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The files for ultrasound diagnosis of gestating mares belonging to the French equine herd recorded for 3 consecutive years were joined with the files for foal birth of these same mares, allowing the statistical analysis of factors of pregnancy loss. For 28 872 positive diagnoses of gestation, 2898 losses were recorded, that is, a global rate of gestation interruption of 9.12%. The etiology of these interruptions is mainly extrinsic: the year and month of insemination, as well as region for climatic reasons. The intrinsic causes that are implicated are breed of the father (heavy breeds except the hypermetric ones lose fewer pregnancies than warm-blooded breeds), age of the mother (losses are lower in mares of 7 to 10 years of age) and status (mares with foals have fewer pregnancy losses than mares not having foaled the previous year), as well as fetuses with consanguinity (when this increases, the pregnancy losses increase as well). However, the additive genetic effect is extremely low; it corresponds to heritability below 5% and few effects of the environment, common to the offspring of the same mare, were identified. This therefore gives little hope of being able to select against the 'gestation loss' trait.

Objective: To present prenatal diagnosis and molecular cytogenetic characterization of trisomy 18 in a monozygotic twin pregnancy, with one structurally abnormal living fetus and one intrauterine fetal demise., Case Report: A 38-year-old woman was referred for amniocentesis at 16 weeks of gestation because of advanced maternal age. Prenatal ultrasound revealed a monozygotic twin pregnancy, with one structurally abnormal living fetus, and one fetal demise. The body structure details of the dead fetus could not be identified, whereas holoprosencephaly and omphalocele were identified in the living fetus on prenatal ultrasound. Quantitative fluorescent polymerase chain reaction assays using polymorphic DNA markers specific for chromosome 21 and chromosome 18, were applied to the uncultured amniocytes in the amniotic cavity of the living fetus and the cultured amniocytes in the amniotic cavity of the fetus with intrauterine fetal demise. The specimen showed a dosage ratio of 2:1 (paternal:maternal) for chromosome 18-specific markers in both twins. The result was consistent with monozygosity and trisomy 18, and the trisomy 18 was possibly caused by a paternal second meiotic division non-disjunction error or a postzygotic mitotic error. Conventional cytogenetic analysis revealed a karyotype of 47,XY,+18 in both twins. The pregnancy was terminated at 19 weeks of gestation, and a 2g small-for-date macerated twin A and a 166g malformed twin B were delivered. Twin A manifested cebocephaly and omphalocele, and twin B manifested premaxillary agenesis and omphalocele., Conclusion: The present case provides evidence that fetal wastage may occur in one of the co-twins in monozygotic twins associated with trisomy 18, and this may in part explain the very rare occurrence of living monozygotic twins with trisomy 18., (Copyright © 2012. Published by Elsevier B.V.)

Objective: Amniocentesis is a popular and effective prenatal diagnostic tool for chromosomal disorders. It is well-established that the risk of chromosomal abnormalities increases with maternal age; however, other related indications are seldom reported. Herein, we report our 30-year experience with amniocentesis from a single medical center, focusing on the indications and rates of abnormality., Material and Methods: A retrospective review of 16,749 pregnant women in the mid-trimester between January 1981 and December 2010 was conducted. The medical records were analyzed., Results: The indications for amniocentesis were advanced maternal age (≥ 34 years old) (n=10,970, 65.5%), increasing-risk maternal triple-marker Down's screening test (≥ 1/270) (n=2090, 12.5%), history of abnormal offspring birth (n=792, 4.7%), abnormal ultrasound findings (n=484, 2.9%), parent with abnormal karyotype (n=252, 1.5%), family history of chromosomal abnormality (n=183, 1.1%), drug and radiation exposure (n=165), abnormal chorionic villus sampling (CVS) results (n=25), intrauterine fetal death (n=50), and other non-specific causes (n=1662, 9.9%). The rate of abnormality for each indication was 16% in the abnormal CVS group, 12% in the intrauterine fetal death group, 11.5% for parental chromosomal abnormality, 8.7% in the abnormal ultrasound finding group, 3.0% in the increasing-risk maternal triple-marker Down's screening test group, 2.5% in the advanced maternal age group, 1.5% for other non-specific causes, 1.4% for history of abnormal offspring birth, and 1.1% for family history of chromosomal abnormality., Conclusions: Both parents with abnormal karyotype and abnormal ultrasound findings are indications for which consideration of further amniocentesis is highly recommended., (Copyright © 2012. Published by Elsevier B.V.)

Background: Plasminogen activator inhibitor type 1 (PAI-1) is an important regulator of fibrinolysis. A common deletion polymorphism that results in a sequence of 4G instead of 5G in the promoter region of the gene is associated with a small increase in the risk of venous thromboembolism. Its potential association with adverse pregnancy events remains controversial., Objective: We aimed to assess the impact of the 4G PAI-1 polymorphism on pregnancy outcomes in women who had no prior history of adverse pregnancy outcomes or personal or family history of venous thromboembolism., Patients/methods: This study represents a secondary investigation of a prior prospective cohort study investigating the association between inherited thrombophilias and adverse pregnancy events in Australian women. Healthy nulliparous women were recruited to this study prior to 22 weeks gestation. Genotyping for the 4G/5G PAI-1 gene was performed using Taqman assays in an ABI prism 7700 Sequencer several years after the pregnancy was completed. Pregnancy outcome data were extracted from the medical record. The primary outcome was a composite comprising development of severe pre-eclampsia, fetal growth restriction, major placental abruption, stillbirth or neonatal death., Results: Pregnancy outcome data were available in 1733 women who were successfully genotyped for this polymorphism. The primary composite outcome was experienced by 139 women (8% of the cohort). Four hundred and fifty-nine women (26.5%) were homozygous for the 4G deletion polymorphism, while 890 (51.4%) were heterozygous. Neither homozygosity nor heterozygosity for the PAI-1 4G polymorphism was associated with the primary composite outcome (homozygous OR = 1.30, 95% CI = 0.81-2.09, P = 0.28, heterozygous OR = 0.84, 95% CI = 0.53-1.31, P = 0.44) or with the individual pregnancy complications., Conclusion: The PAI-1 4G polymorphism is not associated with an increase in the risk of serious adverse pregnancy events in asymptomatic nulliparous women., (© 2012 International Society on Thrombosis and Haemostasis.)

The histone acetyltransferases (HATs) of the MYST family include TIP60, HBO1, MOZ/MORF, and MOF and function in multisubunit protein complexes. Bromodomain-containing protein 1 (BRD1), also known as BRPF2, has been considered a subunit of the MOZ/MORF H3 HAT complex based on analogy with BRPF1 and BRPF3. However, its physiologic function remains obscure. Here we show that BRD1 forms a novel HAT complex with HBO1 and regulates erythropoiesis. Brd1-deficient embryos showed severe anemia because of impaired fetal liver erythropoiesis. Biochemical analyses revealed that BRD1 bridges HBO1 and its activator protein, ING4. Genome-wide mapping in erythroblasts demonstrated that BRD1 and HBO1 largely colocalize in the genome and target key developmental regulator genes. Of note, levels of global acetylation of histone H3 at lysine 14 (H3K14) were profoundly decreased in Brd1-deficient erythroblasts and depletion of Hbo1 similarly affected H3K14 acetylation. Impaired erythropoiesis in the absence of Brd1 accompanied reduced expression of key erythroid regulator genes, including Gata1, and was partially restored by forced expression of Gata1. Our findings suggest that the Hbo1-Brd1 complex is the major H3K14 HAT required for transcriptional activation of erythroid developmental regulator genes.

