Non-invasive risk assessment of fetal sex chromosome aneuploidy through directed analysis and incorporation of fetal fraction.

OBJECTIVE: To assess the performance of a directed chromosomal analysis approach in the prenatal evaluation of fetal sex chromosome aneuploidy. METHODS: We analyzed 432 frozen maternal plasma samples obtained from patients prior to undergoing fetal diagnostic testing. The cohort included women greater than 18 years of age with a singleton pregnancy of greater than 10 weeks gestation. Samples were analyzed using a chromosome-selective approach (DANSR(TM) ) and a risk algorithm that incorporates fetal fraction (FORTE(TM) ). RESULTS: The cohort included 34 cases of sex chromosome aneuploidy. The assay correctly identified 26 of 27 (92.6%) cases of Monosomy X, one case of XXX, and all six cases of XXY. There were four false positive cases of sex chromosome aneuploidy among 380 euploid cases for an overall false positive rate of less than 1%. DISCUSSION: Analysis of the risk for sex chromosome aneuploidies can be accomplished with a targeted assay with high sensitivity.

c-mos and cdc2 cooperate in the translational activation of fibroblast growth factor receptor-1 during Xenopus oocyte maturation.

During oocyte maturation in Xenopus, previously quiescent maternal mRNAs are translationally activated at specific times. We hypothesized that the translational recruitment of individual messages is triggered by particular cellular events and investigated the potential for known effectors of the meiotic cell cycle to activate the translation of the FGF receptor-1 (XFGFR) maternal mRNA. We found that both c-mos and cdc2 activate the translation of XFGFR. However, although oocytes matured by injection of recombinant cdc2/cyclin B translate normal levels of XFGFR protein, c-mos depletion reduces the level of XFGFR protein induced by cdc2/cyclin B injection. In oocytes blocked for cdc2 activity, injection of mos RNA induced low levels of XFGFR protein, independent of MAPK activity. Through the use of injected reporter RNAs, we show that the XFGFR 3' untranslated region inhibitory element is completely derepressed by cdc2 alone. In addition, we identified a new inhibitory element through which both mos and cdc2 activate translation. We found that cdc2 derepresses translation in the absence of polyadenylation, whereas mos requires poly(A) extension to activate XFGFR translation. Our results demonstrate that mos and cdc2, in addition to functioning as key regulators of the meiotic cell cycle, cooperate in the translational activation of a specific maternal mRNA during oocyte maturation.

Induction of anteroposterior neural pattern in Xenopus: evidence for a quantitative mechanism.

The developing vertebrate nervous system arises from ectoderm in response to inductive signals from the dorsal mesoderm, or Spemann organizer. It displays pronounced anteroposterior (AP) pattern, but the mechanism that generates this pattern is poorly understood. We demonstrate that the inducing ability of dorsal mesoderm is regionalized along the AP axis at the early gastrula stage, using the homeodomain-encoding genes Xanf-2 and en-2 as markers of anterior and mid-neural pattern, respectively. In addition, we show that changing the size ratio of posterior dorsal mesoderm to responding ectoderm affects the type of AP pattern induced. A low ratio leads to induction of anterior neural pattern, while a high ratio leads to expression of only mid-neural pattern. These and other results indicate that a quantitative mechanism specifies AP neural pattern.

Partial characterization of a novel growth factor from the blood of women with preeclampsia.

Sera obtained before delivery from women with preeclampsia contain greater mitogenic activity than sera drawn from the same women 24-48 h after parturition or sera from normal parturients. These studies describe the initial characterization of the blood-borne mitogenic factor(s) from preeclamptic women which we have named ELMER (Endogenous Ligand conferring MitogEnic Response). ELMER appears to be a unique mitogen with characteristics that are not identical to those of other known growth factors. ELMER is present in serum as an acid- and heat-labile protein, approximately 160,000 daltons in size, which is a potent mitogen for human fibroblasts but not for human endothelial cells. Its presence in plasma suggests that it is a circulating factor rather than a product of blood coagulation ex vivo. We believe that ELMER represents a potential serum marker of preeclampsia and that it may play roles in the vasospasm and proliferative vascular lesion, termed atherosis, frequently associated with the preeclamptic syndrome.

