Assessment of an AI-based tool for population-wide collection of placental morphological data.

OBJECTIVES: Automated placental assessment could allow accurate and timely morphological/pathological measurements at scale. We undertook a pilot study using an artificial intelligence-based assessment system (AI-PLAX) to ascertain the potential of a state-wide rollout as part of Generation Victoria, assessing the impact of time post-delivery, user, and technology used for image capture, on a range of derived placental data. STUDY DESIGN: Ten placentas were imaged by three different users and imaging technologies (iPad, iPhone, Samsung) at (0 h), 24 h, and 48 h post-delivery. Using AI-PLAX, disc size (short and long length, perimeter, area), shape (normal, abnormal), cord insertion type (central, eccentric), cord coiling, abruption (retroplacental hematoma), and meconium staining were determined. RESULTS: When analysing the maternal surface of the placenta, time in cold storage post-delivery had modest effects on placental dimensions, with decreases in the short length (24-48 h: -3.7 %), disc area (0-24 h: 4.7 % and 0-48 h: -7.4 %), and perimeter (0-48 h: -3.8 %) observed. There was marginal impact on placental dimensions when the placenta was imaged by different users, including long length (+1.9 %), disc area (+2.9 %), and perimeter (+2.0 %). Measures of placental size were not impacted by the type of technology used to capture the images. When analysing the fetal surface of the placenta, more variance in placental size measures were observed between users. Abruption detection was not affected by any parameter. Time between delivery and imaging impacted apparent meconium staining - likely reflecting changes in fetal surface colour over time. Meconium staining was not affected by technology or user. CONCLUSIONS: This study supports the feasibility of the collection of placenta images for later morphological analysis by AI-PLAX, with measures obtained minimally influenced by time in cold storage, user imaging the placenta, or technology to capture the images.

Placental-specific sFLT-1: role in pre-eclamptic pathophysiology and its translational possibilities for clinical prediction and diagnosis.

Pre-eclampsia is a common obstetric complication globally responsible for a significant burden of maternal and perinatal morbidity and mortality. Central to its pathophysiology is the anti-angiogenic protein, soluble fms-like tyrosine kinase-1 (sFLT-1). sFLT-1 is released from a range of tissues into the circulation, where it antagonizes the activity of vascular endothelial growth factor and placental growth factor leading to endothelial dysfunction. It is this widespread endothelial dysfunction that produces the clinical features of pre-eclampsia including hypertension and proteinuria. There are multiple splice variants of sFLT-1. One, known as sFLT-1 e15a, evolved quite recently and is only present in humans and higher order primates. This sFLT-1 variant is also the main sFLT-1 secreted from the placenta. Recent work has shown that sFLT-1 e15a is significantly elevated in the placenta and circulation of women with pre-eclampsia. It is also biologically active, capable of causing endothelial dysfunction and the end-organ dysfunction seen in pre-eclampsia. Indeed, the over-expression of sFLT-1 e15a in mice recapitulates the pre-eclamptic phenotype in pregnancy. Therefore, here we propose that sFLT-1 e15a may be the sFLT-1 variant primarily responsible for pre-eclampsia, a uniquely human disease. Furthermore, this placental-specific sFLT-1 variant provides promise for use as an accurate biomarker in the prediction or diagnosis of pre-eclampsia.

Transcription factors E2F1 and E2F3 are expressed in placenta but do not regulate MMP14.

INTRODUCTION: Preeclampsia is a serious complication of pregnancy for which there are no efficacious medical treatments. Soluble endoglin is as an anti-angiogenic factor that contributes to the pathogenesis of the disease, however little is known about its molecular regulation in placenta. Recent data has demonstrated E2F transcription factors directly regulate MMPs in metastatic disease. Of particular interest was the capacity of E2F1 and E2F3 to up-regulate MMP14, a protease that cleaves and releases soluble endoglin from placenta. The aim of this study was to characterize E2F1 and E2F3 in preeclamptic placenta and assess whether silencing affects soluble endoglin release. METHODS: E2F1 and E2F3 mRNA, protein expression and localization were assessed in severe early onset preeclamptic and preterm control placentas (delivered <34 weeks gestation). E2F siRNA was administered to primary trophoblast and primary endothelial cells and effect on MMP14 mRNA expression and soluble endoglin secretion assessed. RESULTS: E2F1 and E2F3 were localized to the syncytiotrophoblast. E2F1 was significantly down regulated in severe preeclamptic placentas, while E2F3 was unchanged. Silencing E2F1 did not decrease MMP14 expression in primary trophoblast or endothelial cells. However, E2F1 silencing resulted in a significant increase in soluble endoglin secretion from both cell types, and silencing of E2F3 also significantly increased soluble endoglin release from primary trophoblast. DISCUSSION: This study demonstrates that E2F1 and E2F3 are present within the syncytiotrophoblast of placenta and that E2F1 is reduced in preeclampsia. Although silencing of either E2F1 or E2F3 does not alter MMP14 expression, both appear to regulate soluble endoglin release.

Placental SEMA3B expression is not altered in severe early onset preeclampsia.