Placental deficiencies are linked with developmental abnormalities in cattle produced by somatic cell nuclear transfer (SCNT). To investigate whether the aberrant expression of imprinted genes in placenta was responsible for fetal overgrowth and placental hypertrophy, quantitative expression analysis of six imprinted genes (H19, XIST, IGF2R, SNRPN, PEG3, and IGF2) was conducted in placentas of: 1) deceased (died during perinatal period) transgenic calves (D group, n = 4); 2) live transgenic calves (L group, n = 15); and 3) conventionally produced (control) female calves (N group, n = 4). In this study, XIST, PEG3 and IGF2 were significantly over-expressed in the D group, whereas expression of H19 and IGF2R was significantly reduced in the D group compared to controls. The DNA methylation patterns in the differentially methylated region (DMR) from H19, XIST, and IGF2R were compared using Bisulfite Sequencing PCR (BSP) and Combined Bisulfite Restriction Analysis (COBRA). In the D group, H19 DMR was significantly hypermethylated, but XIST DMR and IGF2R ICR were significantly hypomethylated compared to controls. In contrast, there were no noticeable differences in the expression and DNA methylation status of imprinted genes (except DNA methylation level of XIST DMR) in the L group compared to controls. In conclusion, altered DNA methylation levels in the DMRs of imprinted genes in placentas of deceased transgenic calves, presumably due to aberrant epigenetic nuclear reprogramming during SCNT, may have been associated with abnormal expression of these genes; perhaps this caused developmental insufficiencies and ultimately death in cloned transgenic calves., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In 3 experimental runs, the influence of genotype × temperature interactions on the reproductive traits (sexual maturity, egg production, fertility, hatchability, and chick production) of hens of a broiler breeder dam line carrying major genes for dwarfism (dw-) and frizzle (F) was investigated. In experiments 1 and 2, the hens were caged individually under hot (30°C) and temperate (19°C) temperatures, from wk 18 to 72 of age, whereas in experiment 3, hens were kept under moderate temperature (24°C). Hens in experiment 1 were heterozygous for the frizzle gene, and those in experiments 2 and 3 were homozygous, both with and without the dwarf gene. Hens without the above-mentioned major genes (ffDw-) served as control lines. In experiment 1, the frizzle gene (Ff) had no significant effect on sexual maturity, egg production, fertility, hatchability, and chick number under the 2 environmental conditions. In experiment 2, there was a significant interaction between feathering genotype (FF) and environmental temperature for all traits except sexual maturity. Under heat stress, there was a distinct reduction in all reproductive traits except sexual maturity for normally feathered hens compared with frizzle-feathered hens, whereas under temperate conditions, egg production and number of chicks of the FF genotype were reduced and sexual maturity was delayed. In experiments 1 and 2, the dw- gene showed a depressive effect on the growth of hens. In experiment 1, the interaction between dwarf genotype and environmental temperature for egg production was significant. Under temperate conditions, the egg production of dwarf hens was inferior to that of normally sized birds, whereas under hot temperatures, the egg production of the 2 body sizes did not differ. In experiment 2, for sexual maturity, egg production and fertility locus × locus interactions could be determined. The genotype combining the 2 major genes (FFdw-) proved to be inferior to the normally feathered dwarf type (ffdw-) for laying performance but superior in fertility. Under the conditions in experiment 3, the egg production of dwarf birds was significant reduced.

Somatic cell nuclear transfer (SCNT) has been successfully used in many species to produce live cloned offspring, albeit with low efficiency. The low frequency of successful development has usually been ascribed to incomplete or inappropriate reprogramming of the transferred nuclear genome. Elucidating the genetic differences between normal fertilized and cloned embryos is key to understand the low efficiency of SCNT. Here, we show that expression of HSPC117, which encodes a hypothetical protein of unknown function, was absent or very low in cloned mouse blastocysts. To investigate the role of HSPC117 in embryo development, we knocked-down this gene in normal fertilized embryos using RNA interference. We assessed the post-implantation survival of HSPC117 knock-down embryos at 3 stages: E9 (prior to placenta formation); E12 (after the placenta was fully functional) and E19 (post-natal). Our results show that, although siRNA-treated in vivo fertilized/produced (IVP) embryos could develop to the blastocyst stage and implanted without any difference from control embryos, the knock-down embryos showed substantial fetal death, accompanied by placental blood clotting, at E12. Furthermore, comparison of HSPC117 expression in placentas of nuclear transfer (NT), intracytoplasmic sperm injection (ICSI) and IVP embryos confirmed that HSPC117 deficiency correlates well with failures in embryo development: all NT embryos with a fetus, as well as IVP and ICSI embryos, had normal placental HSPC117 expression while those NT embryos showing reduced or no expression of HSPC117 failed to form a fetus. In conclusion, we show that HSPC117 is an important gene for post-implantation development of embryos, and that HSPC117 deficiency leads to fetal abnormalities after implantation, especially following placental formation. We suggest that defects in HSPC117 expression may be an important contributing factor to loss of cloned NT embryos in vivo., (Copyright 2010 Elsevier Inc. All rights reserved.)

The major obstacle to genetic research in SIUD (sudden intrauterine unexplained death) and SIDS (sudden infant death syndrome) cases is the complex characteristics of the human anatomic samples available. In fact, in Italy autopsies are performed at least 24 h post-mortem and tissues can be left in formalin for long fixation times (>4/5 days), thus compromising nucleic acids integrity. In this study we compared the quality of DNA and RNA extracted from tissues differently preserved. As expected, the DNA and RNA from formalin-fixed and paraffin-embedded tissues, formalin-acetic acid-alcohol tissues and ethanol tissues were of poor quality and not adequate for subsequent molecular analysis. The best results were obtained with RNAlater preserved tissues: this buffer was equivalent, if not superior, to freezing method for preservation of postmortem DNA and RNA. In addition, we introduce a new protocol for the amplification of the serotonin transporter gene promoter region (5-HTT) ideal to obtain the increase of specific product, avoiding artifacts formation., (Copyright 2009 Elsevier Inc. All rights reserved.)

