Maternal first trimester serum levels of free-beta human chorionic gonadotrophin and male genital anomalies.

STUDY QUESTION: Are maternal first trimester levels of serum free-beta hCG associated with the development of hypospadias or undescended testis (UDT) in boys? SUMMARY ANSWER: Overall, first trimester maternal levels of serum free-beta hCG are not associated with hypospadias or UDT. However, elevated levels were found in severe phenotypes (proximal hypospadias and bilateral UDT) suggesting an altered pathway of hormonal release in early pregnancy. WHAT IS KNOWN ALREADY: Human chorionic gonadotrophin peaks in first trimester of pregnancy stimulating fetal testosterone production, which is key to normal male genital development. Endocrine-disrupting insults early in pregnancy have been associated with increased risk of common genital anomalies in males such as hypospadias and UDT. One plausible etiological pathway is altered release of hCG. STUDY DESIGN, SIZE, DURATION: We conducted a record-linkage study of two separate populations of women attending first trimester aneuploidy screening in two Australian states, New South Wales (NSW) and Western Australia (WA), in 2006-2009 and 2001-2003, respectively. PARTICIPANTS/MATERIALS, SETTING, METHODS: Included were women who gave birth to a singleton live born male infant. There were 12 099 boys from NSW and 10 518 from WA included, of whom 90 and 77 had hypospadias; and 107 and 109 UDT, respectively. Serum levels of free-beta hCG were ascertained from laboratory databases and combined with relevant birth outcomes and congenital anomalies via record linkage of laboratory, birth, congenital anomalies and hospital data. Median and quartile levels of gestational age specific free-beta hCG multiple of the median (MoM) were compared between affected and unaffected boys. Logistic regression was used to evaluate the association between levels of free-beta hCG MoM and hypospadias or UDT, stratified by suspected placental dysfunction and co-existing anomalies. Where relevant, pooled analysis was conducted. MAIN RESULTS AND THE ROLE OF CHANCE: There was no difference in median hCG levels amongst women with an infant with hypospadias (NSW = 0.88 MoM, P = 0.83; WA = 0.84 MoM, P = 0.76) or UDT (NSW = 0.89 MoM, P = 0.54; WA = 0.95 MoM, P = 0.95), compared with women with an unaffected boy (NSW = 0.92 MoM; WA = 0.88 MoM). Low (<25th centile) or high (>75th centile) hCG levels were not associated with hypospadias or UDT, nor when stratifying by suspected placental dysfunction and co-existing anomalies. However, there was a tendency towards high levels for severe types, although confidence intervals were wide. When combining NSW and WA results, high hCG MoM levels (>75th centile) were associated with increased risk of proximal hypospadias (odds ratio (OR) 4.34; 95% CI: 1.08-17.4) and bilateral UDT (OR 2.86; 95% CI: 1.02-8.03). LIMITATIONS, REASONS FOR CAUTION: There were only small numbers of proximal hypospadias and bilateral UDT in both cohorts and although we conducted pooled analyses, results reported on these should be interpreted with caution. Gestational age by ultrasound may have been inaccurately estimated in small and large for gestational age fetuses affecting hCG MoM calculation in those pregnancies. Despite the reliability of our datasets in identifying adverse pregnancy outcomes, we did not have pathology information to confirm tissue lesions in the placenta and therefore our composite outcome should be considered as a proxy for placental dysfunction. WIDER IMPLICATIONS OF THE FINDINGS: This is one of the largest population-based studies examining the association between maternal first trimester serum levels of free-beta hCG and genital anomalies-hypospadias and UDT; and the first to compare specific phenotypes by severity. Overall, our findings does not support the hypothesis that alteration in maternal hCG levels is associated with the development of male genital anomalies; however, high hCG free-beta levels found in severe types suggest different underlying etiology involving higher production and secretion of hCG. These findings require further exploration and replication. STUDY FUNDING/COMPETING INTERESTS: This work was funded by the National Health and Medical Research Council (NHMRC) grant APP1047263. N.N. is supported by a NHMRC Career Development Fellowship APP1067066. C.B. was supported by a NHMRC Principal Research Fellowship #634341. The funding agencies had no role in the design, analysis, interpretation or reporting of the findings. There are no competing interests. TRIAL REGISTRATION NUMBER: Not applicable.

