Maternal uterine spiral arteries (SpA) undergo significant structural changes in early pregnancy, resulting in increased blood flow to the developing fetus. Endothelial cells (EC) and vascular smooth muscle cells (VSMC) are lost from the SpA wall and are replaced by trophoblasts. We have previously shown that matrix metalloproteinase 10 (MMP-10) and Heparin binding-EGF like growth factor (HB-EGF) gene expression is increased in a 3D EC/VSMC co-culture system in response to trophoblast secreted factors. This study investigated trophoblast mediated MMP-10 and HB-EGF expression and determined if there was a relationship between the secretion of MMP-10 and the release of soluble HB-EGF (sHB-EGF) from EC. MMP-10 was widely expressed in first trimester decidual tissue including trophoblast, and EC, but not VSMC. MMP-10 expression was significantly lower in decidual tissue from pregnancies at increased risk of developing pre-eclampsia compared to low-risk pregnancies. In vitro, SGHEC-7 cells, a human EC line, but not SGHVMC-9, a human VSMC cell line, secreted MMP-10 in response to trophoblast conditioned medium (TCM). TCM contains several growth factors and cytokines, but only interleukin-1β (IL1β) significantly stimulated MMP-10 secretion by SGHEC-7 cells. Interleukin-1 receptor antagonist (IL-1Ra) significantly inhibited TCM-induced MMP-10 secretion. Interrogation of intracellular pathways established the involvement of MEK and JNK in TCM and IL-1β stimulated MMP-10 secretion. Although IL-1β also significantly increased sHB-EGF, inhibition of MMP-10 activity using a broad spectrum MMP inhibitor had no effect on sHB-EGF. Western blot analysis indicated that MMP-10 secreted by EC in response to IL-1β stimulation was the enzymatically inactive pro form.

BACKGROUND: Stanniocalcin-1 (STC-1) is a widely expressed glycoprotein hormone involved in a diverse spectrum of physiological and pathophysiological processes including angiogenesis, mineral homeostasis, cell proliferation, inflammation and apoptosis. Over the last 20years, numerous studies have reported STC-1 expression within female reproductive tissues including the uterus, ovaries and placenta and implicated STC-1 in processes such as ovarian follicular development, blastocyst implantation, vascular remodelling in early pregnancy and placental development. Notably, dysregulation of STC-1 within reproductive tissues has been linked to the onset of severe reproductive disorders including endometriosis, polycystic ovary syndrome, poor trophoblast invasion and placental perfusion in early pregnancy. Furthermore, significant changes in tissue expression and in maternal systemic concentration take place throughout pregnancy and further substantiate the vital role of this protein in reproductive health and disease. OBJECTIVE AND RATIONALE: Our aim is to provide a comprehensive overview of the existing literature, to summarise the expression profile and roles of STC-1 within the female reproductive system and its associated pathologies. We highlight the gaps in the current knowledge and suggest potential avenues for future research. SEARCH METHODS: Relevant studies were identified through searching the PubMed database using the following search terms: 'stanniocalcin-1', 'placenta', 'ovary', 'endometrium', 'pregnancy', 'reproduction', 'early gestation'. Only English language papers published between 1995 and 2020 were included. OUTCOMES: This review provides compelling evidence of the vital function that STC-1 plays within the female reproductive system. The literature presented summarise the wide expression profile of STC-1 within female reproductive organs, as well as highlighting the putative roles of STC-1 in various functions in the reproductive system. Moreover, the observed link between altered STC-1 expression and the onset of various reproductive pathologies is presented, including those in pregnancy whose aetiology occurs in the first trimester. This summary emphasises the requirement for further studies on the mechanisms underlying the regulation of STC-1 expression and function. WIDER IMPLICATIONS: STC-1 is a pleiotropic hormone involved in the regulation of a number of important biological functions needed to maintain female reproductive health. There is also growing evidence that dysregulation of STC-1 is implicated in common reproductive and obstetric disorders. Greater understanding of the physiology and biochemistry of STC-1 within the field may therefore identify possible targets for therapeutic intervention and/or diagnosis.

