After treatment of tissue sections with proteinase K, HSP105 but not HSC70 was also found to be localized in condensed cells and condensed bodies which showed the typical characteristics of apoptotic cells and apoptotic bodies at the interdigital regions of limbs. These findings suggest that HSP105 plays important roles during mouse embryo development.

Background Although calcium channel blockers have not been shown to be beneficial for the treatment of patients with heart failure, a recent clinical trial demonstrated a favorable effect of amlodipine on the survival of patients with heart failure resulting from nonischemic dilated cardiomyopathy. We investigated the effects of amlodipine on a murine model of congestive heart failure induced by the M variant of encephalomyocarditis virus (EMCV). Methods and Results Four-week-old male DBA/2 mice were inoculated with EMCV and administered amlodipine, diltiazem, or vehicle PO for 2 weeks. The heart weight–to–body weight ratio and the histopathological grades of myocardial lesions were significantly lower and survival was significantly increased in the amlodipine-treated group ( P P P N G -monomethyl- l -arginine, an inhibitor of NO synthesis, decreased myocardial lesions significantly in this murine model. Immunohistochemistry revealed that the number of cells stained with antibody against an inducible NO synthase decreased significantly in the amlodipine-treated group compared with that in the control group ( P Conclusions Amlodipine appears to have a protective effect against myocardial injury in this animal model of congestive heart failure. The therapeutic effect of amlodipine may be in part resulting from inhibition of overproduction of NO.

It has been recognized in mammals that after pancreatic duct obstruction, acinar cells progressively disappear and pancreatic islets are preserved. Previous studies by electron microscopy have suggested the involvement of apoptosis in acinar cell deletion. In the present study, we employed molecular biological methods and investigated whether acinar cell deletion is due to apoptosis. In male Sprague-Dawley rats, pancreatic duct ligation was performed through a left paramedian incision. Pancreatic tissue was studied at each of the following intervals after ligation: 3, 6, 12, 18, and 24 h and 2, 3, 5, and 7 days. DNA fragmentation was determined by in situ labeling of DNA strand breaks on tissue sections [fluorescein-labeled terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method (TUNEL)] and by electrophoretic detection of the fragments of extracted DNA. Tissue sections were also examined by hematoxylin/eosin staining and immunohistochemical staining of insulin. Pancreatic duct ligation induced acinar cell deletion by day 5. Pancreatic tissue from control rats demonstrated no TUNEL-positive nuclei. In contrast, acinar cells from rats 12 h to 5 days after duct ligation showed TUNEL-positive nuclei. The number of TUNEL-positive nuclei was maximum 2 days after duct ligation. Electrophoresis showed DNA fragmentation after duct ligation. Control rats showed a genomic DNA pattern. Islets were preserved throughout the experimental period in duct-ligated rats and control rats. The results suggest that apoptosis may be the dominant form of acinar cell death in the rat pancreatic duct ligation model.

Fetal development of the hands and feet of rats was investigated to determine the feasibility of using rats as an experimental model for studying the factors influencing early development of the hands and feet, and especially the dermatoglyphics in humans. Eighty rats fetuses of 14-21 days gestational age and 80 newborn rats of 0-7 days of age were used to study the morphological features of the palmar, plantar, and digital areas and to determine the timing of appearance and the location of the volar pads and flexion creases. Comparisons between analoguous developmental stages of rat and human fetuses demonstrate striking similarities in overall fetal development. Marked differences, however, were found between rat and human fetuses in the timing of developmental milestones and in some morphological features. The results indicate that rats can serve as a useful experimental model in studies of the utility of the epidermal ridge configurations and flexion creases in medical disorders, provided that the differences in the timing of development are taken into consideration.