Heme oxygenase (HO) is the rate-limiting enzyme in the heme catabolic pathway and highly expressed in the placenta. Deficiencies in HO-1, the inducible isoform, have been associated with pregnancy disorders, such as recurrent miscarriages, intrauterine growth retardation, and pre-eclampsia. The aim of this study was to identify if a deficiency in HO-1 affects placental development using a mouse model. When HO-1 heterozygote (Het, HO-1(+/-)) mice were cross-bred, an extremely low birth rate in homozygote (Mut, HO-1(-/-)) offspring (2.4%) and small litter sizes were observed. Placentas and fetuses from Het cross-breedings were relatively smaller and weighed less than those from wild-type (WT) cross-breedings at E12.5 and E15.5. Furthermore, Het placentas had significantly less HO-1 mRNA and protein levels than WT placentas, but no significant differences in placental HO activity. Interestingly, HO-2, the constituitive HO isoform, as well as iNOS and eNOS expression were significantly upregulated in Het placentas. Histological examination showed that the junctional zone (JZ) of Het placentas were markedly thinner than those of WT placentas and appeared to be due to an increase in apoptosis. Immunohistochemistry revealed that HO-1-expressing cells were located primarily in the JZ of Het placentas, specifically in the spongiotrophoblast layer. In addition, diastolic blood pressures and plasma soluble VEGFR-1 (sFlt-1) levels were significantly elevated in pregnant Het mice. We conclude that a partial deficiency in HO-1 is associated with morphological changes in the placenta and elevations in maternal diastolic blood pressure and plasma sFlt-1 levels, despite a compensatory increase in HO-2 expression.

Protein S (PS) is an important natural anticoagulant with potentially multiple biologic functions. To investigate further the role of PS in vivo, we generated Pros(+/-) heterozygous mice. In the null (-) allele, the Pros exons 3 to 7 have been excised through conditional gene targeting. Pros(+/-) mice did not present any signs of spontaneous thrombosis and had reduced PS plasma levels and activated protein C cofactor activity in plasma coagulation and thrombin generation assays. Tissue factor pathway inhibitor cofactor activity of PS could not be demonstrated. Heterozygous Pros(+/-) mice exhibited a notable thrombotic phenotype in vivo when challenged in a tissue factor-induced thromboembolism model. No viable Pros(-/-) mice were obtained through mating of Pros(+/-) parents. Most E17.5 Pros(-/-) embryos were found dead with severe intracranial hemorrhages and most likely presented consumptive coagulopathy, as demonstrated by intravascular and interstitial fibrin deposition and an increased number of megakaryocytes in the liver, suggesting peripheral thrombocytopenia. A few E17.5 Pros(-/-) embryos had less severe phenotype, indicating that life-threatening manifestations might occur between E17.5 and the full term. Thus, similar to human phenotypes, mild heterozygous PS deficiency in mice was associated with a thrombotic phenotype, whereas total homozygous deficiency in PS was incompatible with life.

The ras/Raf/Mek/Erk pathway plays a central role in coordinating endothelial cell activities during angiogenesis. Transcription factors Ets1 and Ets2 are targets of ras/Erk signaling pathways that have been implicated in endothelial cell function in vitro, but their precise role in vascular formation and function in vivo remains ill-defined. In this work, mutation of both Ets1 and Ets2 resulted in embryonic lethality at midgestation, with striking defects in vascular branching having been observed. The action of these factors was endothelial cell autonomous as demonstrated using Cre/loxP technology. Analysis of Ets1/Ets2 target genes in isolated embryonic endothelial cells demonstrated down-regulation of Mmp9, Bcl-X(L), and cIAP2 in double mutants versus controls, and chromatin immunoprecipitation revealed that both Ets1 and Ets2 were loaded at target promoters. Consistent with these observations, endothelial cell apoptosis was significantly increased both in vivo and in vitro when both Ets1 and Ets2 were mutated. These results establish essential and overlapping functions for Ets1 and Ets2 in coordinating endothelial cell functions with survival during embryonic angiogenesis.

We have generated a syntaxin 1A knockout mouse by deletion of exons 3 through 6 and a concomitant insertion of a stop codon in exon 2. Heterozygous knockout animals were viable with no apparent phenotype. In contrast, the vast majority of homozygous animals died in utero, with embryos examined at day E15 showing a drastic reduction in body size and development when compared to WT and heterozygous littermates. Surprisingly, out of a total of 204 offspring from heterozygous breeding pairs only four homozygous animals were born alive and viable. These animals exhibited reduced body weight, but showed only mild behavioral deficiencies. Taken together, our data indicate that syntaxin 1A is an important regulator of normal in utero development, but may not be essential for normal brain function later in life.

TGFbeta/BMP signaling pathways are essential for normal development of neural crest cells (NCCs). Smad4 encodes the only common Smad protein in mammals, which is a critical nuclear mediator of TGFbeta/BMP signaling. In this work, we sought to investigate the roles of Smad4 for development of NCCs. To overcome the early embryonic lethality of Smad4 null mice, we specifically disrupted Smad4 in NCCs using a Cre/loxP system. The mutant mice died at mid-gestation with defects in facial primordia, pharyngeal arches, outflow tract and cardiac ventricles. Further examination revealed that mutant embryos displayed severe molecular defects starting from E9.5. Expression of multiple genes, including Msx1, 2, Ap-2 alpha, Pax3, and Sox9, which play critical roles for NCC development, was downregulated by NCC disruption of Smad4. Moreover, increased cell death was observed in pharyngeal arches from E10.5. However, the cell proliferation rate in these areas was not substantially altered. Taken together, these findings provide compelling genetic evidence that Smad4-mediated activities of TGFbeta/BMP signals are essential for appropriate NCC development.

Background: Activated factor X (FXa) is a vitamin K-dependent serine protease that plays a pivotal role in blood coagulation by converting prothrombin to thrombin. There are no reports of humans with complete deficiency of FX, and knockout of murine F10 is embryonic or perinatal lethal., Objective: We sought to generate a viable mouse model of FX deficiency., Methods: We used a socket-targeting construct to generate F10-knockout mice by eliminating F10 exon 8 (knockout allele termed F10(tm1Ccmt), abbreviated as '-'; wild-type '+'), and a plug-targeting construct to generate mice expressing a FX variant with normal antigen levels but low levels of FX activity [4-9% normal in humans carrying the defect, Pro343-->Ser, termed FX Friuli (mutant allele termed F10(tm2Ccmt), abbreviated as F)]., Results: F10 knockout mice exhibited embryonic or perinatal lethality. In contrast, homozygous Friuli mice [F10 (F/F)] had FX activity levels of approximately 5.5% (sufficient to rescue both embryonic and perinatal lethality), but developed age-dependent iron deposition and cardiac fibrosis. Interestingly, F10 (-/F) mice with FX activity levels of 1-3% also showed complete rescue of lethality. Further study of this model provides evidence supporting a role of maternal FX transfer in the embryonic survival., Conclusions: We demonstrate that, while complete absence of FX is incompatible with murine survival, minimal FX activity as low as 1-3% is sufficient to rescue the lethal phenotype. This viable low-FX mouse model will facilitate the development of FX-directed therapies as well as investigation of the FX role in embryonic development.