Iron deficiency in early pregnancy using serum ferritin and soluble transferrin receptor concentrations are associated with pregnancy and birth outcomes.

BACKGROUND/OBJECTIVES: There are several biomarkers for measuring iron deficiency (ID) in pregnancy, but the prevalence of ID and its association with inflammation and adverse pregnancy outcomes is inconclusive. The aim of this work was to describe the prevalence and determinants of first trimester ID and associations with pregnancy and birth outcomes. SUBJECTS/METHODS: A record-linkage cohort study of archived serum samples of women attending first trimester screening and birth and hospital data to ascertain maternal characteristics and pregnancy outcomes. Sera were analysed for iron stores (ferritin; µg/l), lack of iron in the tissues (soluble transferrin receptor (sTfR); nmol/l) and inflammatory (C-reactive protein (CRP); mg/dl) biomarkers. Total body iron (TBI) was calculated from serum ferritin (SF) and sTfR concentrations. Multivariate logistic regression analysed risk factors and pregnancy outcomes associated with ID using the definitions: SF<12 µg/l, TfR ⩾ 21.0 nmol/l, and TBI<0 mg/kg. RESULTS: Of the 4420 women, the prevalence of ID based on ferritin, sTfR and TBI was 19.6, 15.3 and 15.7%, respectively. Risk factors of ID varied depending on which iron parameter was used and included maternal age <25 years, multiparity, socioeconomic disadvantage, high maternal body weight and inflammation. ID, defined by SF and TBI but not TfR, was associated with reduced risk of gestational diabetes mellitus (GDM). ID defined using TBI only was associated with increased risk of large-for-gestation-age (LGA) infants. CONCLUSIONS: Nearly one in five Australian women begin pregnancy with ID. Further investigation of excess maternal weight and inflammation in the relationships between ID and GDM and LGA infants is needed.

High maternal iron status, dietary iron intake and iron supplement use in pregnancy and risk of gestational diabetes mellitus: a prospective study and systematic review.

AIM: High iron measured using dietary intake and biomarkers is associated with Type 2 diabetes. It is uncertain whether a similar association exists for gestational diabetes mellitus. The aim of this systematic review was to conduct a cohort study examining first trimester body iron stores and subsequent risk of gestational diabetes, and to include these findings in a systematic review of all studies examining the association between maternal iron status, iron intake (dietary and supplemental) and the risk of gestational diabetes. METHODS: Serum samples from women with first trimester screening were linked to birth and hospital records for data on maternal characteristics and gestational diabetes diagnosis. Blood was analysed for ferritin, soluble transferrin receptor and C-reactive protein. Associations between iron biomarkers and gestational diabetes were assessed using multivariate logistic regression. A systematic review and meta-analysis, registered with PROSPERO (CRD42014013663) included studies of all designs published in English from January 1995 to July 2015 that examined the association between iron and gestational diabetes and included an appropriate comparison group. RESULTS: Of 3776 women, 3.4% subsequently developed gestational diabetes. Adjusted analyses found increased odds of gestational diabetes for ferritin (OR 1.41; 95% CI 1.11, 1.78), but not for soluble transferrin receptor (OR 1.00; 95% CI 0.97, 1.03) per unit increase of the biomarker. Two trials of iron supplementation found no association with gestational diabetes. Increased risk of gestational diabetes was associated with higher levels of ferritin and serum iron and dietary haem iron intakes. CONCLUSIONS: Increased risk of gestational diabetes among women with high serum ferritin and iron levels and dietary haem iron intakes warrants further investigation.

Source of angiopoietin-2 in the sera of women during pregnancy.

