Placental steroid receptors and sex differences in fetal growth Professor Vicki Clifton Mater Medical Research Institute University of Queensland Brisbane, Australia
Sex differences start in utero Lying-in Hospital in Dublin from 1757-1784 recording the birth outcomes of more than 20 000 deliveries -greater mortality of males than females -males were generally larger than females -females were more likely to be growth restricted Clarke (1786)
Females are proportionally smaller Clifton et al; 2009 ANZJOG
Verburg PE, et al PLoS One. 2016 Jul 11;11(7):e0158807. Sexual Dimorphism in Adverse Pregnancy Outcomes - A Retrospective Australian Population Study 1981-2011. Reporting males more likely to deliver preterm, associated with pregnancy induced hypertension and pregnancies complicated by GDM
Main question What are the mechanisms that drive sex specific differences in fetal growth in an adverse environment?
Prevalence of asthma worldwide 7% 3-10% 0.7% 1-3% 8% 12% A common pre-existing disease to affect pregnancy
What is asthma?
Asthma control and exacerbations
Perinatal outcomes in pregnancies complicated by asthma Congenital malformations: RR 1.11 [1.02-1.21] Growth restriction RR: 1.46 [1.22-1.75] Preterm delivery OR 2.3 [2.03-2.63] Neonatal death: RR 1.49 [1.11-2.00] Neonatal hospitalisation: RR 1.50 [1.03-2.20] Murphy et al 2013 BJOG, Hodyl et al 2014
Asthma and pregnancy in Qld Outcomes for women with asthma at Mater, Brisbane from 2008-2017 16.4% (13,882/84,695) of pregnant women had asthma. Asthmatic mums are significantly more hypertensive, overweight or obese Neonates of asthmatic mums more likely to have a congenital malformation, preterm delivery, NICU admission, respiratory distress, ventilation More likely to be >90 th and >95 th BWC at birth
How does asthma affect fetal growth?
Pregnancies complicated by asthma Mother Asthma Mild Moderate Or Severe Or Uncontrolled Chronic Inflammation Placenta Altered placental structure and function Fetus Males Females
Mother Asthma Controlled Pregnancies complicated by asthma Placenta Fetus Males Normal placental structure and function Chronic Inflammation Inhaled corticosteroids Females
Pregnancies complicated by asthma exacerbation Mother Placenta Fetus Males Asthma Exacerbation Decreased oxygen and nutrients Acute Inflammation Decreased lung function Decreased oxygen Females
Conclusion Male and female fetuses implement different strategies to control growth in response to asthma during pregnancy. Females reduce growth in response to maternal asthma to confer a survival advantage if there are inflammatory events that result in a further restriction of nutrient or oxygen supply Male growth is targeted towards increasing size in the presence of maternal asthma but increases risk of growth restricted, preterm delivery or death in utero in presence of an acute exacerbation.
Are there sex specific differences in growth in the presence of other pregnancy complications?
Birth weight centiles of males and females of pregnancies complicated by pre-eclampsia 80 * * Birth Weight Centile 60 Normotensive Mother What 40 are the mechanisms? Pre-eclamptic Mother 20 0 Female Male Infant Sex Stark et al 2009 Paediatric Res
Sex specific placental mechanisms influencing growth Males Females Minimal adjustments Greater risk of adverse outcome All targeted towards increased or continued growth Eg: minimal gene changes increased IGF-1 No change in blood flow Multiple adaptations in placental function Reduced growth and increased survival Eg: multiple gene changes Multiple protein changes Reduced blood flow Different strategies for the same maternal environment
Cortisol In pregnancy is essential for fetal development and survival Vascular tone Fat deposition Immune function Kidney development mitosis HPA function Pancreatic function Lung development Reproductive function Brain development
Cortisol nmol/l Sex specific difference in the response to glucocorticoids Female Male 1000 1200 800 600 R = 0.28 * P=0.017 1000 800 NS 400 600 400 200 200 0 1500 2000 2500 3000 3500 4000 4500 5000 0 1500 2000 2500 3000 3500 4000 4500 5000 Female birth weight (g) Male birth weight (g) Hodyl et al 2011 Thorax
GRα isoforms have different translation initiation sites
GR isoforms are generated by alternate splicing 85 kda 81 kda 86 kda 65 kda 74 kda