The alpha7beta1 integrin is a heterodimeric transmembrane receptor that links laminin in the extracellular matrix to the cell cytoskeleton. Loss of the alpha7 integrin chain results in partial embryonic lethality. We have previously shown that alpha7 integrin null embryos exhibit vascular smooth muscle cell defects that result in cerebral vascular hemorrhaging. Since the placenta is highly vascularized, we hypothesized that placental vascular defects in alpha7 integrin null embryos may contribute to the partial embryonic lethality. Placentae from embryonic day (ED) 9.5 and 13.5 alpha7 integrin knockout embryos showed structural defects including infiltration of the spongiotrophoblast layer into the placental labyrinth, a reduction in the placental labyrinth and loss of distinct placental layers. Embryos and placentae that lacked the alpha7 integrin weighed less compared to wild-type controls. Blood vessels within the placental labyrinth of alpha7 integrin null embryos exhibited fewer differentiated vascular smooth muscle cells compared to wild-type. Loss of the alpha7 integrin resulted in altered extracellular matrix deposition and reduced expression of alpha5 integrin. Together our results confirm a role for the alpha7beta1 integrin in placental vascular development and demonstrate for the first time that loss of the alpha7 integrin results in placental defects.

Background: The relationship between the G20210A prothrombin variant (PT-G20210A) and adverse pregnancy outcome has been studied by several groups in the last few years. However, because of the different design and sample sizes of these studies the estimated risks have varied., Objective: In this retrospective, multi-center, cohort study we assessed the risk of thromboembolic or obstetric complications in women belonging to families of probands with isolated PT-G20210A and that were symptomatic for venous thromboembolism (VTE)., Methods: Two hundred and eighty-three female family members that had been pregnant at least once were enrolled. The occurrence of VTE and obstetric complications during pregnancy and postpartum were assessed in carriers of PT-G20210A and compared with non- carriers., Results: One thromboembolic event occurred during the postpartum period in the carriers group. In the same group, 48 out of 359 pregnancies resulted in unexplained fetal loss as compared with 50 out of 357 pregnancies in the non-carriers (RR 0.9; 95% CI: 0.7-1.4). After adjustment, carriers of PT-G20210A showed a trend towards a higher risk of late fetal loss as compared with non-carriers (RR 2.2; 95% CI: 0.8-6.2). Furthermore, in pregnancies subsequent to those with previous fetal loss there was not a different risk of adverse outcome regardless of the carrier status., Conclusions: Female family members who are heterozygous carriers of isolated PT-G20210A do not seem to be at significant increased risk for fetal loss as compared with non-carriers. Screening for PT-G20210A of fertile age women belonging to these families is not warranted in this situation.

Preterm birth is a major contributor of adverse perinatal outcome. Clinical data suggest that an inflammatory response is important in the process leading to preterm labor. By using a recently introduced mouse model of localized intrauterine lipopolysaccharide-induced inflammation, the effect of interleukin (IL)-18 gene disruption and/or IL-18 neutralization as well as combined IL-1alpha/beta gene disruption on inflammation-induced fetal loss was investigated. The frequency of preterm fetal loss was significantly higher in IL-18 knockout mice (58.9%) and in mice administered IL-18-binding protein (59.7%) compared to wild-type controls (34.7%). The rate of fetal loss was not affected by IL-1alpha/beta gene deficiency (38.7%). Decreased IL-18 protein expression combined with elevated IL-12 protein expression in uterine tissue of IL-18 knockout mice and IL-18-binding protein-treated animals was noticed. These data demonstrate that preterm pregnancy loss in response to intrauterine inflammation was enhanced by disruption of the IL-18 gene and/or IL-18 neutralization, events that may relate to exaggerated Th1 responses because of an increased IL-12/IL-18 ratio.

Background: Activated protein C (APC) resistance with or without factor V Leiden (FVL) is a major risk factor for venous thromboembolism. Many previous pregnancy studies have been focused on APC resistance caused by FVL. Very few have investigated APC resistance in the absence of FVL (APCR(FVL-))., Material and Methods: In a prospective study of 2480 unselected gravidae, blood was drawn in early pregnancy (mean = 12 weeks of gestation). APC resistance was analyzed by an APTT-based method (Coatest) APC-resistance) and the presence of FVL was determined by PCR. The APCR(FVL-) group had similar mean APC resistance ratio as the heterozygous carriers of FVL. The analyses were carried out no earlier than 3 months after delivery when all data were recorded. Small-for-gestational age (SGA) was used as a proxy for intrauterine growth restriction., Results: When compared with the control group, women with APCR(FVL-) had no increased risk of SGA, pre-eclampsia, first trimester fetal loss or venous thromboembolism. However, they had an increased risk of second trimester fetal loss (7.3% vs. 2.7%, P = 0.01), and a tendency of being overweight (17.3% vs. 12.6%, P = 0.19) and of delivering extremely preterm (2.8% vs. 1.0%, P = 0.11)., Conclusion: Women with APC resistance not caused by FVL were not at increased risk for SGA, pre-eclampsia, first trimester fetal loss, or abnormal blood loss. However, they showed an increased prevalence of second trimester fetal loss.

Objective: In spite of extensive clinical examinations or autopsies, as many as 15% to 40% of stillbirths remain unexplained. A systemic fetal inflammatory response is an independent risk factor for severe neonatal morbidity, mediated by proinflammatory cytokines. As a major anti-inflammatory cytokine, interleukin-1 receptor antagonist (IL-1ra) plays a crucial role modulating the proinflammatory response. The gene coding for IL-1ra (IL1RN) is polymorphic. We hypothesized that fetal possession of a specific allele, IL-1RN*2, associated with increased proinflammatory responses, may increase susceptibility to intrauterine fetal death., Study Design: Fetal kidney cells were obtained from paraffin blocks of 27 unexplained stillbirths. DNA was isolated and tested for IL-1RN genotypes by polymerase chain reaction. As a control group, DNA from 302 live births was also tested., Results: There was an enhanced rate of IL-1RN*2 homozygocity, 41%, among unexplained stillbirths compared with the control group, 8.6% (P < .001). Histologic analysis of fetal tissues demonstrated a predominant proinflammatory response in IL-1RN*2 homozygote fetuses. Extensive screening (microbiology, maternal serology, placenta histology) did not identify any specific trigger agent., Conclusion: There is an association between unexplained stillbirth and fetal homozygous IL1RN*2 carriage.

The XAB2 protein (XPA-binding protein 2) with 15 tetratricopeptide repeat motifs has been isolated by virtue of its ability to interact with xeroderma pigmentosum group A (XPA) protein in the yeast two-hybrid system. It has been shown that XAB2 interacted with Cockayne syndrome groups A and B (CSA and CSB) proteins and RNA polymerase II, which are known to be involved in transcription-coupled repair (TCR) and transcription, and that the antibodies against XAB2 protein inhibited the recovery of RNA synthesis after UV irradiation and normal RNA synthesis when microinjected into living fibroblasts. These results have indicated that XAB2 is involved in TCR and transcription. In this report, to elucidate the function of XAB2 in vivo, two types of mutations were introduced into the XAB2 gene in mice: a deletion of the region encompassing the promoter and exons 1-4, and a deletion of the C-terminal 162 amino acids. Both types of XAB2-heterozygous mice appeared normal physiologically and behaviorally. However, XAB2-homozygotes were selectively absent among the newborn mice. A detailed analysis of embryos at different stages of development indicated that the XAB2-homozygous mutants could survive until the morula stage, but could not develop to the blastocyst stage. These results indicate that XAB2 has an essential function in mouse embryogenesis.

