Placentae of small appropriately-grown-for-gestational-age neonates exhibit sexually dimorphic transcriptomic changes representative of placental insufficiency.

INTRODUCTION: Previous studies have reported that neonates less than the 25th BWC especially if they were male, were more likely to be associated with birth complications suggesting small neonates often identified as appropriately grown are at risk of adverse outcomes. We have questioned whether smaller neonates not typically categorized as "small for gestational age" may not reach their genetically determined growth due to placental insufficiency. METHODS: RNA-Seq was performed on the Illumina NovaSeq 600 using term placentae from neonates that were less than the 10th birthweight centile (BWC) (n = 39), between the 10th and the 30th BWC (n = 15) or greater than the 30th BWC (n = 23). Bioinformatic analyses were conducted and statistical significance was assessed at a level of P < 0.05 for single comparisons or FDR <0.05 unless otherwise noted. RESULTS: Gene set enrichment analysis revealed differences between BWC groups and in relation to the sex of the placenta. Genes associated with hypoxia, inflammatory responses, estrogen responsive genes, and androgen responsive genes were enriched (FDR <0.1) for in placentae of neonates <10th BWC regardless of sex and also in male placentae of neonates between the 10th-30th BWC. Female placenta of neonates between the 10th-30th BWC were comparable to placentae of neonates >30th BWC. DISCUSSION: These findings provide evidence that small male neonates may be at a greater risk of an adverse outcome than females due to changes in gene expression that are associated with placental dysfunction. The current data raises questions of whether placental pathology for smaller appropriately grown neonates should be scientifically and clinically examined in more depth.

The impact of obesity and uncontrolled asthma during pregnancy on metabolic and inflammatory pathways.

OBJECTIVE: Asthma and obesity are both inflammatory complications of pregnancy and when combined contribute to an increased risk of uncontrolled asthma during pregnancy and poor perinatal outcomes. Our previous work has identified the presence of maternal asthma is associated with a proinflammatory milieu in the placenta and reduced fetal growth. The current study was designed to determine the relationships between immunomodulatory metabolic pathways and inflammation and establish whether these pathways are associated with uncontrolled asthma in obese pregnant women. METHODS: Fifty-three obese (BMI >30) pregnant women were recruited prospectively. Participants were classified as having no asthma, controlled asthma, and uncontrolled asthma based on a doctor diagnosis and assessment using the Asthma Control Questionnaire (ACQ). Circulating plasma concentrations of metabolic hormones leptin, adiponectin, insulin, glucose, and extracellular vesicle (EVs) associated cytokines were measured at 18- and 36-weeks gestation. RESULTS: Concentrations of metabolic and inflammatory markers among obese participants with or without asthma were not significantly different throughout gestation. However total adiponectin concentrations increased as gestation progressed in obese, non-asthmatic women but did not increase in women with asthma. Plasma adiponectin and leptin levels in women with uncontrolled asthma were positively correlated with EV inflammatory markers including GM-CSF, IL-6, TNFα and IFNγ protein. CONCLUSIONS: This study demonstrated that most metabolic markers remain unchanged with the presence and severity of asthma in obese pregnant women. However, differences in the associations between metabolic and inflammatory pathways were observed in women with asthma and may be one of the mechanisms contributing to uncontrolled asthma in obese pregnant women.

Sex-dependent differential transcript expression in the placenta of growth restricted infants.