Developmental gonadal expression of the transcription factor SET and its target gene, P450c17 (17alpha-hydroxylase/c17,20 lyase).

Cytochrome P450c17 catalyzes the 17alpha-hydroxylase/17,20 lyase activity needed for sex steroid synthesis. We recently characterized the nuclear phosphoprotein SET as a novel transcriptional regulator that binds to the -447/-399 region of the rat P450c17 gene, along with the transcription factors COUP-TF II, NGF-IB, and SF-1. Gel shift studies localized SET binding to nucleotides -410/-402. We have shown that SET activates transcription of the rat P450c17 gene in neuronal precursor cells and now show that it also activates transcription from the -418/-399 region of the rat P450c17 gene in mouse Leydig MA-10 cells. Studying the ontogenic expression of SET and P450c17 in the rodent gonad, we found that SET expression preceded P450c17 expression in the embryonic genital ridge, suggesting that SET may be important for initiating P450c17 expression in this region. Expression of SET also preceded P450c17 expression in the testis and ovary, and its expression was much greater during embryogenesis than in the adult gonad. In the adult rat testis, P450c17 was expressed only in Leydig cells, while SET was expressed in Leydig cells and in spermatocytes. In the adult rat ovary, P450c17 was expressed only in theca cells, while SET was expressed in theca cells and also in oocytes. Because SET is expressed early in development in the genital ridge and in the testis and ovary, and because SET has many functions in addition to its activity as a transcription factor, we determined whether SET acts a transcription factor in oocytes. The SET protein was detected by Western blots in Xenopus oocytes from stages II through VI and in mature oocytes. Using extracts of Xenopus oocytes in gel shift assays, we detected a protein that bound to the -418/-399 region of the rat P450c17 gene, to which SET binds. Nuclear injection of either a -418/-399TK32LUC wildtype reporter construct or a construct containing a mutant SET site into Xenopus oocytes from stages III through VI resulted in activation of luciferase activity with the wildtype but not the mutant construct in all stages. These data suggest that Xenopus SET is able to bind to specific DNA sequences to activate transcription at all stages of Xenopus oogenesis. These data indicate that SET is an evolutionarily conserved transcription factor that participates in the early ontogenesis of the gonadal system, regulates P450c17 gene transcription in Leydig cells, and may also activate other genes expressed in immature oocytes, thus playing a role in oocyte development.

Fast MR imaging of fetal CNS anomalies in utero.

BACKGROUND AND PURPOSE: Although sonography is the primary imaging technique for evaluating the developing fetus, significant limitations exist in the sonographic prenatal diagnosis of many brain disorders. Fast MR imaging is increasingly being used to determine the underlying cause of nonspecific fetal CNS abnormalities detected sonographically and to confirm or provide further support for such anomalies. Our goal was to determine the value of MR imaging in establishing the diagnosis of fetal CNS anomalies, to ascertain how this information might be used for patient counseling, and to assess its impact on pregnancy management. METHODS: We prospectively performed MR examinations of 73 fetuses (66 pregnancies) with suspected CNS abnormalities and compared these with available fetal sonograms, postnatal images, and clinical examinations. Retrospectively, the impact on patient counseling and pregnancy management was analyzed. RESULTS: Images of diagnostic quality were routinely obtained with in utero MR imaging, which was particularly valuable in detecting heterotopia, callosal anomalies, and posterior fossa malformations, and for providing excellent anatomic information. We believe that 24 (46%) of 52 clinical cases were managed differently from the way they would have been on the basis of sonographic findings alone. In every case, the referring physicians thought that MR imaging provided a measure of confidence that was not previously available and that was valuable for counseling patients and for making more informed decisions. CONCLUSION: Sonography is the leading technique for fetal assessment and provides reliable, inexpensive diagnostic images. Fast MR imaging is an important adjunctive tool for prenatal imaging in those instances in which a complex anomaly is suspected by sonography, when fetal surgery is contemplated, or when a definitive diagnosis cannot be determined.

Treatment of thyroid disease in pregnancy.