Placental development requires coordinated angiogenesis regulated by multiple factors including angiopoietins. Previously we demonstrated that the concentration of angiopoietin-2 (Ang-2) in the sera of women rises markedly in pregnancy in early gestation. This increase is reduced in pregnancies subsequently complicated by intrauterine growth restriction (IUGR). We now show that the concentration of Ang-2, but not Ang-1, in maternal serum is increased during normal pregnancy, peaking at the end of the first trimester. We also demonstrate that a key source of the elevated Ang-2 levels during pregnancy is decidual endothelial cells (DECs) but not cytotrophoblasts. Secretion of Ang-2 by DECs relies on the release from intracellular stores and the synthesis of new Ang-2 protein and is regulated by serum factors at a translational level. Further studies on the role of Ang-2 during pregnancy are warranted as well as the evaluation of Ang-2 as a marker to predict adverse pregnancy outcomes.

First trimester screening of maternal placental protein 13 for predicting preeclampsia and small for gestational age: in-house study and systematic review.

OBJECTIVE: To describe normative levels of PP13 in first trimester of pregnancy and determine the accuracy of PP13 in predicting preeclampsia and small for gestational age (SGA) infants. METHODS: We measured PP13 in archived first trimester serum samples from an unselected maternal cohort of 2989 women. Associations of PP13 levels and diagnostic accuracy in predicting adverse pregnancy outcomes were assessed using multivariate logistic regression models. Due to inadequate number of cases we then conducted a systematic review and subsequent meta-analysis of predictive accuracy. Structured searches including all languages were completed in electronic databases and supplemented by cross-checking reference lists of relevant publications. Characteristics, data extraction and quality assessment of studies was conducted by independent assessors. RESULTS: Overall, 2678 women were included in the in-house study with 71 (2.7%) preeclampsia cases, 5 (0.2%) early-onset preeclampsia (≤34 weeks) cases; and 191 (7.1%) and 41 (1.5%) infants SGA<10th and <3rd centile. Median (IQR) normative level of PP13 in unaffected pregnancies was 53.5 (37.7-71.8) pg/ml. The area under the receiver operating characteristic curve (AUC) for multivariate models was 0.72 (95%CI 0.66-0.78) for preeclampsia; 0.82 (95%CI 0.63-0.99) for early-onset preeclampsia; 0.73 (95%CI 0.69-0.77) for SGA<10th centile; and 0.83 (95%CI 0.78-0.88) for SGA<3rd centile. Eight studies were included in the systematic review, normative levels of PP13 were assessed in four studies but these were variable; and meta-analysis was performed on seven studies. Sensitivity rates of PP13 based on 5% fixed false positive rates were 24%, 45% and 26% for preeclampsia, for early-onset preeclampsia and SGA, respectively. There was no evidence of between-study heterogeneity. CONCLUSIONS: First trimester PP13, in combination with maternal characteristics and other serum biomarkers was inadequate for screening purposes and predicting women at risk.

Reduced maternal serum concentrations of angiopoietin-2 in the first trimester precede intrauterine growth restriction associated with placental insufficiency.

The aim of this study was to investigate whether maternal serum levels of angiopoietin-2 (Ang-2) and pregnancy-associated plasma protein A (PAPP-A) are associated with subsequent intrauterine growth restriction (IUGR). Ang-2 was measured in 29 nonpregnant and 44 pregnant women at 10-13 weeks of gestation. The median concentration of Ang-2 was 26.61 ng/ml in normal pregnant women compared with 1.71 ng/ml in nonpregnant controls (P < 0.01). Women who subsequently developed severe IUGR had lower levels of Ang-2 compared with normal pregnant controls (P < 0.01). PAPP-A levels were similar in all pregnant groups. These findings suggest that Ang-2 should be evaluated for its ability to predict pregnancies that later are affected by IUGR.

Influence of mechanical and biological signals on gene expression in human MG-63 cells: evidence for a complex interplay between hydrostatic compression and vitamin D3 or TGF-beta1 on MMP-1 and MMP-3 mRNA levels.