INTRODUCTION: The pathological decrease of fetal growth during gestation can lead to subsequent poor health outcomes for the fetus. This process is commonly controlled by the placenta, the interface between mother and baby during gestation. Sex-specific gene expression has been implicated in placental function, therefore, there is a need to determine if it is important during reduced fetal growth. We therefore aimed to characterise placental gene expression at term to evaluate sex-specific genetic changes that occur in small for gestational age (SGA) infants. METHODS: RNA-sequencing of twelve human placental tissue samples collected from pregnancies yielding either term appropriate for gestational age (AGA) or SGA infants identified at delivery. Candidate genes associated with fetal size and fetal sex were identified using differential gene expression and weighted gene co-expression network analyses. Single-cell sequencing data was used for candidate validation and to estimate candidate transcript expression in specific placental cell populations. RESULTS: Differential gene expression and weighted gene co-expression network analyses identified 403 candidate transcripts associated with SGA infants. One hundred and three of these transcripts showed sex-specific expression. . Published placental sequencing datasets were used to validate the key expression results from the twelve placental samples initially studied; the sex-independent transcript expression for genes involved in cell cycle processes in males (7 transcripts) and endoplasmic reticulum stress in females (17 transcripts). DISCUSSION: This study identified the activation of multiple molecular mechanisms involved in the placental response to an adverse environmental stressor. Mechanisms such as disrupted protein synthesis were shared between infant biological sex when comparing AGA to SGA, whilst other pathways such as cell cycle and endoplasmic reticulum stress appear as independent/specific to either males or females when investigating reduced fetal growth. This data suggests that sexual dimorphism is an important consideration when examining placental dysfunction and poor fetal growth.

An ex vivo approach to understanding sex-specific differences in placental androgen signalling in the presence and absence of inflammation.

INTRODUCTION: The mechanisms that contribute to continued male intrauterine growth in response to an adverse maternal environment, such as those brought on by maternal asthma, remain largely undefined but may, in part, be mediated by androgen-mediated signalling. We previously reported the expression of multiple AR protein isoforms in the human placenta and proposed the novel AR-45 isoform to be integral in mediating male-specific androgen-dependent signalling in the presence of maternal asthma. In the current study we have used an ex vivo approach to further understand sex-specific differences in placental androgen signalling in the presence and absence of inflammation using human term villous placental explants. METHODS: Explants were cultured in the presence and absence of 0.1 nM dihydrotestosterone (DHT), 1 µg/ml lipopolysaccharide (LPS), or DHT + LPS for 24hr. Tissue was used for gene expression and subcellular AR protein isoform expression. RESULTS: Cytoplasmic and nuclear AR protein isoforms expression did not vary between culture conditions in either sex. AR-45 activity was upregulated in male placentae cultured in DHT, LPS and DHT + LPS only. There were no changes in the expression of androgen-mediated downstream targets in males in response to culture conditions, but females had significantly reduced IGF1R expression in response to LPS. DISCUSSION: Increased AR-45 activity in the presence of inflammation may drive continued male feto-placental growth via maintained expression of downstream growth targets. Our findings build on previous work suggesting an important role for AR-45 in regulating male-specific adaptations to placental inflammation and underscores the need to further characterise the function of this AR isoform.

A preferential switch between placental GR exon 1 promoter variants in the presence of maternal asthma or inflammation upregulates GRα D isoforms.

INTRODUCTION: The human placenta expresses multiple glucocorticoid receptor (GR) isoforms that may be partially regulated by the untranslated 5' exon 1 GR gene promoter region which consists of 9 different promoters and 13 splice variants. The objective of this study was to determine which GR exon 1 variants are expressed in the human placenta and relate these findings to GR mRNA and protein expression. METHODS: Placental extracts from pregnancies with or without the complication of maternal asthma and trophoblast cells exposed to an inflammatory challenge in vitro were examined using PCR and Western blot to measure GR exon 1 variants, GR splice variant mRNA and GR protein isoforms, respectively. RESULTS: All 9 GR exon 1 variants were detectable in the human placenta and included GR exons 1A, 1B, 1C, 1D, 1E, 1F, 1H, 1I and 1J. In the presence of maternal asthma and a male fetus there was preferential expression of GR exon 1B, 1C, IF and 1J (KW-ANOVA, P < 0.05) which were positively correlated with GRα D3 protein isoform. In female placentae from pregnancies complicated by asthma there was no upregulation of any exon 1 variant (KW-ANOVA, P < 0.05). Exposure of BeWo trophoblast cell line to an inflammatory challenge, lipopolysaccharide, in vitro, resulted in preferential expression of GR exon 1B, 1D, 1E and 1H and associated with GRα-D1 protein upregulation. DISCUSSION: The preferential expression of different GR exon 1 promoters drive the upregulation of GRα D isoforms and contribute to glucocorticoid resistance observed in male placentae of pregnancies complicated by asthma.