Various studies found an association of different renin-angiotensin system (RAS) components with gestational duration and preterm birth, as well as with preeclampsia. Approximately 25% of first-time pregnant women develop a mild to severe hypertension in pregnancy or even preeclampsia. Based on recently published single-cell RNA-sequencing, we hypothesized an alternative RAS function in placenta and furthermore, an implication in hypertensive disorders in pregnancy. Placental RAS expression and localization was analyzed via quantitative polymerase chain reaction and in situ mRNA padlock probes. Tissue was collected from first-trimester elective termination (n=198), from healthy third-trimester controls (n=54), from early-onset preeclamptic (n=54) and age-matched controls (n=29), as well as first-trimester placentae from women with a high uterine artery resistance index (high-risk for preeclampsia, n=9) and controls (n=8). Serum levels of Ang (angiotensin) I to IV from women before and after conception were measured via mass spectrometry (n=10). Placental explants were cultured in 2.5% oxygen with Ang II, candesartan, and leptin. Seahorse XF96 MitoStress assays assessed trophoblast metabolism. Here, we show that maternal angiotensin acts on placental LNPEP (leucine aminopeptidase), that is, angiotensin IV-receptor and fetal angiotensin on placental AGTR1 (angiotensin II receptor type 1). Maternal circulating RAS shifts towards Ang IV in pregnancy. Ang IV decreases trophoblastic mitochondrial respiration and increases placental leptin via placental LNPEP. Lower placental LNPEP in preeclampsia and in first-trimester patients at high-risk for preeclampsia suggests a new alternative route in maternal RAS signaling and may contribute to hypertension and disease in pregnancy. The study shows how hypertensive disorders in pregnancy may be connected metabolic alterations that finally seem to contribute to the multifactorial disease in pregnancy, preeclampsia.

During the first trimester of pregnancy the decidua is comprised of decidual stromal cells (DSC), invading fetal trophoblast cells and maternal leukocytes, including decidual natural killer (dNK) cells and macrophages. dNK cells are distinct from peripheral blood NK cells and have a role in regulating trophoblast invasion and spiral artery remodelling. The unique phenotype of dNK cells results from the decidual environment in which they reside, however the interaction and influence of other cells in the decidua on dNK phenotype is unknown. We isolated first trimester DSC and decidual macrophages and investigated the effect that DSC and decidual macrophage secreted factors have on CD56+ decidual lymphocyte receptor expression and cytokine secretion (including dNK cells). We report that DSC secreted factors induce the secretion of the cytokines IL-8 and IL-6 from first trimester CD56+ cells. However, neither DSC nor decidual macrophage secreted factors changed CD56+ cell receptor expression. These results suggest that secreted factors from DSC influence CD56+ decidual lymphocytes during the first trimester of pregnancy and therefore may play a role in regulating the unique phenotype and function of dNK cells during placentation.

Stanniocalcin-1 (STC-1) is a multi-functional glycosylated peptide present in the plasma of healthy women postpartum and increased further in pregnancies complicated by preeclampsia. Although the STC-1 gene is expressed by the placenta what regulates its secretion and from which cells at the feto-maternal interface is unknown. Here, we demonstrate for the first time that the syncytiotrophoblast and cytotrophoblast are a major site of STC-1 protein expression in first trimester placental tissue. Further, in response to low oxygen, first trimester chorionic villous tissue from pregnancies at increased risk of developing preeclampsia secreted significantly more STC-1 than normal tissue under the same conditions. Using the human trophoblast cell line BeWo we have shown that low oxygen increased the secretion of STC-1 but it required co-stimulation with the Adenosine-3', 5'-cyclic monophosphate (cAMP) analogue, 8-Bromo adenosine-3', 5'-cyclic monophosphate cAMP (8 Br-cAMP) to reach significance. Inhibition of Hypoxia inducible factor 2α (HIF-2α) and the Phosphatidylinositol-3 kinase (PI3-Kinase)/AKT/Serum and glucocorticoid-induced kinase-1(SGK-1) pathway resulted in significant inhibition of STC-1 secretion. As both low oxygen and cAMP are known to play a central role in placental function, their regulation of STC-1 points to a potentially important role in the maintenance of a normal healthy pregnancy and we would hypothesize that it may act to protect against prolonged placental hypoxia seen in preeclampsia.