Biological mediators can influence the activity and differentiation of bone cells. 1,25-dihydroxy-vitamin D3 (1,25-(OH)2D3) is known to induce differentiation of precursors into mature osteoblasts, and transforming growth factor-beta1 (TGF-beta1) can modulate the activity of bone cells leading to alterations in proliferation and gene expression patterns. Bone-derived cells were loaded via intermittent cyclic hydrostatic pressure (icHP) on cells under basal conditions and in the presence of 1,25-(OH)2D3 or TGF-beta1. Evaluating the effects of loading on the cells allowed for a comparison to be made between responsiveness to biomechanical and biochemical stimuli and their potential interplay. The effects of icHP on mRNA levels for the specific genes involved in bone remodelling and differentiation were measured in MG-63 cells using reverse transcription-polymerase chain reaction (RT-PCR). The mRNA levels for matrix metalloproteinase-1 and -3 (MMP-1 and MMP-3) were significantly, and uniquely, increased (p < 0.001) in cells exposed to icHP under serum-free conditions for 4-12 h. However, mRNA levels for MMP-3, but not MMP-1, were significantly enhanced in cells subjected to static hydrostatic pressure (HP). Treatment of cells with 1,25-(OH)2D3 resulted in increased (p < 0.001) mRNA levels for osteocalcin and decreased (p < 0.001) mRNA levels for both MMP-1 and MMP-3. In cells exposed to icHP and 1,25-(OH)2D3, the mRNA levels for both MMP-1 and MMP-3 were elevated (p < 0.001) compared with hormone alone, but not to the same degree (p < 0.01) as cells subjected to icHP alone. Addition of TGF-beta1 to cells led to increases in cell proliferation and expression of collagen I, as well as decreases in expression of osteocalcin and MMP-1 and MMP-3. Exposure of cells to icHP and TGF-beta1 again led to unique and significant increases in expression of MMP-1 and MMP-3. No changes in mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or any of the other 9 genes assessed, including those for MMP-2 and MMP-13, were detected under any of the conditions described. Therefore, icHP can induce alterations in mRNA levels for a specific subset of genes in both premature and mature osteoblasts. Such stimuli can modulate the impact of potent biological mediators in defining patterns of gene expression by bone cells and potentially modify function in vivo.

P2Y(11) receptor expression by human lymphocytes: evidence for two cAMP-linked purinoceptors.

The effects of extracellular ATP, ADP, AMP and adenosine on cAMP accumulation have been studied in freshly isolated B-lymphocytes from patients with chronic lymphocytic leukemia. Extracellular ATP and several nucleotide analogs stimulated cAMP accumulation with the following order of potency: ATP (EC(50)=120+/-20 microM)>ADP>>AMP. ADP was less effective than ATP and may be a partial agonist. AMP exhibited variable but generally weak activity. The stable analog of ATP, alpha,beta-methylene ATP (EC(50)=110+/-15 microM) also stimulated cAMP accumulation and exhibited similar efficacy to ATP. The P2Y(2) receptor agonist, UTP had no effect on intracellular cAMP levels. Adenosine and the A(2A)/A(2B) receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) also stimulated cAMP accumulation in CLL lymphocytes. Adenosine deaminase inhibited the cAMP response to adenosine but had no effect on the ATP-induced cAMP response. On the other hand, the AMP analog, adenosine 5'-thiomonophosphate, (AMPS; 1.0 mM) inhibited ATP-induced and alpha,beta-methylene ATP-induced cAMP production but had no effect on adenosine-induced cAMP production. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the presence of P2Y(11) receptor as well as A(2A) and A(2B) receptor mRNA in chronic lymphocytic leukemia lymphocytes. However, A(2B) receptors would appear to be relatively ineffective because the A(2A) selective agonist, CGS-21680 exhibited comparable efficacy to NECA. Furthermore, the A(2A)-selective antagonist 8-(3-chlorostyryl)-caffeine (CSC) right-shifted the concentration-response curve for NECA. Taken together, the data indicate that ATP induces cAMP accumulation via the activation of P2Y(11) receptors whereas adenosine induces cAMP accumulation via the activation of A(2A) receptors. Coordinate activation of P2Y(11) and A(2A) receptors may influence the developmental fate of normal B-lymphocytes.

