title: An investigation into expression of hydrogen sulfide synthesising enzymes in placentas from normal and complicated pregnancies
Creators: name: Saleh, Fatema
Publisher: name: University of Glasgow
Year: 2022

description: Hydrogen sulfide (H2S) has recently attracted substantial interest as an endogenous gaseous signalling molecule. Like nitric oxide (NO) and carbon monoxide (CO), it promotes vasodilation and exhibits cytoprotective and anti-inflammatory properties. It is involved in diverse physiological and pathophysiological processes such as neurogenesis, regulation of blood pressure, atherosclerosis and inflammation. Endogenous H2S is synthesised predominantly by three enzymes: cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST). Recently, the endogenous production of H2S in human myometrium and placental tissues and its role in pathophysiology of complicated pregnancies with pre-eclampsia (PE) and fetal growth restriction (FGR) has been studied. In PE, inadequate remodelling of spiral arteries by trophoblasts causes ischemia-reperfusion insult of the placenta which is one source for oxidative stress, and it causes reductions in utero-placental blood flow, often resulting in FGR. The limited research undertaken to study expression of H2S synthesising enzymes in anormal and abnormal placental tissues is characterised by contradictory findings in mRNA and protein expressions of these enzymes. All studies used a random placental sampling method which could mask spatial variation in gene expression. Therefore, this study aimed to examine expression of these enzymes using a systematic sampling method in which placental samples were taken from different identified zones of each placenta. The expression of these enzymes was studied at both mRNA and protein levels using quantitative polymerase chain reaction (qPCR) and western blotting techniques. Extensive testing of several anti-CBS and CSE antibodies for western blotting revealed persistent non-specific binding of these antibodies to multiple unidentified proteins in all sample types. Antibodies tested included all those used in the previous studies in western blotting and in situ methods. These all showed highly images of CBS and CSE bands and lacked any supplementary data about testing antibodies specificity raising concerns about the previous research findings and identifying another potential source of the lack of consistency in conclusions they reached. CRISPR knockout clones of each protein were generated using two different cell lines to test specificity of these antibodies which confirmed the antibodies did detect proteins of the expected size. This enabled identification the correct CBS and CSE bands to study the expression at protein level in the comparative groups. Additionally, these clones identified isoforms not predicted by gene databases, allowed retrospective specificity testing of in situ procedures used and revealed an intriguing regulation of CBS, CSE by 3-MST which may be of relevance to placenta. CBS, CSE and 3-MST spatial expressions were examined in normal placentas obtained from women who delivered by caesarean section (CS) and were not in labour, in placentas from women who delivered spontaneously and in placentas from women with complicated pregnancies with PE, FGR and high body mass index (BMI). The study showed that there were significant spatial differences in expression of CSE and 3-MST, with an up-regulation in labour when compared to non-labour group at a particular placental site. Also, the study showed a significant increase in 3-MST mRNA and protein abundances in FGR group compared to control healthy group at the outer placental site. Only CBS and CSE mRNA abundances were significantly increased in PE compared to healthy controls at inner and middle placental sites, respectively. The spatial difference in gene expression in labour or complicated pregnancies at precise zones suggests that there is a controlled spatial change in expression or susceptibility to change which may be due to the vascular biology of the placenta. The physiological and pathological significances of these differences remain to be elucidated but oxidative stress and inflammatory pathway are the common links. Also, the reduction in CBS and CSE protein abundances in absence of 3-MST may suggest that there is a complex of regulatory mechanisms on these enzymes. Taken together, these results suggest that H2S involves in labour and pathophysiology of PE and FGR. However, further investigations with highly specific antibodies are required especially the data in this study showed significant differences in expression between controls and the targeted groups. To conclude, the present study highlights the possibility that ordinary placental sampling methods may mask the altered expression of some genes, and therefore, this study represent a further step toward developing a systematic way for sampling in placental research. In addition to this, the present study illustrates how using simple CRISPR knockout technology could help testing specificity of primary antibodies, and presents a real example of how incomplete reporting of antibodies research antibodies compromise the reducibility of research results. Furthermore, it emphasises on full documentation of the experimental procedure describing the data supporting the specificity of antibodies validation to help researchers to collaborate and build on each other’s work.