Urinary thiosulphate levels over the first three days of life.

<p>H₂S turnover was stable across the first three days of life in term and preterm neonates. In very preterm neonates, levels rose significantly over the first 72 hours of life (median±IQR). ^a-b-cp<0.0001 significant difference across days in very preterm gestational age group (Friedman repeated measures ANOVA for non-parametric data).</p

Clinical Characteristics of Neonates.

<p>Data presented as median (minimum-maximum) or number (%). APGAR Score – scores 7 and above are generally regarded as normal, 4 to 6 fairly low and 3 and below critically low; CRIB II Score – Clinical Risk Index for Babies II, higher scores reflect poorer physiological stability; CPAP – Continuous Positive Air Pressure respiratory support; Patent Ductus Arteriosus refers to a hemodynamically significant duct diagnosed in first 72 hrs; IVH – intraventricular hemorrhage greater than grade II (significant IVH); Mean Blood Pressure reported is that at 24 h postnatal age and was not assessed in term controls; Death is those infants that survived to 72 h postnatal age but died prior to discharge.</p><p>*significantly different from females of the same gestational age group p<0.05;</p>†<p>significantly different from preterm neonates, within sex.</p

A preabsorption control was conducted to identify nonspecific binding of the antibody.

<p>GR antibody was preabsorbed with control peptide prior to Western Blot exposure. The left panel represents binding after GR antibody preabsorption and the right panel is the blot exposed to GR antibody alone. Human placental tissues was included as a positive control.</p

Maternal and Neonatal Characteristics.

<p>Maternal and Neonatal Characteristics.</p

Sex differences in thiosulphate levels in very preterm neonates in early postnatal life (median±IQR).

<p>H₂S turnover, measured as urinary thiosulphate excreted per day per kg body weight, was significantly higher in males than females on both day 1 (*p = 0.01) and day 2 (**p = 0.04) of postnatal life (Friedman repeated measures ANOVA for non-parametric data).</p

Summary of GR isoform expression in guinea pig placenta in relation to fetal sex, gestational age and betamethasone exposure.

<p>The circles represent a cell with the smaller circle being the nucleus and the larger being the cytoplasm. Isoforms that were present under each condition are in small font while isoforms that were significantly altered by treatment, or gestational age are in bold. Fig 4a represents female placentae and 4b represents male placentae. This figure was generated from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148226#pone.0148226.t002" target="_blank">Table 2</a> and isoforms were included in the figure if the median value was greater than 0 regardless of whether GR expression was significantly altered or not.</p

Identification of Eight Different Isoforms of the Glucocorticoid Receptor in Guinea Pig Placenta: Relationship to Preterm Delivery, Sex and Betamethasone Exposure

<div><p>The placental glucocorticoid receptor (GR) is central to glucocorticoid signalling and for mediating steroid effects on pathways associated with fetal growth and lung maturation but the GR has not been examined in the guinea pig placenta even though this animal is regularly used as a model of preterm birth and excess glucocorticoid exposure. Guinea pig dams received subcutaneous injections of either vehicle or betamethasone at 24 and 12 hours prior to preterm or term caesarean-section delivery. At delivery pup and organ weights were recorded. Placentae were dissected, weighed and analysed using Western blot to examine GR isoform expression in nuclear and cytoplasmic extracts. A comparative examination of the guinea pig GR gene identified it is capable of producing seven of the eight translational GR isoforms which include GRα-A, C1, C2, C3, D1, D2, and D3. GRα-B is not produced in the Guinea Pig. Total GR antibody identified 10 specific bands from term (n = 29) and preterm pregnancies (n = 27). Known isoforms included GRγ, GRα A, GRβ, GRP, GRA and GRα D1-3. There were sex and gestational age differences in placental GR isoform expression. Placental GRα A was detected in the cytoplasm of all groups but was significantly increased in the cytoplasm and nucleus of preterm males and females exposed to betamethasone and untreated term males (KW-ANOVA, P = 0.0001, P = 0.001). Cytoplasmic expression of GRβ was increased in female preterm placentae and preterm and term male placentae exposed to betamethasone (P = 0.01). Nuclear expression of GRβ was increased in all placentae exposed to betamethasone (P = 0.0001). GRα D2 and GRα D3 were increased in male preterm placentae when exposed to betamethasone (P = 0.01, P = 0.02). The current data suggests the sex-specific placental response to maternal betamethasone may be dependent on the expression of a combination of GR isoforms.</p></div

Relationship between baseline microvascular blood flow at 24 hr and H₂S turnover on day 2 of postnatal life.

<p>H₂S turnover (measured as urinary thiosulphate) was significantly correlated with baseline microvascular blood flow in preterm male neonates 29–36 wk GA (Pearson correlation; p = 0.04, <i>r</i> = 0.43). No relationship was observed for females of the same gestational age, very preterm neonates (24–28 wk GA) or term neonates (37+wk GA).</p

Structural equation model of predicted interactions of the gasotransmitters and their contribution to the regulation of microvascular blood flow at 24h postnatal age in the preterm human.

<p>The overall model (males and females combined) is presented and has a Goodness of Fit of χ² = 1.02 and RMSEA value of 0.017 (CI 0.00–0.28). Structural equation modelling examines linear causal relationships among variables, while simultaneously accounting for measurement error. The measurement error, or variance, determined in the model is 0.66 for microvascular blood flow, 0.77 for hydrogen sulphide, 0.24 for nitric oxide and 0.07 for carbon monoxide. NO was positively correlated with H₂S (p = 0.002, z = 3.05). There was an inverse correlation between CO and H₂S (p = 0.18, z = -1.34). There was a significant relationship between H₂S and microvascular blood flow (p = 0.012, z = 2.52) when the input of NO and CO to H₂S was included in the model.</p

Physical Characteristics of Neonates.

<p>Data presented as median (range) or number (percentage) as appropriate. PU laser Doppler perfusion units</p><p>Physical Characteristics of Neonates.</p