Unlabelled: The deletion of chromosome 22q11.2 is involved in the majority of DiGeorge or velo-cardiofacial syndrome. The phenotypic variability was noted in the "CATCH 22" acronym. This acronym doesn't recapitulate the full spectrum of the symptoms. The diagnosis of this syndrome can be done with the prenatal diagnosis, with fetal pathology or with a child alive., Methods: Review of 52 cases with the microdeletion 22q11. Six cases were diagnosed during the prenatal period, 12 cases at fetal pathology examination, and 34 cases during infancy., Results: Cardiac malformations were the major indications (75%) to search for the microdeletion. The facial dysmorphy was difficult to diagnose during the antenatal period or in dead foetus, thereby it was not often recognized. The renal anomalies usually present in 35% of cases, were diagnosed in only 6 to 16% of the cases in our study., Conclusion: Phenotypic diversity of the DiGeorge syndrome is important. Its knowledge allows to better determine the indications of the research of the microdeletion. 22q11.2.

Objective: Interleukin-6 (IL-6)-mediated inflammatory processes have been proposed to be involved in the pathogenesis of pregnancy-associated complications such as late unexplained intrauterine fetal death (IUFD). Therefore we determined whether a common guanine/cytosine polymorphism at position -174 of the promoter of the IL-6 gene (IL6) known to affect in vivo protein activity can serve as candidate gene for this condition., Methods: In a multicenter case-control study, we evaluated the IL6 promoter polymorphism by pyrosequencing in 92 women with IUFD. Ninety-four healthy women with at least one uncomplicated full-term pregnancy and no history of IUFD served as the control group., Results: No significant association was found between the presence of at least one mutant allele of the IL6 promoter polymorphism (P = .2; odds ratio = 1.5 [95% confidence interval, 0.8-2.7]) and the incidence of IUFD. In women with IUFD, the presence of at least one mutant allele of the IL6 promoter polymorphism did not influence timing of fetal death (33.9 [5.1] gestational weeks vs 34.1 [4.9] gestational weeks, P = .8) or birth weight (2055 [1119] g vs 1963 [992] g, P = .7)., Conclusion: To our knowledge, we are the first to report on a common polymorphism of the IL6 promoter gene in women with late IUFD. The investigated IL6 promoter polymorphism can not be seen as candidate gene for IUFD in Caucasian women.

Choroideremia (CHM) is a hereditary eye disease caused by mutations in the X-linked CHM gene. Disruption of the Chm gene in mice resulted in prenatal death of Chm-/Y males and Chm-/Chm+ females that had inherited the mutation from their mothers. Male chimeras and Chm+/Chm- females with paternal transmission of the mutation were viable and had photoreceptor degeneration reminiscent of human choroideremia. Here, we show that Chm-/Y males and Chm-/Chm+ females were retarded at e7.5 and died before e11.5 due to multiple defects of the extra-embryonic tissues. Mutant embryos exhibited deficiency of diploid trophoblasts associated with overabundance of giant cells. In yolk sac and placenta, severe defects in vasculogenesis were obvious. Chm-/Y males exhibited more pronounced phenotypes than Chm-/Chm+ females. The lethal genotypes could be rescued by tetraploid aggregation. Chm-/Chm+ females, but not Chm-/Y males, could also be rescued when their Chm+/Chm- mothers were mated with Mus spretus males. Backcross analysis suggested that the viability of interspecies hybrid Chm-/Chm+ females may be due to expression from the Chm allele on the M. spretus X-chromosome rather than a modifier effect. Our results demonstrate that Chm is essential for diploid trophoblast development and plays a role in the vascularization in placenta and yolk sac.

The availability of the relevant mutant mouse lines provided an opportunity to test the doctrine that platelet activation and fibrin formation account for the importance of thrombin for hemostasis. Prothrombin-deficient mice that survive to birth exsanguinate in the perinatal period. By contrast, protease-activated receptor 4 (PAR4)-deficient mice, which have platelets that fail to respond to thrombin, survive to adulthood with only a mild bleeding diathesis, and fibrinogen-deficient mice show perinatal bleeding but those that survive this period can have a relatively normal life expectancy. We now report that mice that lacked both PAR4 and fibrinogen exsanguinated at birth like prothrombin-deficient mice. However, while approximately half of prothrombin-deficient embryos die during midgestation, mice lacking both PAR4 and fibrinogen developed normally. At face value, these results suggest that platelet activation and fibrin formation are together sufficient to account for the importance of thrombin for hemostasis but not for its importance for embryonic development.

Objective: We determined whether gene polymorphisms associated with thrombophilia and vascular disease as etiologic factors were involved in the pathogenesis of pregnancy-associated complications., Methods: We conducted a multicenter case-control study in which we studied 94 women with late unexplained intrauterine fetal death (IUFD) and 94 healthy women with at least one uncomplicated full-term pregnancy and no history of IUFD. We obtained blood samples from all subjects and analyzed their DNA for 12 common polymorphisms of thrombophilic and vascular genes (factor V Leiden, factor V H1299R, prothrombin G20210A, factor XIII V34L, MTHFR C677T, MTHFR A1298C, beta-fibrinogen-455 G to A, PAI-1 4G/5G, GPIIIa L33P, HFE C282Y, apolipoprotein B R3500Q, and apolipoprotein E2/E3/E4)., Results: We found no significant association between any of the polymorphisms investigated and IUFD. Subgroup analyses involving various combinations of polymorphisms and in which gestational age and fetal weight were corrected for also showed no significant results., Conclusions: Our data represent the largest study to date with respect to thrombophilic and vascular gene polymorphisms in IUFD. In accordance with others, we challenge the importance of thrombophilic and vascular gene polymorphisms in the pathogenesis of this condition.

There are two general pathways by which multicellular eukaryotes repair double-strand DNA breaks (DSB): homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). All mammalian mutants in the NHEJ pathway demonstrate a lack of B and T lymphocytes and ionizing radiation sensitivity. Among these NHEJ mutants, the DNA-PK(cs) and Artemis mutants are the least severe, having no obvious phenotype other than the general defects described above. Ku mutants have an intermediate severity with accelerated senescence. The XRCC4 and DNA ligase IV mutants are the most severe, resulting in embryonic lethality. Here we show that the lethality of DNA ligase IV-deficiency in the mouse can be rescued when Ku86 is also absent. To explain the fact that simultaneous gene mutations in the NHEJ pathway can lead to viability when a single mutant is not viable, we propose a nuclease/ligase model. In this model, disrupted NHEJ is more severe if the Artemis:DNA-PK(cs) nuclease is present in the absence of a ligase, and Ku mutants are of intermediate severity, because the nuclease is less efficient. This model is also consistent with the order of severity in organismal phenotypes; consistent with chromosomal breakage observations reported here; and consistent with the NHEJ mutation identified in radiation sensitive human SCID patients., (Copyright 2002 Elsevier Science B.V.)