Mitogenic effect of lithium in FRTL-5 cells can be reversed by blocking de novo cholesterol synthesis and subsequent signal transduction.

Lithium therapy is the therapeutic mainstay for bipolar disorder and has been associated in the thyroid with euthymic goiter, hyper and hypothyroidism as well as thyroid autoimmune disease. The FRTL-5 cell line is a well known model of thyroid cell physiology, where lithium has been shown to increase 3H-thymidine uptake at concentrations of 2 mM. This mitogenic effect was not associated with adenylate cyclase as measured by cyclic adenosine monophosphate (cAMP) production. The de novo synthesis of cholesterol is an important signal transduction pathway in FRTL-5 cells, where newly synthesized Rho GTPase is geranylgeranylated, enabling membrane localization of the G-protein and subsequent G1 to S-phase transition, resulting from extracellular stimulation. Here we confirm lithium mitogenicity at therapeutically relevant concentrations (1 mM) and demonstrate a lithium-associated accumulation of FRTL-5 cells in S-phase of the cell cycle. These effects could be abolished by Pravastatin, a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA), the rate-limiting enzyme in the formation of intermediates (de novo cholesterol synthesis) required for G-protein prenylation. Pravastatin, similar to lithium, showed no effect on cAMP production either under basal or thyroid stimulating hormone (TSH)-stimulated conditions indicating that de novo cholesterol synthesis is not involved with adenylate cyclase. The inhibitory effect of pravastatin could be overcome by reinitiating de novo cholesterol synthesis. This was achieved by the addition of the cell permeable, first metabolite (mevalonate) after HMG-CoA, which allowed the cycle to continue, leading eventually to protein prenylation, despite the presence of Pravastatin. These novel findings demonstrate lithium involvement in de novo cholesterol synthesis and G-protein prenylation, an important signal transduction pathway in FRTL-5 cells.

Pharmacological profile of a novel cyclic AMP-linked P2 receptor on undifferentiated HL-60 leukemia cells.

1. Extracellular ATP (EC50=146+/-57 microM) and various ATP analogues activated cyclic AMP production in undifferentiated HL-60 cells. 2. The order of agonist potency was: ATPgammaS (adenosine 5'-O-[3-thiotriphosphate]) > or = BzATP (2'&3'O-(4-benzoylbenzoyl)-adenosine-5'-triphosphate) > or = dATP > ATP. The following agonists (in order of effectiveness at 1 mM) were all less effective than ATP at concentrations up to 1 mM: beta,gamma methylene ATP > or = 2-methylthioATP > ADP > or = Ap4A (P1, P4-di(adenosine-5') tetraphosphate) > or = Adenosine > UTP. The poor response to UTP indicates that P2Y2 receptors are not responsible for ATP-dependent activation of adenylyl cyclase. 3. Several thiophosphorylated analogs of ATP were more potent activators of cyclic AMP production than ATP. Of these, ATPgammaS (EC50=30.4+/-6.9 microM) was a full agonist. However, adenosine 5'-O-[1-thiotriphosphate] (ATPalphaS; EC50=45+/-15 microM) and adenosine 5'-O-[2-thiodiphosphate] (ADPbetaS; EC50=33.3+/-5.0 microM) were partial agonists. 4. ADPbetaS (IC50=146+/-32 microM) and adenosine 5'-O-thiomonophosphate (AMPS; IC50=343+/-142 microM) inhibited cyclic AMP production by a submaximal concentration of ATP (100 microM). Consistent with its partial agonist activity, ADPbetaS was estimated to maximally suppress ATP-induced cyclic AMP production by about 65%. AMPS has not been previously reported to inhibit P2 receptors. 5. The broad spectrum P2 receptor antagonist, suramin (500 microM), abolished ATP-stimulated cyclic AMP production by HL-60 cells but the adenosine receptor antagonists xanthine amine congener (XAC; 20 microM) and 8-sulpho-phenyltheophylline (8-SPT; 100 microM) were without effect. 6. Extracellular ATP also activated protein kinase A (PK-A) consistent with previous findings that PK-A activation is involved in ATP-induced differentiation of HL-60 cells (Jiang et al., 1997). 7. Taken together, the data indicate the presence of a novel cyclic AMP-linked P2 receptor on undifferentiated HL-60 cells.