Failure of the placental capillary network to develop normally is associated with early onset fetal growth restriction (FGR) and pre-eclampsia (PE). Although the symptoms are observed at term, the problem begins in the first trimester. However, investigations at this clinically relevant time are hindered by difficulties in identifying earlystage pregnancies that are at risk of developing FGR/PE. Using uterine artery Doppler ultrasound in the first trimester as a proxy measure of poor placentation, we have identified pregnancies at increased risk of developing early onset FGR/PE. Placental endothelial cells (PEC) isolated from pregnancies at increased risk of developing FGR/PE grew more slowly and their basal rate of apoptosis was significantly higher than that seen in the normal group. The pro-apoptotic stimulus, TNFα, induced apoptosis in cells from both groups but this was significantly greater in the high risk group. TNF receptor expression was unaffected. Inhibition of nitric oxide (NO) production significantly increased the sensitivity of cells from the normal pregnancies to TNFα but not in the high risk group establishing a functional role for NO in this system. In conclusion, first trimester PEC from pregnancies at increased risk of developing early onset FGR/PE were inherently more sensitive to apoptotic stimuli and this was functionally linked to the synthesis of NO. This may contribute to the poor placental vascular development seen in on going pregnancies.

Equine chorionic gonadotrophin (eCG) is a placental glycoprotein critical for early equine pregnancy and used therapeutically in a number of species to support reproductive activity. The factors in trophoblast that transcriptionally regulate eCG beta-subunit (LHB), the gene which confers the hormones specificity for the receptor, are not known. The aim of this study was to determine if glial cells missing 1 regulates LHB promoter activity. Here, studies of the LHB proximal promoter identified four binding sites for glial cells missing 1 (GCM1) and western blot analysis confirmed GCM1 was expressed in equine chorionic girdle (ChG) and surrounding tissues. Luciferase assays demonstrated endogenous activity of the LHB promoter in BeWo choriocarcinoma cells with greatest activity by a proximal 335 bp promoter fragment. Transactivation studies in COS7 cells using an equine GCM1 expression vector showed GCM1 could transactivate the proximal 335 bp LHB promoter. Chromatin immunoprecipitation using primary ChG trophoblast cells showed GCM1 to preferentially bind to the most proximal GCM1-binding site over site 2. Mutation of site 1 but not site 2 resulted in a loss of endogenous promoter activity in BeWo cells and failure of GCM1 to transactivate the promoter in COS-7 cells. Together, these data show that GCM1 binds to site 1 in the LHB promoter but also requires the upstream segment of the LHB promoter between -119 bp and -335 bp of the translation start codon for activity. GCM1 binding partners, ETV1, ETV7, HOXA13, and PITX1, were found to be differentially expressed in the ChG between days 27 and 34 and are excellent candidates for this role. In conclusion, GCM1 was demonstrated to drive the LHB promoter, through direct binding to a predicted GCM1-binding site, with requirement for another factor(s) to bind the proximal promoter to exert this function. Based on these findings, we hypothesize that ETV7 and HOXA13 act in concert with GCM1 to initiate LHB transcription between days 30 and 31, with ETV1 partnering with GCM1 to maintain transcription.

The pathologies of the pregnancy complications pre-eclampsia (PE) and fetal growth restriction (FGR) are established in the first trimester of human pregnancy. In a normal pregnancy, decidual spiral arteries are transformed into wide diameter, non-vasoactive vessels capable of meeting the increased demands of the developing fetus for nutrients and oxygen. Disruption of this transformation is associated with PE and FGR. Very little is known of how these first trimester changes are regulated normally and even less is known about how they are compromised in complicated pregnancies. Interactions between maternal and placental cells are essential for pregnancy to progress and this review will summarise the challenges in investigating this area. We will discuss how first trimester studies of pregnancies with an increased risk of developing PE/FGR have started to provide valuable information about pregnancy at this most dynamic and crucial time. We will discuss where there is scope to progress these studies further by refining the ability to identify compromised pregnancies at an early stage, by integrating information from many cell types from the same pregnancy, and by improving our methods for modelling the maternal-fetal interface in vitro.