Objective: The purpose of this study was to investigate the possible role of inherited and acquired thrombophilia in women with unexplained abortions and intrauterine fetal death., Study Design: We included 75 women with >/=1 unexplained fetal loss, and 75 control subjects with at least 1 healthy term infant and without gestational complications. All of these women were tested for mutations of factor V Leiden, methylenetetrahydrofolate reductase, and prothrombin gene; deficiencies of antithrombin-III, protein C, and protein S; and the presence of antiphospholipid antibodies and fasting homocysteine concentration. A placental histologic study was also carried out., Results: Thirty-five percent of the 75 patients had thrombophilia (control subjects, 16%; P =.008; odds ratio, 2.78). This prevalence was more prominent in second and third trimesters (P =.0002; odds ratio, 6.3), and the presence of combined genetic defects was associated with intrauterine fetal death (P =.04; odds ratio, 12; 95% CI, 1.44-102). When we analyzed the overall gestations of the patients, we observed an increase of intrauterine fetal death in patients with thrombophilia (P =.01) and early pregnancy loss in patients without thrombophilia (P =.02). The analysis of the correlation between extensive placental infarctions and thrombophilic defects rendered values in the boundaries of significance (P =.05)., Conclusion: The significant high prevalence of biologic causes in patients with late fetal loss suggests that a study of thrombophilia should be carried out, together with an assessment of a preventive treatment.

Objective: To describe the relationship between the degree of placental histologic villous mineralization (VM) and stillbirth in aneuploid and euploid fetuses., Methods: The extent of VM for aneuploid and gestational age-matched euploid placentas was graded semiquantitatively on a 0 to 3 scale based on the number of terminal or stem villi containing mineralizations in forty x10 fields of view. The extent of VM was analyzed in relation to fetal status at delivery (liveborn or stillborn) for both aneuploid and euploid fetuses., Results: For 14 available aneuploid placentas, grade 0 or 1 VM was recorded for seven aneuploid specimens, of which two were stillborn. Grade 2 or 3 VM was recorded for seven aneuploid specimens, of which six were stillborn. Fourteen gestational age-matched euploid placentas served as controls. Grade 0 or 1 VM was observed in nine euploid specimens, of which four were stillborn. Grade 2 or 3 VM was observed in five euploid specimens, of which four were stillborn. For aneuploid fetuses, stillbirth was significantly more frequent with grade 2 or 3 VM compared with grade 0 or 1 VM (chi(2) = 4.667, P <.05). This relationship did not exist for euploid fetuses (chi(2) = 1.659, P >.05)., Conclusion: Histologic VM is not a universal finding in, or exclusive to, stillbirths. Aneuploid but not euploid stillbirths show increased histologic VM compared with livebirths. This may implicate impaired placental or circulatory function as a mechanism for death in aneuploid fetuses.

In mammals, both maternal and paternal genomes are required for a fetus to develop normally to term. This requirement is due to the epigenetic modification of genomes during gametogenesis, which leads to an unequivalent expression of imprinted genes between parental alleles. Parthenogenetic mouse embryos that contain genomes from nongrowing (ng) and fully grown (fg) oocytes can develop into 13.5-day-old fetuses, in which paternally and maternally expressed imprinted genes are expressed and repressed, respectively, from the ng oocyte allele. The H19 gene, however, is biallelically expressed with the silent status Igf2 in such parthenotes. In this study, we examined whether the regulation of H19 monoallelic expression enhances the survival of parthenogenetic embryos. The results clearly show that the ng(H19-KO)/fg(wt) parthenogenetic embryos carrying the ng-oocyte genome that had been deleted by the H19 transcription unit successfully developed as live fetuses for 17.5 gestation days. Control experiments revealed that this unique phenomenon occurs irrespective of the genetic background effect. Quantitative gene expression analysis showed that day 12.5 ng(H19-KO)/fg(wt) parthenogenetic fetuses expressed Igf2 and H19 genes at <2 and 82% of the levels in the controls. Histological analysis demonstrated that the placenta of ng(H19-KO)/fg(wt) parthenotes was afflicted with atrophia with severe necrosis and other anomalies. The present results suggest that the cessation of H19 gene expression from the ng-allele causes extended development of the fetus and that functional defects in the placenta could be fatal for the ontogeny., ((C)2002 Elsevier Science (USA).)

Objective: To review animal and human data available regarding the etiology, maternal and fetal impact, and treatment of intrahepatic cholestasis of pregnancy (ICP)., Methods: Pertinent studies on human and animal models of ICP were selected through a MEDLINE database search, focusing on etiology and clinical impact of the disease. Analytic and descriptive studies were included, and the data were analyzed looking for crude numbers., Results: Intrahepatic cholestasis of pregnancy is a pregnancy-specific disorder. Its prevalence is higher in Chile and Sweden compared with any other population. Its etiology is largely unknown, although endocrine, genetic, and environmental factors have been postulated as responsible for the appearance of the disease. Maternal effects of ICP are mild; however, there is a clear association between ICP and poor perinatal outcome, including a higher frequency of fetal distress, preterm labor and delivery, and unexplained fetal death. The treatment is mainly symptomatic. Recent data suggest that oral use of ursodeoxycholic acid improves maternal condition and might prevent the fetal complications of ICP., Conclusions: Intrahepatic cholestasis of pregnancy should be considered a high-risk condition, and careful fetal assessment and appropriate medical intervention might improve perinatal outcome.

The aim of the study was to carry out cytogenetic analyses in pregnancy losses. Samples of cartilage and placenta tissue were obtained prospectively from 237 pregnancy losses of more than 16 weeks of gestation (130 stillbirths, 97 induced abortions and 10 early neonatal deaths). Cartilage culture was performed in 222 samples and placental culture was initiated in 224. The overall culture success rate was 83.5%, 72.3% in stillbirths, 97% in induced abortions and 100% in early neonatal death. An abnormal karyotype was detected in 52 cases: 6.9% in stillbirths, 43.6% in induced abortions and 20% in early neonatal deaths. The rate of discrepancy between the prenatal cytogenetic results in amniotic fluid and the post-termination karyotype was 3%. The tissue of choice for cytogenetic analysis was cartilage in induced abortions and early neonatal death, and placenta in stillbirth. The majority of cases had a chromosome abnormality: multiple congenital anomalies in 74.6%, and a single major anomaly in 9.7%.

The survival of the preimplantation mammalian embryo depends not only on providing the proper conditions for normal development but also on acquiring the mechanisms by which embryos cope with adversity. The ability of the early conceptus to resist stress as development proceeds may be regulated by diverse factors such as the attainment of a cell death program and protective mechanisms involving stress-induced genes and/or cell cycle modulators. This paper reviews the recent research on the genetic regulation of early embryo cell death and senescence focussing on the bovine species where possible. The different modes of cell death will be explained, clarifying the confusing cell death terminology, by advocating the recommendations set forth by the Cell Death Nomenclature Committee to extend to the embryology research field. Specific pro-death and anti-death genes will be discussed with reference to their expression patterns during early mammalian embryogenesis.