At thetimethattheVersion2terminologymanuscriptwaspublished,theauthorsinvitedalluserstosubmitcorrectionsandcomments.As of the current date, only 3 minor corrections have been identified. These are listed as follows:1. There was a misspelling in the company name for Yojiro Ooshima on page 371; the corrected affiliation is: Shin Nippon BiomedicalLaboratories, Ltd., Kagoshima, Japan.2. The company name for Meg Parkinson was mistakenly listed as GlaxoSmithKline Services on page 371; the correct company name isGlaxoSmithKline Pharmaceuticals.3. In the acknowledgements of the contributions of MARTA/MTA colleagues on page 375 the surname of Joyce Rendemonti was misspelled.The authors would like to apologise for any inconvenience this may have caused to the readers of the journal.

Mammalian hairy and Enhancer of split homolog-1 (HES-1) encodes a helix-loop-helix (HLH) factor that is thought to act as a negative regulator of neurogenesis. To directly investigate the functions of HES-1 in mammalian embryogenesis, we performed a targeted disruption of the HES-1 locus. Mice homozygous for the mutation exhibited severe neurulation defects and died during gestation or just after birth. In the developing brain of HES-1-null embryos, expression of the neural differentiation factor Mash-1 and other neural HLH factors was up-regulated and postmitotic neurons appeared prematurely. These results suggest that HES-1 normally controls the proper timing of neurogenesis and regulates neural tube morphogenesis.

The process of palate fusion was examined in 13- and 14-day-old mouse fetuses by using in situ staining for nuclear DNA fragmentation (TUNEL method) and immunofluorescent staining for keratin, with special reference to the disruption of the midline epithelial seam. TUNEL-positive cells were found in the disappearing midline seam and the oral and nasal epithelial triangles at some late stages of palate fusion, but not in the palatal shelves prior to contact or in the intact midline epithelial seam. It seems that DNA fragmentation or apoptosis is required for the midline epithelial seam to disrupt, but may not be necessary for initial contact of palatal shelves or for the epithelial fusion of opposing palatal shelves. A similar sign of apoptotic cell death was observed in the disappearing epithelial seam between the fusing nasal septum and dorsal palate. We have demonstrated that apoptotic programmed cell death does occur at some stages of palate fusion, although the present results do not exclude the possibility of epithelial-mesenchymal transformation and the oral and nasal migration of midline epithelial cells.

Maxillary regions of day-12.5 ICR mouse fetuses were dissected and cultured in a chemically defined serumless medium, and the effects of anticonvulsant drugs on in vitro palatogenesis were studied. The explants were treated for 72 h in vitro with 50 to 200 micrograms/mL diphenylhydantoin (DPH), 200 to 800 micrograms/mL sodium phenobarbital (PB), 12.5 to 400 micrograms/mL sodium valproate (VPA), and 3 to 100 micrograms/mL diazepam (DAZ). During the culture, the secondary palatal shelves of control explants elevated, grew medially, and fused after 72-h culture in a manner similar to the palatogenetic process in vivo. The fusion of palatal shelves was inhibited dose-dependently by treatments with DPH, VPA, and DAZ. PB showed no significant inhibitory effects on palatal fusion at concentrations up to 800 micrograms/mL. The in vitro toxicity of the anticonvulsants tested appeared to correlate with the relative in vivo teratogenic potential of the drugs. The present study demonstrated that the in vitro organ culture system should be useful for screening teratogenic agents, especially those causing cleft palate, and for exploring the mechanisms of cleft palate formation.

In the developing mammalian central nervous system, neural precursor cells present in the ventricular zone determine their fate to become neurons or glial cells, migrate towards the outer layers and undergo terminal differentiation. The transcriptional repressor HES-1, a basic helix-loop-helix (bHLH) factor structurally related to the Drosophila hairy gene, is expressed at high levels throughout the ventricular zone, but the level decreases as neural differentiation proceeds. Because of this negative correlation, we tested whether continuous expression of HES-1 inhibits neural differentiation. A HES-1 and lacZ-transducing retrovirus (SG-HES1) and a control lacZ-transducing retrovirus (SG) were injected into the lateral ventricles of mouse embryos, and the fate of the infected neural precursor cells was examined by X-gal staining. The SG virus-infected cells migrated and differentiated into neurons and glial cells. In contrast, the cells infected with SG-HES1 virus remained in the ventricular/subventricular zone, decreased to approximately 10% in number as compared with that of the newborn during the postnatal 4-5 weeks and, when they survived, were present exclusively in the ependymal layer. Furthermore, whereas cultured neural precursor cells infected with SG virus became immunoreactive for neuronal and glial markers, the cells infected with SG-HES1 virus did not. These results show that persistent expression of HES-1 severely perturbs neuronal and glial differentiation.