What do different GR isoforms do? Different isoforms confer specificity between cells and organs GRα A- inhibition of inflammation, growth and nutrient transport GRα C- apoptosis GRα D- cell differentiation GRβ- antagonises the response to cortisol by binding to GRα A and preventing it from binding to DNA GR A, P and γ low trans-activational activity act as antagonists to the translational isoforms
Human placental glucocorticoid receptor isoforms Male Female Female Male Male Female Male M Nuc Cyto Nuc Cyto Nuc Cyto Nuc Cyto Nuc Cyto Nuc Cyto Nuc Cyto 100 kda 75 kda 50 kda 37 kda GRα GRα-C1-3 GR-P 69 KDa 68 KDa GR-A GRα-D1 GRα-D2 GRα-D3 48 KDa 38 KDa Beta-Actin N=131 Known isoforms: GRα A, C, D1-3, GR P, GR A and GRβ Unknowns: 69, 68, 60, 45-48, 38 kda proteins
Placental GR protein expression in relation to sex and asthma Female Asthma Placental tissue Male Cytoplasm GRα-D Cytoplasm GRβ GRα A GRα-D GRα-C GRα D Nucleus Nucleus Anti-proliferative Anti-inflammatory Apoptosis Glucocorticoid resistance Proliferation Published in Placenta 2014 Saif et al
Birth weight centile Maternal asthma affects fetal growth sexspecifically 80 70 60 50 40 30 20 10 0 Control No Inhaled Corticosteroid Inhaled Corticosteroid n = 16 n = 15 n = 24 n = 22 n = 43 n = 47 Non-asthmatic Asthmatic * Male Female Murphy et al. 2003, Am J Respir Crit Care Med
Placental glucocorticoid receptor (GR) isoforms interact with androgen pathways Female placentae remain responsive to cortisol due to increased GR-α A expression Males induce a state of glucocorticoid resistance via GRβ Cortisol GR- A GR-β Antiproliferation?Androgen? Growth Reduced growth
The androgen receptor (AR) Characterised by three major functional domains - NTD, DBD, and CTD (which consists of the hinge region and LBD) Each domain is involved in ensuring appropriate transcriptional regulation of target genes
AR isoforms Androgen-dependent isoform (AR-FL) Constitutive C-terminally truncated AR isoforms (AR-Vs) N-terminally truncated AR isoform (AR-45)
The human placenta expresses multiple AR isoforms Seven protein bands detected in human placenta (N=60) AR-FL, AR-V1, AR-V7, AR-45 Proteins: 120, 90, 55 kda
AR expression varies with fetal sex and maternal asthma status Multiple AR isoforms were decreased in male placentae from asthmatic pregnancies compared to control no such changes were observed in females (data not shown)
AR regulated downstream targets are increased in male placentae from asthmatic pregnancies Presence of asthma appears to have a greater effect on expression of androgenmediated downstream targets in males What isoform is transcriptionally regulating these target genes?
Nuclear AR-45 is associated with increased AR target gene expression in male placentae from asthmatic pregnancies Nuclear AR-FLprotein Nuclear AR-45 protein Control Asthma Control Asthma Male Male Male Male r 2 p r 2 p r 2 p r 2 p IGF-1 0.240 n.s -0.132 n.s 0.225 n.s 0.458 * IGF-1R -0.394 n.s -0.440 n.s 0.261 n.s 0.561 * IGFBP-1 0.152 n.s -0.332 n.s -0.067 n.s 0.343 n.s IGFBP-3 0.217 n.s -0.077 n.s 0.000 n.s 0.247 n.s IGFBP-5 0.055 n.s 0.139 n.s 0.442 n.s 0.489 * VEGF -0.209 n.s -0.140 n.s 0.092 n.s 0.327 n.s Placental AR-45 may have a significant physiological role in mediating in utero male growth in the presence of maternal asthma
Current hypothesis-gr-β and AR-45 influence continued male growth in the presence of a complication of pregnancy AR-45 AR-FL AR-V1 AR-V7 AR-45 Growth factors AR-FL GR-β
Implications No clear understanding of how the fetal placental unit responds to glucocorticoids or androgens because we do not understand the role of the different steroid receptor isoforms and how they influence steroid responsivity. Growth restricted fetuses are often delivered preterm and administered betamethasone prior to delivery for lung maturation but can result in detrimental systemic cardiovascular changes. It may be possible to avoid these adverse side effects of glucocorticoid administration with the use of selective GR modulators or improve growth through AR receptor modulators Approaches such as this may be applicable to obstetrics and neonatology but the fundamental biology needs to be conducted
NHMRC SRF University of Adelaide Robinson Research Institute Lyell McEwin Hospital