Embryonic mortality in both farm animals and humans occurs most frequently during the first few weeks after conception. It can be attributed to abnormalities in the earliest developmental processes during embryogenesis that include implantation, maternal recognition of pregnancy, and formation of the placenta and cardiovascular system. The molecular mechanisms that are essential for all of these early processes are being elucidated at a rapid pace using transgenic and gene knockout approaches in mice. Two important general conclusions have emerged from this work. First, placental defects can occur by a number of different molecular mechanisms and can result from defects in the development or function of its trophoblast, mesenchymal or vascular components. Second, placental and cardiovascular functions are intimately linked. Cells of the placenta, for example, produce hormones that have profound effects on maternal and fetal cardiac and vascular function. In addition, development of the two is linked mechanistically through the use of some genes that are essential for development of both. Understanding the molecular basis of these processes should help to address the major limits to the success of embryo transfer, IVF and embryo cloning practices in livestock species.

The MEN/ELL gene was cloned as a fusion partner of the MLL gene in the t(11;19)(q23;p13.1) translocation, which is found in adult myeloid leukemia. MEN belongs to a family of RNA polymerase II elongation factors and dysregulated production of MEN through the MLL promoter could cause malignant transformation of myeloid cells. To pursue the physiological role and determine the requirement of the MEN gene product in mouse development, we generated knockout mice (MEN-/-) by gene targeting in embryonic stem cells. After intercrossing heterozygous mice to generate homozygous mutants, we identified no homozygotes (MEN-/-) even at E9.5, as well as after birth, by Southern analysis. Moreover, histological examinations revealed degenerative changes in nearly one-fourth of E6.5 embryos, which were gradually resorbed by E8.5. Our findings demonstrated that MEN-/- mice are embryonic lethal, and die before E6.5 and after implantation. MEN should play a nonredundant role in postimplantation development of mice., (Copyright 2000 Academic Press.)

CREB-binding protein (CBP) is a transcriptional co-activator which is required by many transcription factors. Rubinstein-Taybi syndrome (RTS), which is an autosomal dominant syndrome characterized by abnormal pattern formation, is associated with mutations in the human CBP gene. Various abnormalities occur at high frequency in the skeletal system of heterozygous Cbp-deficient mice, but some features of RTS such as cardiac anomalies do not, suggesting that some symptoms of RTS are caused by a dominant-negative mechanism. Here we report the characterization of homozygous Cbp-deficient mice. Homozygous mutants died around E10.5-E12.5, apparently as a result of massive hemorrhage caused by defective blood vessel formation in the central nervous system, and exhibited apparent developmental retardation as well as delays in both primitive and definitive hematopoiesis. Cbp-deficient embryos exhibited defective neural tube closure which was similar to those observed in twist-deficient embryos. However, a decrease in the level of twist expression was not observed in Cbp-deficient embryos. Anomalous heart formation, a feature of RTS patients and mice mutated in the CBP-related molecule, p300, was not observed in Cbp-deficient embryos. Since both Cbp and p300 are ubiquitously expressed in embryonic tissues including the developing heart, these results suggest that cardiac anomalies observed in RTS patients may be caused by a dominant negative effect of mutant CBP.

Rel transcription factors function in flies and vertebrates in immunity and development. Although Rel proteins regulate diverse processes, the control of their function is conserved. In a two-hybrid screen for additional components of the pathway using the Drosophila I-kappaB protein Cactus as a bait, we isolated a novel coiled-coil protein with N-terminal Arg-Asp (RD)- like motifs that we call Cactin. Like the other components of this pathway, Cactin is evolutionarily conserved. Over-expression of cactin in a cactus(A2) heterozygous background results in the enhancement of the cactus phenotype. Both the embryonic lethality and ventralization are strongly increased, suggesting that cactin functions in the Rel pathway controlling the formation of dorsal-ventral embryonic polarity.

Mice lacking the erythroid Kruppel-like factor (EKLF) die in utero at embryonic day 15 (E15) from severe anemia. EKLF(-/-) embryos display a marked deficit in beta-globin gene expression. To test whether beta-globin deficiency was solely responsible for the anemia and intrauterine death, we corrected the globin chain imbalance in EKLF(-/-) embryos by breeding with a strain of mice that express high levels of human gamma-globin. Despite efficient production of hybrid malpha(2)-hgamma(2) hemoglobin in the fetal livers of EKLF(-/-) animals, hemolysis was not corrected and survival was not prolonged. We concluded that deficiency of nonglobin EKLF target genes is a major contributor to the definitive red blood cell abnormalities and prenatal death in EKLF(-/-) embryos. These results suggest that strategies designed to antagonize EKLF function in adults with hemoglobinopathy, in an attempt to reactivate gamma-globin gene expression, may adversely affect other essential aspects of red blood cell physiology. (Blood. 2000;95:1827-1833)

Mice lacking both c-Jun-NH(2)-terminal kinases (JNK1 and JNK2) were generated to define their roles in development. Jnk1/jnk2 double mutant fetuses die around embryonic day 11 (E11) and were found to display an open neural tube (exencephaly) at the hindbrain level with reduced apoptosis in the hindbrain neuroepithelium at E9.25. In contrast, a dramatic increase in cell death was observed one day later at E10.5 in both the hindbrain and forebrain regions. Moreover, about 25% of jnk1-/-jnk2+/- fetuses display exencephaly probably due to reduced levels of JNK proteins, whereas jnk1+/-jnk2-/- mice are viable. These results assign both pro- and anti-apoptotic functions for JNK1 and JNK2 in the development of the fetal brain.

Objective: Recently, it has been proposed that hereditary coagulation abnormalities leading to an increased venous thrombosis risk may play a role in the development of preeclampsia. We tested this hypothesis in women who have had preeclampsia compared with matched control subjects., Study Design: We conducted a case-control study of 163 women with preeclampsia during 1991-1996. Control subjects were matched for age and delivery date. Patients and control subjects were tested for the presence of factor V Leiden, prothrombin 20210A allele, protein C, protein S, and antithrombin deficiency. Logistic regression methods were used for data analysis., Results: The prevalence of these genetic risk factors was similar in the patient group (12.9%) and the control group (12.9%; odds ratio, 1.0; 95% confidence interval, 0.5-3.9). Unexpectedly, we found a high prevalence of factor V Leiden in the control group (9.2%)., Conclusion: We found no differences in the prevalence of genetic risk factors of thrombosis in women with preeclampsia compared with control subjects.