With appropriate therapy, complications related to thyroid disease in pregnancy can be minimized. Although the diagnosis of thyroid endocrinopathy may be difficult in pregnancy, few therapies are contra-indicated. Because medications may cross the placenta, however, clinicians need always to be mindful of potential fetal effects and should work to use the minimal dose necessary to achieve maternal euthyroidism. Thyroid function tests, in particular free T4 and TSH, remain good measures of thyroid function and therapy in pregnancy.

Translational activation and cytoplasmic polyadenylation of FGF receptor-1 are independently regulated during Xenopus oocyte maturation.

FGF signaling is critical for establishing the Xenopus laevis embryonic body plan and requires the expression of functional FGF receptor during early embryogenesis. FGF receptor-1 (XFGFR) maternal mRNA is present in immature oocytes, but the protein is not expressed until oocyte maturation. In this report we demonstrate that endogenous XFGFR translation begins just prior to germinal vesicle breakdown and that translation depends on completion of earlier meiotic events. We show that the previously identified XFGFR 3'UTR translation inhibitory element (TIE), which is necessary and sufficient for repressing translation in the immature oocyte, also regulates the onset of translation during oocyte maturation. In addition we demonstrate that cytoplasmic polyadenylation of XFGFR RNA is regulated independently of TIE-mediated translation and it not sufficient to activate the translation of XFGFR. These experiments reveal that polyadenylation and translational activation are separable events in this mRNA, each of which is timed and regulated independently.

Interphase FISH with chromosome-specific protelomere probes for rapid prenatal diagnosis in a reciprocal translocation carrier.

Interphase fluorescence in situ hybridization (FISH) analysis has become an accepted practice for rapid preliminary analysis of chromosome aneuploidy from direct amniocyte preparations. The use of dual-color interphase FISH analysis with chromosome-specific protelomere probes for the rapid exclusion of chromosomally unbalanced segregants in the pregnancy of a reciprocal translocation carrier is reported. Amniocentesis was performed at 16 weeks gestation on the carrier of a t(5;14)(p14.2;p13), who was ascertained after the birth of a son with the der(5) chromosome. Interphase FISH analysis with probes for 5pter, 5qter and 14qter showed two signals for each, consistent with alternate segregation of the maternal translocation. Subsequent metaphase analysis confirmed a 46,XY,t(5;14)(p14.2;p13)mat karyotype in the fetus. This case illustrates the utility of interphase FISH analysis with protelomere probes for rapid prenatal diagnosis in cases of parental reciprocal translocation.

FGF is a prospective competence factor for early activin-type signals in Xenopus mesoderm induction.

Normal pattern formation during embryonic development requires the regulation of cellular competence to respond to inductive signals. In the Xenopus blastula, vegetal cells release mesoderm-inducing factors but themselves become endoderm, suggesting that vegetal cells may be prevented from expressing mesodermal genes in response to the signals that they secrete. We show here that addition of low levels of basic fibroblast growth factor (bFGF) induces the ectopic expression of the mesodermal markers Xbra, MyoD and muscle actin in vegetal explants, even though vegetal cells express low levels of the FGF receptor. Activin, a potent mesoderm-inducing agent in explanted ectoderm (animal explants), does not induce ectopic expression of these markers in vegetal explants. However, activin-type signaling is present in vegetal cells, since the vegetal expression of Mix.1 and goosecoid is inhibited by the truncated activin receptor. These results, together with the observation that FGF is required for mesoderm induction by activin, support our proposal that a maternal FGF acts at the equator as a competence factor, permitting equatorial cells to express mesoderm in response to an activin-type signal. The overlap of FGF and activin-type signaling is proposed to restrict mesoderm to the equatorial region.

Incidence of persistent birth injury in macrosomic infants: association with mode of delivery.