In order to examine the effects of an alcohol dehydrogenase inhibitor on the embryotoxic effects of ethanol, pregnant ICR mice were treated with 100 mg/kg pyrazole prior to ethanol injection. I.p. treatment with 2 or 4 g/kg ethanol on day 7 of gestation increased the prenatal mortality rate and produced external and skeletal malformations in the offspring, and the embryotoxic effects were potentiated by pyrazole pretreatment, suggesting that ethanol rather than its metabolites is mainly responsible for the embryotoxicity. In the second experiment, we housed pregnant mice in an ethanol-vapor box for 3 or 6 days in order to examine the effects of prolonged low level exposure to alcohol. The maternal blood alcohol concentration was maintained approximately 0.03 mg/mL during inhalation. The inhalation treatment with ethanol increased the prenatal mortality rate, although teratogenicity was not shown.

Limb buds of day 14 rat fetuses were cut into pieces and transplanted into the subcutaneous tissue of athymic (nude) mice. In day 14 fetal limbs, mesenchymal cells have begun to condense to form cartilaginous anlage, but no cartilage has been formed. Within 7 days after grafting, masses of hyaline cartilage developed. Numerous osteoblasts appeared, and new bone formation began by 14 days. By 20 days, osteoclasts appeared, and the formation of bone trabeculae and marrow cavities progressed. The cytological characteristics of chondrocytes, osteoblasts and osteoclasts were essentially the same as those seen in vivo. Many grafts developed into long bones having the diaphysis and epiphysis. The mode of chondrogenesis and osteogenesis in the grafts was histologically similar to the corresponding process in vivo, although the differentiation was slower in the grafted limbs. Since the grafted limb buds showed remarkable growth and tissue differentiation for at least several weeks, this heterotransplantation system would be of potential use for the study of bone formation and resorption as well as for developmental toxicological studies.

Forelimb buds of day-14 rat fetuses were cut into pieces and transplanted subcutaneously into athymic (nude) mice. On the 7th, 9th, and 11th days after grafting, the nude mice were treated with various drugs including rat teratogens. On the 20th day, the grafted tissue was examined macroscopically and histologically. While control grafts showed substantial growth and tissue differentiation similar to that observed in vivo, the differentiation of grafts was significantly inhibited by the treatment with 5-fluorouracil, cyclophosphamide, hydroxyurea, cycloheximide, mitomycin C, caffeine, aspirin, retinol palmitate, all-trans-retinoic acid, and ascorbic acid. Hydrocortisone, tetracycline, and thalidomide did not adversely affect the differentiation of grafts. Thus, the susceptibility of transplanted rat limb buds was generally close to the teratologic sensitivity of rat fetuses in vivo. The heterotransplantation method of embryonic tissues may be useful as a new experimental system in developmental toxicology.