The Fischer rat thyroid cell line FRTL-5 exhibits a nondiploid karyotype.

The FRTL-5 cell line is a stable thyroid cell line derived from the thyroid gland of the Fischer rat under defined culture conditions, which has been widely adopted as a model system for the study of thyroid cell function and for bioassay. While characterizing by flow cytometry FRTL-5 cells that were supplied to this laboratory by ATCC (American Type Cell Collection), we discovered that the cells (ATCC CRL8305) were not diploid, having approximately twofold the DNA content relative to a diploid control. The increase in DNA content also applied to cells originally supplied by the ATCC (described as passage 14) that when counted in metaphase had a modal chromosomal count of 84, indicating tetraploid status, double the expected 42 of a diploid rat cell. In a private communication, the ATCC confirmed these findings which nevertheless are contrary to previous literature reports where they were reported to be diploid. Tetraploid cells are less sensitive to thyrotropin (TSH) as measured by cyclic adenosine monophosphate (cAMP) production, compared with diploid cells (p = < 0.001). Despite similar 3H-thymidine uptake in 0.2% fetal calf serum, tetraploid cells show increased 3H-thymidine uptake in 5% fetal calf serum in the absence of TSH (p = 0.001). The origin of these chromosomal changes is unclear, but these findings must raise doubts regarding the suitability of the tetraploid FRTL-5 cell line as a model for studies of human or animal thyroid physiology.

Extracellular ATP triggers cyclic AMP-dependent differentiation of HL-60 cells.

Extracellular ATP and ATPgammaS (1-1000 microM) stimulated cyclic AMP (cAMP) production in undifferentiated HL-60 cells. The potency order for adenine nucleotides and adenosine was ATPgammaS > ATP >> ADP > or = AMP = Adenosine. Indomethacin (50 microM) had no effect on ATP-induced cAMP production. ATP and ATPgammaS also suppressed cell growth and induced differentiation as revealed by fMLP-stimulated beta-glucuronidase release 48 h after exposure. The potency order for the induction of fMLP-stimulated beta-glucuronidase release by adenine nucleotides and adenosine was ATPgammaS > or = ATP > ADP > AMP = Adenosine approximately 0. The protein kinase A inhibitor Rp-8-Br-cAMPS (10-200 microM) suppressed ATP-induced differentiation but had no effect on ATP-dependent growth suppression. UTP which, like ATP, activates P2U receptors on HL-60 cells, had no effect on cAMP production, cell growth, or differentiation. The data suggest the existence of a novel receptor for ATP on undifferentiated HL-60 cells that is coupled to the activation of adenylate cyclase and cAMP-dependent differentiation.

Extracellular ATP triggers cyclic AMP-dependent differentiation of HL-60 cells.

Extracellular ATP and ATP gamma S (1-1000 microM) stimulated cyclic AMP (cAMP) production in undifferentiated HL-60 cells. The potency order for adenine nucleotides and adenosine was ATP gamma S > ATP > > ADP > 3 AMP = Adenosine. Indomethacin (50 microM) had no effect on ATP-induced cAMP production. ATP and ATP gamma S also suppressed cell growth and induced differentiation as revealed by fMLP-stimulated beta-glucuronidase release 48 h after exposure. The potency order for the induction of fMLP-stimulated beta-glucuronidase release by adenine nucleotides and adenosine was ATP gamma S > 3 ATP > ADP > AMP = Adenosine approximately 0. The protein kinase A inhibitor Rp-8-Br-cAMPS (10-200 mM) suppressed ATP-induced differentiation but had no effect on ATP-dependent growth suppression. UTP which, like ATP, activates P2U receptors on HL-60 cells, had no effect on cAMP production, cell growth, or differentiation. The data suggest the existence of a novel receptor for ATP on undifferentiated HL-60 cells that is coupled to the activation of adenylate cyclase and cAMP-dependent differentiation.