Identification of placental androgen receptor isoforms in a sheep model of maternal allergic asthma.

Maternal asthma is known to impact intrauterine growth outcomes, which may be mediated, in part, by altered androgen signalling. Our aim was to explore whether the sheep placenta expresses androgen receptor (AR) isoforms and determine if the differential expression of AR protein isoforms is altered by maternal asthma. Four known AR isoforms were detected (AR-FL, AR-v1, AR-v7, and AR-45), and their expression and subcellular distribution was altered in the presence of maternal allergic asthma. These findings underscore the importance for in vivo models of maternal asthma to delineate molecular patterns that may contribute to feto-placental growth and development.

The impact of maternal asthma during pregnancy on fetal growth and development: a review.

INTRODUCTION: Asthma is a highly prevalent co-morbidity during pregnancy that can worsen as gestation progresses and is associated with several adverse perinatal outcomes. These adverse outcomes often result from uncontrolled asthma during pregnancy and acute asthma exacerbations that are associated with alterations in placental function and fetal growth. AREAS COVERED: This paper will discuss how maternal asthma in pregnancy affects fetal growth and development which may alter future offspring health. Changes in placental function occur in a sex-specific manner in pregnancies complicated by asthma and result in differences in fetal growth and development which may influence child health. The follow up of children from mothers with asthma suggests they are at greater risk of developing asthma, have alterations in microvascular structure that may contribute to a future risk of cardiovascular disease and epigenetic modifications in immune cell function. The current evidence suggests that appropriately managed asthma during pregnancy results in normal fetal growth and development. EXPERT OPINION: Clinical management of asthma during pregnancy needs significant improvement to prevent adverse outcomes for the fetus. The key to improving maternal and fetal outcomes is through education of health professionals and parents about controlling asthma during pregnancy.

Placental glucocorticoid receptor isoforms in a sheep model of maternal allergic asthma.

Maternal asthma increases the risk of adverse pregnancy outcomes and may affect fetal growth and placental function by differential effects on the expression of glucocorticoid receptor (GR) isoforms, leading to altered glucocorticoid signalling. Our aim was to examine the effect of maternal asthma on placental GR profiles using a pregnant sheep model of asthma. Nine known GR isoforms were detected. There was a significant increase in the expression of placental GR isoforms that are known to have low trans-activational activity in other species including GR A, GR P and GRγ which may result in a pro-inflammatory environment in the presence of allergic asthma.

Human placental androgen receptor variants: Potential regulators of male fetal growth.

INTRODUCTION: Numerous studies show that males have increased intrauterine growth compared to females, and that pregnancy complications may further these growth differences, but the regulatory mechanisms underlying these differences remain unknown. We propose that these growth outcomes may be due to sex-specific differences in androgen sensitivity - giving rise to altered growth signalling pathways - mediated by the differential expression of placental androgen receptor (AR) variants. METHODS: Placental protein and mRNA were used to identify AR protein variant levels and AR-downstream target gene expression, and were then analysed against neonatal measurements. Dihydrotestosterone (DHT)-induced AR protein variant expression and downstream growth factors were examined in vitro. RESULTS: Four known AR variants (AR-FL, AR-V1, AR-V7, and AR-45), and three unknown proteins (120, 90 and 55 kDa) immunoreactive to the anti-AR antibody were identified in human placentae. Male placentae from controlled asthmatic pregnancies had increased AR-45 and decreased AR-V1 and AR-V7 nuclear expression. Increased nuclear AR-45 expression was associated with increased insulin-like growth factor 1 (IGF-1), IGF-1 receptor (IGF-1R), and IGF-binding protein 5 (IGFBP-5) mRNA expression and normal male growth. AR-45 mRNA and protein did not change in the presence of uncontrolled maternal asthma and associated with an increase in small for gestational (SGA) male fetuses. In vitro DHT stimulation increased AR-45 protein and IGF-1R and IGFBP-5 mRNA expression. CONCLUSIONS: Collectively, our data shows altered AR protein expression and downstream signalling targets may contribute to sex-specific fetal growth outcomes in response to an adverse environment, and that AR-45 appears central in mediating these changes.