Hyperthermia was induced in ICR mice on day 8.5 of gestation by immersing them in hot water. Control mice were immersed in water at 38 degrees C for 15 min. In dams exposed to 42 degrees C for 12.5-15 min or to 43 degrees C for 7.5-10 min, externally malformed fetuses increased significantly and in a dose-related manner. Anterior neural tube defects (exencephaly, anencephaly, encephalocele, and cranial neural tube defect with facial cleft) were induced most frequently. Embryonic and fetal death and skeletal malformations also increased following heat stress. At intervals after heat exposure, sections of embryonic prosencephalon were prepared for light microscopy. At 2 h after heat stress, mitotic figures disappeared almost completely in embryonic tissues. Mitotic activity was inhibited for at least 3 h. At 6 h, there was a burst of new mitotic activity. Some damaged cells became pyknotic and abnormal cells were encountered in the neuroepithelial tissue after 3-12 h. Thus, neural tube defects in mouse embryos following maternal hyperthermia may result from a temporary cessation of cell proliferation and partial necrosis of the embryonic neuroepithelium.

Di(2-ethylhexyl)phthalate (DEHP) and mono-(2-ethylhexyl)phthalate (MEHP) were administered PO or IP to pregnant ICR mice at varying doses on days 7, 8, and 9 of gestation. In groups given DEHP orally, resorptions and malformed fetuses increased significantly at 1,000 mg/kg. Fetal weights were also significantly suppressed. Anterior neural tube defects (anencephaly and exencephaly) were the malformations most commonly produced. No teratogenic effects were revealed by IP doses of DEHP and PO or IP doses of MEHP, although high doses were abortifacient and lethal to pregnant females. Thus DEHP is highly embryotoxic and teratogenic in mice when given PO but not IP. The difference in metabolism, disposition, or excretion by the route of administration may be responsible for the difference in DEHP teratogenicity. Although MEHP is a principal metabolite of DEHP and is several times more toxic than DEHP to adult mice, it seems that MEHP and its metabolites are not teratogenic in ICR mice.

Morphological data from 3,614 well-preserved human embryos derived from artificial termination of pregnancy were used to determine whether ectopic implantation or enlarged myomas could enhance the prevalence of localized malformations of the embryo. The frequency of malformed embryos was 11.6% among 43 recovered from ectopic pregnancies, 6.2% among 97 from myomatous pregnancies, and 3.3% among 3,474 from normally implanted pregnancies not complicated by myomas. Unilateral amelia in the ectopic cases and caudal dysplasia in the myoma cases were significantly increased. Both malformations were quite unusual in the control group. It is argued that spatial restrictions could be a teratogenic agent in human embryos. Mothers of both ectopic and myoma cases were on average much older than mothers of the control specimens, and those with myomas had a higher frequency of previous pregnancy wastages. Ectopic pregnancy was found to be associated with lowered parity, previous ectopic pregnancy, and maternal smoking and drinking. These associations are discussed and interpreted in relation to etiology of each of the two conditions.

Important data relative to organogenesis and teratogenesis can be learned from the study of early embryos derived from induced abortions, which are legal in Japan. Nearly 1/3 of the conceptuses from the unrecognized period of pregnancy (less than 35 days) possess abnormalities, most of which would normally cause them to be aborted. The commonest findings were degeneration and necrosis and neural tube defects. The timetables for normal organogenesis were included in the "Atlas of Prenatal Histology," published in 1983. The relationship between normal organogenesis (or teratogenesis) and gestational age exhibits a certain amount of variability; also it is not usually possible to determine the exact ovulation age of the embryo. The occurrence of abnormalities in the early intrauterine embryo is far more common than the number of such abnormalities seen in newborns, again indicating that most abnormal embryos are aborted. Early intrauterine embryos also showed a high correspondence of internal to external abnormalities, especially in the case of midline defects, indicating that the embryonic midline acts as a developmental unit. Embryonic tissues from human embryos grafted subcutaneously or intratesticularly into athymic mice were accepted and grew and differentiated to a considerable extent, showing that such tissues could be effectively used experimentally.