OBJECTIVE: Our purpose was to determine the incidence of birth injury in a cohort of macrosomic infants (birth weight >4000 gm) and analyze the association between persistent injury and delivery method. STUDY DESIGN: Deliveries of 2924 macrosomic infants were reviewed. Outcomes were compared with those of 16,711 infants with birth weights between 3000 and 3999 gm. RESULTS: Macrosomic infants had a sixfold increase in significant injury relative to controls (relative risk 6.7,95% confidence interval 6.5 to 6.9). Risk of trauma correlated with delivery mode: forceps were associated with a fourfold risk of clinically persistent findings compared with spontaneous vaginal delivery or cesarean section. However, the overall incidence of persistent cases remained low (0.3%); a policy of elective cesarean section for macrosomia would necessitate 148 to 258 cesarean sections to prevent a single persistent injury. Avoidance of operative vaginal delivery would require 50 to 99 cesarean sections per injury prevented. CONCLUSIONS: These findings support a trial of labor and judicious operative vaginal delivery for macrosomic infants.

Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos.

Peptide growth factors may play a role in patterning of the early embryo, particularly in the induction of mesoderm. We have explored the role of fibroblast growth factor (FGF) in early Xenopus development by expressing a dominant negative mutant form of the FGF receptor. Using a functional assay in frog oocytes, we found that a truncated form of the receptor effectively abolished wild-type receptor function. Explants from embryos expressing this dominant negative mutant failed to induce mesoderm in response to FGF. In whole embryos the mutant receptor caused specific defects in gastrulation and in posterior development, and overexpression of a wild-type receptor could rescue these developmental defects. These results demonstrate that the FGF signaling pathway plays an important role in early embryogenesis, particularly in the formation of the posterior and lateral mesoderm.

Regulation of the fibroblast growth factor receptor in early Xenopus embryos.

Recent evidence suggests that fibroblast growth factor (FGF) is a primary mesoderm inducer in Xenopus development. We have isolated a full-length cDNA clone for the Xenopus FGF receptor. Like other FGF receptors, the Xenopus homolog is a membrane-spanning protein with a split intracellular tyrosine kinase domain. The Xenopus FGF receptor mRNA is present as a maternal message whose levels are constant through early development. There is no specific regional localization of the transcript by analysis of FGF receptor mRNA levels in microdissected embryonic tissue. In isolated animal-pole blastomeres, FGF receptor mRNA declines over 16 hr in culture and this loss can be prevented by incubation with FGF or activin. Despite the presence of the FGF receptor mRNA in the oocyte, oocytes in culture do not respond to added FGF. However, injection of exogenous Xenopus FGF receptor transcripts into oocytes does generate a functional response to FGF. Our data suggest that posttranscriptional response to FGF. Our data suggest that posttranscriptional mechanisms regulate the FGF receptor in the oocyte and early embryo and further suggest that mesoderm-inducing factors influence receptor mRNA levels during the time of early tissue formation.

Mitogenic activity is increased in the sera of preeclamptic women before delivery.

There is increasing evidence that endothelial cell injury and altered endothelial cell function play an important role in the pathogenesis of preeclampsia. Endothelial cell injury can lead to the secretion of potent mitogens by activating platelets and directly through the increased production of peptide growth factors by endothelial cells themselves. This study was undertaken to test the hypotheses that increased secretion of mitogenic factors is a feature of preeclampsia and that this activity could be detected in the serum of preeclamptic women. Paired serum samples were collected in early labor and again at 24 to 48 hours post partum from term patients with preeclampsia (n = 15) and normal pregnant controls (n = 14). A bioassay was used to quantify mitogenic activity in these paired samples by assessing their ability to stimulate the incorporation of tritiated thymidine into deoxyribonucleic acid of confluent, quiescent (GO stage) human fibroblasts in monolayer culture. Mitogenic activity was significantly increased in prepartum, preeclamptic sera compared with normal controls and diminished rapidly postpartum to levels equivalent to normal pre- and postpartum serum. These findings are consistent with endothelial cell injury, a process that we believe plays a central role in the pathophysiology of the preeclamptic syndrome.

FGF signalling in the early specification of mesoderm in Xenopus.