TREB5 (hXBP-1) is a basic region leucine zipper protein which binds to a CRE-like element in both human T-cell leukemia virus type 1 and MHC class II genes. To study the function(s) of TREB5 in normal development, we have generated TREB5 deficient mice by gene targeting. Heterozygous mutant mice have not exhibited any obvious abnormalities; however, homozygous mutant embryos die between embryonic days 10.5 and 14.5. The major defect responsible for lethality is cellular necrosis of cardiac myocytes located at the atrium and the truncus arteriosus with its following ventricle. Necrotic alteration was not observed in either the endocardial cushion or the conotruncal ridge. These results indicate that TREB5 plays an essential role in maintenance and/or growth of cardiac myocytes during cardiogenesis., (Copyright 1999 Academic Press.)

Hypoxia-inducible factor 1 (HIF-1) is a dimeric transcription factor composed of HIF-1alpha and HIF-1beta subunits that plays an essential role in mammalian O2 homeostasis. In Hif1a-/- knockout mice, complete deficiency of HIF-1alpha resulted in cardiac and vascular malformations and embryonic lethality at E10.5. Between E8. 75 and E9.25 striking vascular regression and abnormal remodeling occurred in the cephalic region concomitant with marked mesenchymal cell death. Similar vascular defects were observed in HIF-1alpha- and VEGF-deficient embryos and VEGF mRNA expression was not induced by hypoxia in Hif1a-/- embryonic stem cells. Surprisingly, Hif1a-/- embryos demonstrated increased VEGF mRNA expression compared to wild-type embryos. In tissue culture cells, VEGF mRNA expression was induced by glucose deprivation independent of HIF-1alpha, providing a mechanism for increased VEGF mRNA expression in Hif1a-/- embryos, in which absence of adequate tissue perfusion resulted in both O2 and glucose deprivation. Rather than being associated with VEGF deficiency, the vascular defects in Hif1a-/- embryos were spatially correlated with cell death, the onset of which preceded vascular regression., (Copyright 1999 Academic Press.)

The three mammalian Raf serine/threonine protein kinases mediate the transduction of proliferative and differentiative signals from a variety of cell surface receptors to the nucleus. We report here that Craf-1 is essential for mouse development, as its mutation results in embryonic lethality. Developmental defects are found in mutant placentas as well as in the skin and in the lungs of mutant embryos. Craf-1 mutants also display a generalized growth retardation which is consistent with the ubiquitous expression of Craf-1 and which could be due to the reduced proliferation of mutant cells. Interestingly, the time-point of embryonal death varies depending on the genetic background. This suggests that Craf-1-mediated signaling is affected by genetic background-specific alleles of other genes., (Copyright 1998 Elsevier Science Ireland Ltd. All Rights Reserved.)

The genetics and the epidemiology of Down syndrome (DS) was studied in the area which is covered by our registry of congenital malformations. For each of the 398 new DS cases which were ascertained during the period 1979 to 1996 more than 50 factors were studied and compared to those from control infants. The prevalence of DS was 1.66 per 1000; 2.2% of the DS cases were stillbirths and 29.4% were induced abortions. Karyotypes were obtained in 391 cases of which all but 23 were 47,+21;9 were mosaics (2.3%), and 14 had translocations (3.6%). Interchromosomal effect was a question in 7 cases. The most common types of associated malformations were cardiac anomalies (46.2%) and intestinal atresias (6.0%). Seasonality or time/space clusters were not observed in spite of the Chernobyl nuclear accident. No paternal age effect was demonstrated; 5.3% of the mothers of DS had 2 previous spontaneous abortions (controls 3.7% p < 0.05). At birth, the DS infants measured and weighted less and their head circumference was lower than in control infants. Weight of placenta was also lower than in control infants. In this material there were 4.5% of consanguineous marriages (P < 0.01). The pregnancies of the DS children were more often complicated by threatened abortions than in the controls, 3.2% of the mothers of the DS children were diabetic controls (1.7%), although the difference was not statistically significant. For all other factors studied no statistically significant difference with respect to controls could be demonstrated.

We report on a malformed stillborn with deletion 3p subsequent to direct paracentric insertion (intraarm shift) in the normal father which had been first mistaken for paracentric inversion. The corrected diagnosis was supported by FISH of mapped markers on metaphase chromosomes. In addition we looked for recombinants in sperm. This observation reminds similar cases that had been considered exceptions to the expected meiotic recombination of paracentric inversions and points to a cytogenetic pitfall. Published deletions and paracentric inversions in 3p are briefly quoted.

A classical notion regarding the expression of murine embryonic zeta- and adult alpha-globin genes holds that there is a switch in globin production from the embryonic to the adult form during fetal development. Our previous in situ hybridization studies challenged this view, since both zeta- and alpha-globin mRNAs can be detected simultaneously in the earliest erythrocyte populations. This finding raises the possibility that zeta-globin production might be wholly or partially redundant in embryos in which the adult alpha-globin is also expressed. To test this possibility, we created a null mutation of the zeta-globin gene using homologous recombination in embryonic stem cells. Many outbred mice homozygous for the zeta-null mutation were able to develop normally, undermining the notion that there is an absolute need for zeta-globin and indicating that alpha-globin alone can serve the survival needs of the fetus. Interestingly, insertion of the PGK-Neo cassette (used to create the null mutation) into the zeta-globin gene appears to influence the expression of the nearby alpha-globin genes, giving rise to reduced alpha-globin production and to an alpha-thalassemia-like syndrome. There is also evidence indicating the strong influence of genetic background on the zeta-null and alpha1-null phenotypes, both of which are much more severe in the 129/SvEv inbred genetic background. These quantitative differences can potentially be exploited to identify genes important for erythropoiesis.

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type proteinase inhibitor that directly inhibits factor Xa and, in a factor Xa-dependent fashion, produces feedback inhibition of the factor VIIa/TF catalytic complex responsible for the initiation of coagulation. To further define the physiologic role of TFPI, gene-targeting techniques were used to disrupt exon 4 of the TFPI gene in mice. This exon encodes Kunitz domain-1 of TFPI, which is required for factor VIIa/TF inhibition. In mice heterozygous for TFPI gene-disruption, TFPI(K1)(+/-), an altered form of TFPI lacking Kunitz domain-1, circulates in plasma at a concentration approximately 40% that of wild-type TFPI. TFPI(K1)(+/-) animals have plasma TFPI activity approximately 50% that of wild-type mice, based on a functional assay that measures factor VIIa/TF inhibition, and have a normal phenotype. Sixty percent of TFPI(K1)(-/-) mice die between embryonic days E9.5 and E11.5 with signs of yolk sac hemorrhage. The extent of structural abnormalities within the yolk sac vascular system appears to mirror the condition of the embryo, suggesting that the embryonic and extra-embryonic tissues are both responding to same insult, presumably circulatory insufficiency. Organogenesis is normal in TFPI(K1) null animals that progress beyond E11.5, but hemorrhage, particularly in the central nervous system and tail, is evident during later gestation and none of the TFPI(K1)(-/-) mice survive to the neonatal period. The presence of immunoreactive fibrin(ogen) in the liver and intravascular thrombi is consistent with the notion that unregulated factor VIIa/TF action and a consequent consumptive coagulopathy underlies the bleeding diathesis in these older embryos. Human TFPI-deficient embryos may suffer a similar fate because an individual with TFPI deficiency has not been identified.