A collaborative study was conducted to investigate the teratological susceptibility of the Pika (Ochotona rufescens rufescens) to selected teratogenic chemicals: cyclophosphamide, 6-mercaptopurine, 5-fluorouracil, 6-aminonicotinamide, actinomycin D, ethylurethan, ampicillin, tetracycline, thalidomide, diphenylhydantoin, hypervitaminosis A, aspirin, dexamethasone, betamethasone and bredinin. Some of the chemicals were shown to be teratogenic in the Pika, but this animal was generally more resistant to their teratogenicity than the rabbit and rodents. In the Pika, thalidomide did not induce any typical limb defects, which have been produced in the rabbit. Pikas reproduce well and appear to have no substantial disadvantages as an animal species for teratological studies. Thus, the Pika may be useful as a new non-rodent species for teratological testings.

Effects of potassium thiocyanate (KSCN) on normal and neoplastic mammary development in female mice were studied. Administration of KSCN as drinking water (0.1 or 0.3%) for 12 weeks to 8-week-old SHN female mice resulted in the dose-related inhibition of development of normal mammary lobulo-alveolar system, hyperplastic alveolar nodules and spontaneous mammary tumors associated with marked decrease in plasma levels of triiodothyronine and thyroxine. On the other hand, the treatment affected little estrous cycles, ovarian structures, plasma prolactin levels and weights of body, anterior pituitary and adrenals. The incidence and number of pregnancy-dependent mammary tumors in GR/A female mice were also significantly suppressed by the similar treatment with KSCN with no influence on reproductivity as well as pituitary levels of prolactin and growth hormone. These results suggest that KSCN does not affect the secretion of pituitary and ovarian mammotropic hormones and that thyroid hormones are principally involved in normal and neoplastic development of mammary glands.

One hundred and twenty-nine embryos with polydactyly were found among 36,380 human conceptuses obtained through induced abortion during the period from 1962 to 1974, giving an overall incidence of 0.35%; this must be taken as an underestimate because the denominator includes a large number of damaged embryos. A significant increase was noted in 1971 and 1972, and some exogenous factors, most probably ascertainment bias, were suspected to be responsible. About three quarters of the present cases had preaxial polydactyly in contrast to the predominance of postaxial type in Negro and Caucasian populations. The ratio unilateral to bilateral affection was approximately 2∶1.

Among 813 women who had obtained a vaginal spermicide within 48 weeks of the estimated date of fertilization (EDF), 47 (5.8%) had an early miscarriage. Among women who had obtained oral contraceptives, 35/1,127 (3.1%) miscarried, and among women who obtained neither, 140/4,231 (3.3%) miscarried. The risk ratio estimate comparing spermicide users with nonusers was 1.8 (90% confidence interval 1.4, 2.3). The association was strongest among women who had obtained a spermicide within 12 weeks of the EDF. Examination of abortus material revealed that the association with spermicides was strongest among those where an abnormal fetus was present.

A causal relationship between neural tube defects and maternal febrile illness in early pregnancy was investigated retrospectively using human embryo data from Japan. The frequency of febrile illness was compared with corresponding data from normal controls, and holoprosencephalic and polydactylous embryos. Within the group of neural tube defects, the prevalence of maternal hyperthermia was significantly higher than in normal control and holoprosencephaly mothers. The association was constant and highly significant specifically for exencephaly, which is an embryonic forerunner of anencephaly. The correlation with myeloschisis (without exencephaly) was not significant. In eight out of the ten cases in which the timing of the fever was known, it occurred during the clinically determined critical period of neural tube closure in human embryos. These results support the hypothesis that maternal hyperthermia in early pregnancy can cause neural tube defects in man, especially anencephaly.

In this chapter some of the information on normal and abnormal development will be summarized. Strong emphasis is given to comparisons between commonly used laboratory mammals and primates, especially human, rather than stressing phylogenetic aspects.

