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.

Maternal plasma cell-free fetal and maternal DNA at 11-13 weeks' gestation: relation to fetal and maternal characteristics and pregnancy outcomes.

OBJECTIVE: To examine the possible relationship between maternal and fetal characteristics and pregnancy outcomes on fetal and maternal cell-free (cf) DNA in maternal plasma at 11-13 weeks' gestation. METHODS: cfDNA was extracted from maternal plasma of 1,949 singleton pregnancies and chromosome-selective sequencing was used to determine the proportion of cfDNA and total cfDNA counts which was of fetal and maternal origin. Multivariate regression analysis was used to determine whether specific maternal and fetal characteristics and pregnancy complications, such as preeclampsia (PE), early spontaneous preterm birth (SPB) and delivery of small for gestational age (SGA) neonates, were significant predictors of fetal and maternal cfDNA in maternal plasma. RESULTS: The fetal and maternal cfDNA plasma concentration increased with serum pregnancy-associated plasma protein-A and free ß-human chorionic gonadotropin level, was higher in women of Afro-Caribbean and East-Asian racial origin than in Caucasians, and lower in smokers, but it was not significantly altered in pregnancies complicated by PE, SPB or SGA. The fetal cfDNA level was inversely related to maternal weight and uterine artery pulsatility index, and maternal cfDNA increased with maternal weight. CONCLUSIONS: The fetal and maternal cfDNA level in maternal plasma is affected by maternal and fetal characteristics, but it is not altered in pregnancy complications.

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.

Clinical course and biochemistry of sialuria.

Sialuria is a rare inborn error of metabolism in which excessive free sialic acid (N-acetylneuraminic acid, NeuAc) is synthesized. A defect in the feedback inhibition of UDP-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase by the end-product of the sialic acid synthetic pathway, CMP-NeuAc, is the mechanism underlying this overproduction. Recent evidence suggests that sialuria is an autosomal dominant disorder. Only five patients have been documented to have such an enzymatic defect. We report a longitudinal study of one of the original sialuria patients, to age 11 years. Although he has coarse features and massive hepatomegaly, he has shown normal growth and relatively normal development. Pulmonary function testing showed minimal small airway obstruction. At 11 years, he developed intermittent abdominal pain and transient transaminase elevation above his baseline. Sialuria should be considered in the differential diagnosis of a patient with a phenotype suggestive of a mucopolysaccharidosis or oligosaccharidosis in the absence of developmental regression or prominent dysostosis multiplex. We recommend close monitoring of liver and pulmonary function in sialuria patients.

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.

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.

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.

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.

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.

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.

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.

The tumor suppressor Smad4/DPC 4 as a central mediator of Smad function.

BACKGROUND: The invertebrate and vertebrate Smad proteins have recently been identified as important mediators of the responses to transforming growth factor beta (TGF-beta) and related factors. We have previously shown that Smad3 and Smad4 (the product of the tumor suppressor gene DPC 4) strongly synergize as mediators of TGF-beta signaling, and that inactive carboxy-terminally truncated mutants of either Smad act as dominant-negative inhibitors of the natural TGF-beta response. The finding that Smad4, unlike Smad3, does not interact with the TGF-beta receptor, coupled with the distinct structural features of Smad4, raises the possibility that Smad4 cooperates not only with Smad3, but also with Smad1 and Smad2 to mediate signaling by TGF-beta family members. RESULTS: Overexpression of Smad4 in 'animal caps' taken from Xenopus embryos induced both ventral and dorsal mesoderm, thereby mimicking the effects of TGF-beta family members - bone morphogenetic protein-2 (BMP-2) or BMP-4 and activin, respectively. Low levels of Smad4 mRNA coinjected with Smad1 or Smad2 mRNA also synergized to induce ventral or dorsal mesoderm, respectively. In addition, Smad4 synergized Smad2, as it does with Smad3, to induce gene expression from the promoter for plasminogen activator inhibitor-1. The carboxy-terminal domains of both Smad3 and Smad4 were required for this synergy. Finally, a short carboxy-terminal truncation of Smad4, previously identified as a mutation of DPC 4 in tumors, blocked nuclear translocation of wild-type Smads 1, 2, 3 and 4, consistent with our observation of a physical interaction between truncated Smad4 and the other Smads. CONCLUSIONS: Our observations indicate that Smad4 cooperates with Smad1, Smad2 and Smad3 to act as a common mediator of signaling by TGF-beta-related factors, and provide a mechanism that explains the dominant-negative interference with receptor signaling that results from expression of the naturally occurring Smad4/DPC 4 truncation mutant.

Trapping genes expressed in the developing mouse inner ear.

Identification of the genes involved in the development of the mouse inner ear and developmental studies of mice that bear mutations in these genes is an important approach to understanding genetically determined human auditory dysfunction. Towards this end, we initiated a gene trap screen designed to simultaneously mark and mutate genes in mouse embryonic stem cells by the insertion of a lacZ reporter gene. Expression of beta-galactosidase in gene trap cell lines was monitored both before and after the addition of factors that are known to affect inner ear development. Gene trap cell lines that expressed beta-galactosidase under one or more culture conditions were used to create chimeric mouse embryos for studies of reporter gene expression in vivo. A high proportion of these gene trap insertions were expressed in the developing inner ear, suggesting that this strategy provides an effective means of identifying genes that may be involved in inner ear development or function.

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.

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.

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.

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.

Cell mixing in the spinal cords of mouse chimeras.

With the aim of determining whether there is significant cell mixing during development of the spinal cord, experimental chimeric mice containing two genetically distinct cell populations were produced by aggregating BALB/c or BALB/c x LPT hybrid embryos with C3H/HeN embryos. The BALB/c and LPT hybrid spinal cord cells were distinguished histochemically from the C3H/HeN spinal cord cells by using beta-glucuronidase as an independent cell genotype marker. BALB/c and LPT hybrid cells have high levels of beta-glucuronidase activity, while the C3H/HeN cells have low levels. The spinal cords of the chimeras were dissected out regionally (i.e., cervical, thoracic, and lumbar areas) and were sectioned serially. Each region was then analyzed by scoring large- and medium-sized neuronal cell bodies (greater than or equal to 10 microns) whose genotypes were distinguished by their beta-glucuronidase levels. Observations of seven chimeric mice, with coat colors that varied from one extreme (5% albino) to the other (90% albino), suggest that each chimeric spinal cord is a relatively homogeneous population throughout its length. On average only 4 to 5 percentage point differences were observed when comparing left-right, cranial-caudal, and dorsal-ventral regions within a given chimera. The cell mixing, however, is not total, and regional variations were noted. Maximum left-right differences between different spinal cord levels never exceeded 18 percentage points, while in the entire cord the maximum left-right difference was 11 percentage points. When considering dorsal-ventral differences, 18 and 15 percentage points were observed within the spinal cord levels and the entire cord, respectively. However, when comparisons were made between smaller subregions (e.g., right-dorsal-cervical vs left-ventral-lumbar), larger differences of up to 30 percentage points were observed. In addition, the genotype proportions in the spinal cord were closely correlated with the visually estimated proportions of coat color genotypes.