We have examined the role of FGF signalling in the development of muscle and notochord and in the expression of early mesodermal markers in Xenopus embryos. Disruption of the FGF signalling pathway by expression of a dominant negative construct of the FGF receptor (XFD) generally results in gastrulation defects that are later evident in the formation of the trunk and tail, though head structures are formed nearly normally. These defects are reflected in the loss of notochord and muscle. Even in embryos that show mild defects and gastrulate properly, muscle formation is impaired, suggesting that morphogenesis and tissue differentiation each depend on FGF. The XFD protein inhibits the expression of the immediate early gene brachyury throughout the marginal zone, including the dorsal side; it does not, however, inhibit the dorsal lip marker goosecoid, which is expressed in the first involuting mesoderm at the dorsal side that will underlie the head. The XFD protein also inhibits Xpo expression, an immediate early marker of ventral and lateral mesoderm. These results suggest that FGF is involved in the earliest events of most mesoderm induction that occur before gastrulation and that the early dorsal mesoderm is already composed of two cell populations that differ in their requirements for FGF.

Temporal regulation of the Xenopus FGF receptor in development: a translation inhibitory element in the 3' untranslated region.

Early frog embryogenesis depends on a maternal pool of mRNA to execute critical intercellular signalling events. FGF receptor-1, which is required for normal development, is stored as a stable, untranslated maternal mRNA transcript in the fully grown immature oocyte, but is translationally activated at meiotic maturation. We have identified a short cis-acting element in the FGF receptor 3' untranslated region that inhibits translation of synthetic mRNA. This inhibitory element is sufficient to inhibit translation of heterologous reporter mRNA in the immature oocyte without changing RNA stability. Deletion of the poly(A) tract or polyadenylation signal sequences does not affect translational inhibition by this element. At meiotic maturation, we observe the reversal of translational repression mediated by the inhibitory element, mimicking that seen with endogenous maternal FGF receptor mRNA at meiosis. In addition, the activation of synthetic transcripts at maturation does not appear to require poly(A) lengthening. We also show that an oocyte cytoplasmic protein specifically binds the 3' inhibitory element, suggesting that translational repression of Xenopus FGF receptor-1 maternal mRNA in the oocytes is mediated by RNA-protein interactions. These data describe a mechanism of translational control that appears to be independent of poly(A) changes.

Does human chorionic gonadotropin have human thyrotropic activity in vivo?

Current evidence indicates that thyroid cells are sensitive to human chorionic gonadotropin (hCG) stimulation. In turn, thyroid hormones appear to influence ovarian and endometrial physiology and reproductive function. Our studies addressed the possible effect of endogenous and exogenous hCG on in vivo thyroid function in normal pregnancy and controlled ovarian hyperstimulation, respectively. Circulating concentrations of hCG in pregnant women during gestation were positively correlated with serum free thyroxine (r = 0.43, p = 0.02) and negatively correlated with thyrotropin levels in the same patients (r = 0.42, p = 0.02). By contrast, exogenous administration of hCG to effect follicular maturation in non-pregnant patients undergoing ovarian hyperstimulation resulted in lower circulating hCG concentrations than seen in pregnancy and failed to alter free thyroxine or thyrotropin levels (p > 0.22). Endogenous isoforms of hCG in early pregnancy appear to have thyrotropic activity in vivo. However, the results indicate that, under clinical conditions of controlled ovarian hyperstimulation for assisted reproduction, exogenous hCG does not affect the hypothalamic-pituitary-thyroid axis.

Preeclamptic sera stimulate increased platelet-derived growth factor mRNA and protein expression by cultured human endothelial cells.

Monolayer cultures of human endothelial cells were incubated with pre- and postdelivery sera from five women with preeclampsia and four matched, normal pregnancies. Conditioned media collected from endothelial cells pretreated in vitro with prepartum sera from preeclamptic women contained greater mitogenic activity and elevated levels of platelet-derived growth factor (PDGF)-like peptides than cells exposed to normal pregnancy sera or postpartum preeclamptic sera. Under the same experimental conditions, predelivery preeclamptic sera stimulated greater expression of endothelial cell PDGF-B-chain mRNA than that accumulated in the presence of matched postdelivery sera. By contrast, no differences in endothelial cell PDGF-B mRNA levels were noted when pre- and postdelivery sera from normal parturients were tested. The results suggest that a factor(s) in the blood of preeclamptic women can stimulate the synthesis and release of a potent growth factor and vasoconstrictor from human endothelial cells.