Two major compounds of PVC plasticizers, di-2-ethylhexyl phthalate (DEHP) and di- n -butyl phthalate (DBP), were mixed with food at levels of 0.05, 0.1, 0.2, 0.4, and 1.0 wt% and given to pregnant mice of ICR-JCL strain throughout gestation. Treatment with 0.2, 0.4, and 1.0% DEHP and 1.0% DBP resulted in decreased maternal weight gain and increased resorption rate. All of the implanted ova died in utero at 0.4 and 1.0% level of DEHP. The malformation rate in term fetuses increased at 0.2% level of DEHP and 1.0% level of DBP, the difference from the control group being at the borderline level of significance. The major malformations observed in the treated groups were neural tube defects (exencephaly and spina bifida), suggesting that the phthalic acid esters could interfere with the closure of neuropores in developing embryos. Treatment with the compounds caused intrauterine growth retardation and delayed ossification with an apparently dose-related response pattern. These results indicate that DEHP and DBP at a high dose level could be embryotoxic and possibly teratogenic mice. The maximum no effect level of the PAEs on mouse fetuses was 70 mg/kg/day, which is far higher than the estimated human current intake from the environment.

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.This paper explored, using induced abortuses both alive and dead in utero in a Nishimura consecutive series, the causal relationships among maternal genital bleeding, supportive hormone therapy, and congenital anomalies, with special reference to the critical organogenesis period. Data from 667 undamaged embryos from induced abortions whose mothers had had early genital bleeding and from 90 embryos with polydactyly and 38 with limb reductions were studied. The following conclusions were drawn from examining this series and the damaged abortuses' case histories: 1) Major malformations such as central nervous system anomalies, cleft lip, polydactyle, and limb reductions were not caused by maternal genital bleeding but that the bleeding was a consequence of conceiving an abnormal embryo. 2) No indication was found that exogenous hormones, especially those used in Japan for preventing miscarriages, induced major malformations at the embryonic stage, including limb reductions, nor that they could salvage embryos already severely malformed. 3) Most of the minor anomalies seen at embryonic stages are suggested to be normal variants without any functional impairment of embryonic development. 4) Though this retrospective investigation could not link estrogens used to prevent miscarriages with major malformations, the authors do not rule out the possibility that progestogen/estrogen intake during early pregnancy may be related to increased incidence at birth of internal anomalies such as cardiac anomalies and esophageal atresia, or external malformations manifested at the fetal or neonatal stage.

Recognized spontaneous abortions in previous pregnancies were significantly more frequent in mothers of 87 Japanese embryos with cleft lip with or without cleft palate (CL(P] than in mothers of normal embryos. The number of prior pregnancies was found to be smaller in mothers of CL(P) embryos with additional defects, suggesting that unrecognized abortions might also occur more frequently in these women. The higher incidence of abortions was associated with more severe forms of CL(P), ie, CL(P) with associated anomalies rather than isolated CL(P), and bilateral rather than unilateral CL. These results are compatible with the "multifactorial two-threshold" model with a lower threshold beyond which the malformation occurs and a higher one beyond which the embryo dies. The present results are in contrast with the reduced spontaneous abortion rates in families of CL(P) probands reported previously. The reduced rates may be due to selection of those families in which CL(P) embryos escaped spontaneous abortion and survived to birth, although the effects of compensatory reproduction or maternal recall bias in postnatal data cannot be ruled out completely.

One hundred fifty embryos with holoprosencephaly were found among the total of 36,380 conceptuses obtained through induced abortion in the period from 1962 to 1974, giving an overall incidence of 0.4 percent. The occurrence was period from 1962 to 1974, giving an overall incidence of 0.4 percent. The occurrence was largely at random through time, and no "epidemic" was noted in particular years or months, but there appeared more cases derived from conceptions in winter than in summer months. The mean maternal age did not differ significantly from that of the general embryonic population, indicating that, although none of our cases were karyotyped, chromosome aberrations such as trisomies 13 and 18 that are closely associated with maternal age may not constitute a major part as causes of holoprosencephaly in human embryos. Materal age did not differ by the presence or absence of associated external anomalies. No association was found with paternal age, parental consanguinity nor with maternal medical history, including irregularity of menstrual cycles, and smoking and drinking habits. There was an indication that the mothers were prone to have repeated miscarriages, supporting the view that some kind of maternal predisposition is responsible for the causation of holoprosencephaly. Argument was made that, apart from various chromosome aberrations well documented as causes of this malformation, polygenic mechanism probably accounts for the majority of the cases with normal karyotype.