Dexamethasone and sex regulate placental glucocorticoid receptor isoforms in mice.

Maternal dexamethasone exposure in the mouse impairs placental development and programs adult disease in a sexually dimorphic manner. Glucocorticoids bind to different glucocorticoid receptor (GR) isoforms to regulate gene transcription and cellular signaling. We hypothesized that sexually dimorphic placental responses to glucocorticoids are due to differences in GR isoforms present in the placenta. Pregnant C57Bl6 mice were exposed to saline or dexamethasone from E12.5 until E14.5 (1 µg/kg/h) before the collection of placentae. Cytoplasmic and nuclear protein fractions were extracted from placentae of male and female fetuses for Western blot analysis of GR isoforms. Eight known isoforms of the GR were detected in the mouse placenta including the translational isoforms GRα-A, B, C and D1-3 and the splice variants GRA and GRP. The expression of GRA, GRP and each of the GRα isoforms were altered by dexamethasone in relation to fetal sex and cellular location. Placentae of female fetuses had higher GRα-A and GRP expression in the cytoplasm than males, and GRα-C was more highly expressed in the nucleus of females than that in males. Dexamethasone significantly increased the cytoplasmic expression of GRα-A, but reduced the expression of GRα-C in placentae of males. Dexamethasone increased the expression of the GRα-C-regulated genes Sgk1 and Bcl2l11, particularly in females. The cleaved caspase-3 staining in placental sections indicated GRα-C may mediate sex differences in dexamethasone-induced apoptosis. These findings may underlie the sex-specific placental adaptations that regulate different growth profiles in males and females and different risks for programmed disease outcomes in offspring.

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

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, GRß, 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 GRß was increased in female preterm placentae and preterm and term male placentae exposed to betamethasone (P = 0.01). Nuclear expression of GRß 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.

FBXO31 is the chromosome 16q24.3 senescence gene, a candidate breast tumor suppressor, and a component of an SCF complex.

A BAC located in the 16q24.3 breast cancer loss of heterozygosity region was previously shown to restore cellular senescence when transferred into breast tumor cell lines. We have shown that FBXO31, although located just distal to this BAC, can induce cellular senescence in the breast cancer cell line MCF-7 and is the likely candidate senescence gene. FBXO31 has properties consistent with a tumor suppressor, because ectopic expression of FBXO31 in two breast cancer cell lines inhibited colony growth on plastic and inhibited cell proliferation in the MCF-7 cell line. In addition, compared with the relative expression in normal breast, levels of FBXO31 were down-regulated in breast tumor cell lines and primary tumors. FBXO31 was cell cycle regulated in the breast cell lines MCF-10A and SKBR3 with maximal expression from late G(2) to early G(1) phase. Ectopic expression of FBXO31 in the breast cancer cell line MDA-MB-468 resulted in the accumulation of cells at the G(1) phase of the cell cycle. FBXO31 contains an F-box domain and is associated with the proteins Skp1, Roc-1, and Cullin-1, suggesting that FBXO31 is a component of a SCF ubiquitination complex. We propose that FBXO31 functions as a tumor suppressor by generating SCF(FBXO31) complexes that target particular substrates, critical for the normal execution of the cell cycle, for ubiquitination and subsequent degradation.