Here we performed direct integration of two independent microarray datasets: third-trimester placental samples obtained after cesarean sections from preeclamptic women (n = 25) and women with uncomplicated pregnancies (n = 23) and placental samples from gestational age matched first-trimester pregnancies with normal uterine artery Doppler resistance indexes (n = 11). By using differential expression analysis, gene set enrichment analysis and reconstruction of gene regulatory networks we obtained two networks that represent broad view of the regulatory pathways involved in normal pregnancy and preeclampsia. The results of integrative analysis highlight important hub genes, that could be involved in preeclampsia underlying mechanisms.

Background: Preeclampsia is a complex and common human-specific pregnancy syndrome associated with placental pathology. The human specificity provides both intellectual and methodological challenges, lacking a robust model system. Given the role of imprinted genes in human placentation and the vulnerability of imprinted genes to loss of imprinting changes, there has been extensive speculation, but no robust evidence, that imprinted genes are involved in preeclampsia. Our study aims to investigate whether disturbed imprinting contributes to preeclampsia. Methods: We first aimed to confirm that preeclampsia is a disease of the placenta by generating and analyzing genome-wide molecular data on well-characterized patient material. We performed high-throughput transcriptome analyses of multiple placenta samples from healthy controls and patients with preeclampsia. Next, we identified differentially expressed genes in preeclamptic placentas and intersected them with the list of human imprinted genes. We used bioinformatics/statistical analyses to confirm association between imprinting and preeclampsia and to predict biological processes affected in preeclampsia. Validation included epigenetic and cellular assays. In terms of human specificity, we established an in vitro invasion-differentiation trophoblast model. Our comparative phylogenetic analysis involved single-cell transcriptome data of human, macaque, and mouse preimplantation embryogenesis. Results: We found disturbed placental imprinting in preeclampsia and revealed potential candidates, including GATA3 and DLX5, with poorly explored imprinted status and no prior association with preeclampsia. As a result of loss of imprinting, DLX5 was upregulated in 69% of preeclamptic placentas. Levels of DLX5 correlated with classic preeclampsia markers. DLX5 is expressed in human but not in murine trophoblast. The DLX5(high) phenotype resulted in reduced proliferation, increased metabolism, and endoplasmic reticulum stress-response activation in trophoblasts in vitro. The transcriptional profile of such cells mimics the transcriptome of preeclamptic placentas. Pan-mammalian comparative analysis identified DLX5 as part of the human-specific regulatory network of trophoblast differentiation. Conclusions: Our analysis provides evidence of a true association among disturbed imprinting, gene expression, and preeclampsia. As a result of disturbed imprinting, the upregulated DLX5 affects trophoblast proliferation. Our in vitro model might fill a vital niche in preeclampsia research. Human-specific regulatory circuitry of DLX5 might help explain certain aspects of preeclampsia.

Searchable abstracts of presentations at key conferences on reproductive biology and medicine ISSN 2052-1472 (online)

The peak number of oocytes is reached during intrauterine development, after which numbers decline until reserves are depleted and a woman enters menopause. In premature ovarian insufficiency (POI), the process of follicle depletion occurs at a young age (generally taken as 40 years); the condition affects about 1% of women. In this study, we investigate whether women with POI had experienced a different perinatal milieu, as reflected in their birth weight or prematurity.In this retrospective case-control study, we evaluated whether women diagnosed with POI had a different birth weight or prematurity rate (<37 weeks) compared with women aged over 40 at natural menopause (the controls). Binary logistic regression models were used to analyze the data and correct for smoking. 59 women with POI and 92 controls were recruited.13.6% of women diagnosed with POI were born prematurely, compared with 6.6% of controls (p = 0.018). Corrected for gestational age, women with POI did not have a different birth weight compared with controls. As a consequence of the design of our study, mean age at time of interview differed significantly between groups, at 37.5 and 46 years respectively for women diagnosed with POI and controls. Years of oral contraception use, smoking, age at menarche, BMI and education levels were similar in the two groups.Our findings indicate that prematurity is a risk factor for POI. Prenatal factors contributing to prematurity, or postnatal factors that are the result of prematurity, may lead to early follicle depletion.