Thirty-seven cases of human embryos at the early postimplantation period were procured after induced abortion and examined histologically. Their developmental stages ranged from Carnegie stages 6 to 11, and their standard ages ranged from 14 to 24 days after fertilization. Five cases (13.5%) were grossly abnormal, and seven (18.9%) were degenerating partially or in toto. Gross abnormalities included distorted embryonic disc, disorganized neural groove or tube, and neural tube dysraphism. The high prevalence rate of defective embryos at the early postimplantation period supports the clinical finding that a substantial proportion of human conceptions are eliminated from an early stage of pregnancy, often without the knowledge of the mother. The fate of undifferentiated pathological embryos is uncertain and remains to be determined.

Pregnant ICR mice were immersed in water at 42 or 43 °C for 10 min once or twice daily on days 12 through 15 of gestation and the postnatal development of their offspring was observed. Postnatal weight gain of the offspring and their brain weight at 11 weeks of age were smaller than the values for control animals. As compared with controls, prenatally heated mice were less active in an open field and learned slower in a water-filled multiple T-maze and in a shuttle box. Thus, prenatal brief hyperthermia in mice was shown to suppress the body and brain growth during the postnatal period and adversely affect their emotionality and learning capacity.

Di(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP) were mixed with diet at graded levels of 0.05, 0.1, 0.2. 0.4 and 1.0 wt-% and given to pregnant ICR mice throughout gestation. Maternal weight gain was suppressed and fetal resorption increased at 0.2, 0.4 and 1.0% levels of DEHP and 1.0% level of DBP. All the implanted ova died early in rats fed 0.4 and 1.0% levels of DEHP. External malformations increased significantly by 0.2% DEHP, and 1.0% DBP showed borderline significance. The major malformations in treated groups were neural tube defects (exencephaly and myeloschisis), suggesting that the phthalic acid esters (PAEs) affect neural tube closure in developing embryos. Treatment with the compounds caused intrauterine growth retardation and delayed ossification with an apparently dose-related response pattern. These results indicate that a high dose of DEHP and DBP might be embryotoxic and teratogenic in mice. The maximum nonembryotoxic doses of PAEs in mice were more than 2000 times the estimated level of human intake through the food chain. Thus it is assumed that the current "normal" exposure level of PAEs dose not pose an imminent threat to human fetal development.

Using data from 3,474 human embryos derived form induced abortion, various maternal factors ascertained prior to morphologic examination of the specimens were analyzed in association with the frequency of malformed embryos. The overall frequency of malformed embryos was 3.3%; polydactyly, holoprosencephaly, exencephaly with or without myeloschisis, and cleft lip accounted for 88% of the total malformations. Mothers of low parity showed an increased frequency of malformed embryos. There were nine embryos form mothers with thyroid disorders, of which one was affected with exencephaly and myeloschisis and another with holoprosencephaly and polydactyly; this finding may support the relation of maternal hyperthyroidism with anencephaly suggested by previoius case reports. The frequency of malformed embryos from mothers who drank alcohol was significantly lower than from those who did not. Several possibilities were suggested to interpret this unexpected finding. No association was verified with maternal age, irregular menstrual cycles, irradiation, contraceptive methods, maternal occupation, and a smoking habit, or with parental consanguinity. The limitation of this study is discussed in relation to both negative and positive results.

A 74 year old man was found at necropsy to have multiple coronary arteriosystemic fistulae involving all three major coronary arteries and both ventricles. This is the second such case reported. All terminal branches of the coronary arteries entered the myocardium and numerous tiny vessels opened into the cardiac chambers. No coronary veins or coronary sinus were recognized. This vascular anomaly is considered to result from the abnormal persistence of intertrabecular spaces within the embryonic myocardium.