Circulating activin A concentrations are significantly higher in pre-eclampsia (PE) compared with those in a normotensive pregnancy. We and others have previously identified ACVR2A, which codes for the activin A binding receptor, as a maternal PE susceptibility gene with reduced expression in PE. The PE-associated rs1424954 genetic polymorphism also results in decreased receptor expression. Given that PE is characterised by systemic vascular endothelial dysfunction, we used an in vitro cell-line model to investigate how reduced ACVR2A expression modifies the response of maternal vascular endothelial cells to normotensive and PE activin A concentrations. The SGHEC-7 cell line was used as a model of vascular endothelial cells. ACVR2A expression was silenced using siRNA transfection and confirmed with real-time PCR and immunoblotting. A non-targeting siRNA was used as a negative control (NC). Following a 72 h transfection, SGHEC-7s were treated for 24 h with activin A concentrations representative of normotensive (0, 10 ng/ml) and PE (50 ng/ml) pregnancies. SGHEC-7 proliferation was determined using the electrical impedance-based xCELLigence system, while permeability was measured with a Millipore fluorescence-based assay. Student’s t test and one-way ANOVA with Bonferroni’s post-hoc test where appropriate were used for statistical evaluations. Each experiment was performed at least n = 3 times. A p-value of < 0.05 was considered statistically significant. ACVR2A siRNA markedly reduced ACVR2A mRNA and protein levels by 78% (p < 0.05) and 54% (p < 0.01) respectively. Activin A at 10 ng/ml significantly increased proliferation of both NC and ACVR2A siRNA transfected cells by at least 43% (p < 0.01) above that of 0 ng/ml. These increases were inhibited by at least 23% (p < 0.01) by the PE 50 ng/ml concentration. Nevertheless, at all tested concentrations of activin A, proliferation of ACVR2A siRNA transfected cells was significantly reduced by at least 32% (p < 0.01) compared with that of NC cells. The PE concentration of 50 ng/ml also significantly increased permeability of NC cells by 39% (p < 0.01) compared with that of the 0 ng/ml baseline. In contrast, permeability of ACVR2A siRNA transfected cells was significantly increased by 36% (p < 0.01) even at 0 ng/ml, with no further increase observed at 50 ng/ml, when compared with that of NC cells. This study demonstrates that decreased expression of the ACVR2A receptor, in addition to activin A, can also result in dysfunctional vascular endothelial proliferation and permeability. Therefore, the altered expression of the ACVR2A receptor, resulting from a genetic predisposition, may modify the threshold required for activin A to impact vascular endothelial function, thereby exacerbating systemic vascular endothelial dysfunction in PE.

Aberrant placental angiogenesis is associated with fetal growth restriction (FGR). In mice, targeted disruption of the homeobox gene, transforming growth β-induced factor ( Tgif-1 ), which is also a transcription factor, causes defective placental vascularisation. Nevertheless, the role of TGIF-1 in human placental angiogenesis is unclear. We have previously reported increased TGIF-1 expression in human FGR placentae and demonstrated localisation of TGIF-1 protein in placental endothelial cells (ECs). However, its functional role remains to be investigated. In this study, we aimed to specifically compare TGIF-1 mRNA expression in placental ECs isolated from human FGR-affected pregnancies with gestation-matched control pregnancies in two independent cohorts from Australia and Canada and to identify the functional role of TGIF-1 in placental angiogenesis using the human umbilical vein endothelial cell-derived cell line, SGHEC-7, and primary human umbilical vein ECs. Real-time PCR revealed that TGIF-1 mRNA expression was significantly increased in ECs isolated from FGR-affected placentae compared with that of controls. The functional roles of TGIF-1 were determined in ECs after TGIF-1 siRNA transfection. TGIF-1 inactivation in ECs significantly reduced TGIF-1 at both the mRNA and protein levels, as well as the proliferative and invasive potential, but significantly increased the angiogenic potential. Using angiogenesis PCR screening arrays, we identified ITGAV , NRP-1 , ANPGT-1 and ANPGT-2 as novel downstream targets of TGIF-1 , after TGIF-1 inactivation in ECs. Collectively, these results show that TGIF-1 regulates EC function and the expression of angiogenic molecules; and when abnormally expressed, may contribute to the aberrant placental angiogenesis observed in FGR.

Searchable abstracts of presentations at key conferences on reproductive biology and medicine ISSN 2052-1472 (online)