Recent evidence suggests that Semaphorin 3B (SEMA3B) is upregulated in severe preeclampsia, and a major driver of cytotrophoblast aberrations in this disease. Here we independently assess whether SEMA3B expression is altered in a large cohort of severe early onset preeclamptic placentas. We demonstrate that SEMA3B relative mRNA expression and copy number are not changed in PE placentas. We confirm this at the protein level by western blot. Interestingly, exposure of term trophoblasts or explants to hypoxia induced a significant down regulation of SEMA3B mRNA, but a trend towards increased SEMA3B protein expression. We conclude that SEMA3B mRNA and protein is not altered in severe early onset preeclamptic placentas.

Stability of absolute copy number of housekeeping genes in preeclamptic and normal placentas, as measured by digital PCR.

Measuring mRNA expression is fundamental to placental research. Ideally, mRNA transcript numbers are directly quantified. However, PCR analysis using the ΔΔCT method relies on the stability of housekeeping genes and only reports relative expression. Digital PCR (dPCR) directly quantifies mRNA copy number and is more accurate than quantitative PCR. We quantified absolute mRNA copy number of housekeeping genes in normotensive pre-term (n = 20), severe preeclamptic (n = 11) and term (n = 12) placenta using dPCR. Whilst there was some variation, we confirm absolute mRNA copy number of GAPDH, TOP1, CYC1 and YWHAZ in placenta does not significantly alter between these cohorts, or across gestation.

Galectin-7 acts as an adhesion molecule during implantation and increased expression is associated with miscarriage.

INTRODUCTION: Galectins are expressed at the fetal-maternal interface and have multiple roles including during blastocyst implantation. The expression of galectin-7 however has not been investigated in the uterus. We aimed to localise galectin-7 to the endometrium of women with normal fertility and with a history of miscarriage and prospectively determine whether serum levels are altered in women who subsequently miscarry. We also investigated the role of galectin-7 on trophoblast-endometrial epithelial cell adhesion. METHODS: Immunohistochemistry localised galectin-7 to endometrium throughout the menstrual cycle in women (normal fertility or with history of miscarriage) and in first trimester implantation sites. Galectin-7 serum levels were determined by ELISA. We used both endometrial epithelial-trophoblast cell lines and primary cells for cell-cell adhesion experiments. RESULTS: Galectin-7 immunolocalized to endometrial luminal and glandular epithelium in normally fertile women and was upregulated in epithelium and stroma of women with a history of miscarriage. Similarly, galectin-7 serum levels were elevated at 6 weeks gestation in women who subsequently miscarried compared to gestation matched controls. Exogenous galectin-7 reduced endometrial epithelial-trophoblast adhesion in cell-line and primary cell assays. However, when endometrial epithelial cells were isolated from women with endometrial disorders, galectin-7 increased epithelial-trophoblast adhesion. CONCLUSIONS: Galectin-7 is produced by endometrial epithelium and is abnormally elevated in the endometrium of women with a history of miscarriage. Serum levels may be useful as a predictive biomarker of miscarriage. Our data suggests that galectin-7 facilitates adhesion of the embryo to the endometrium and elevated galectin-7 may result in abnormal adhesion.

PLAC4 is upregulated in severe early onset preeclampsia and upregulated with syncytialisation but not hypoxia.

INTRODUCTION: Severe early onset preeclampsia is one of the most serious complications of pregnancy. Placental specific 4 (PLAC4) is very highly expressed in placenta relative to all other tissues. Recently in a biomarker screening study, we found PLAC4 mRNA was significantly upregulated in maternal whole blood and placenta obtained from cases of severe preeclampsia. Intriguingly however, very little is known about its expression or functional role in either normal pregnancy or pregnancies complicated by preeclampsia. METHODS: The objective of this study was to characterize the protein expression and localization of PLAC4 in severe early onset preeclamptic placenta. Given so little of the biology of PLAC4 is known, we also examined whether the expression of PLAC4 alters with syncytialisation or placental hypoxia. RESULTS: We found PLAC4 protein expression was significantly (p < 0.05) upregulated in severe early onset preeclamptic placentas (n = 24) compared to gestationally matched preterm controls (n = 12). PLAC4 protein was specifically localized to the syncytiotrophoblast of preterm, preeclamptic and term placentas. Functional analysis of PLAC4 mRNA and protein expression revealed a significant (p < 0.05) increase with syncytialisation of BeWo cells. However, exposure of either syncytialised BeWo cells or primary term placental explants to hypoxia (1% oxygen) did not alter the expression of either PLAC4 mRNA or protein. CONCLUSION: In conclusion, we have found PLAC4 is significantly upregulated in association with severe preterm preeclampsia. Furthermore, it is upregulated with syncytialisation, but not hypoxia. It is possible PLAC4 may have a role in the pathogenesis of preeclampsia, and its biology merits further investigation.

MT-MMPs in pre-eclamptic placenta: relationship to soluble endoglin production.

INTRODUCTION: Pre-eclampsia is a serious complication of pregnancy, characterized by severe endothelial dysfunction resulting in hypertension, proteinuria and maternal end-organ damage. Soluble endoglin is an anti-angiogenic factor released from placenta that has been linked to severe pre-eclampsia. We recently reported MMP-14 is capable of cleaving endoglin to release soluble endoglin from placenta, however inhibition studies only partially repressed production. To this end we have sought to identify other proteases that mediate endoglin shedding from placenta. MMP-14 is one of six-membrane-type (MT-) MMPs, a sub-family of the MMP superfamily, so named because they are membrane bound. MMP-15 is phylogenetically the closest MMP relative to MMP-14, however its inhibition has no effect on soluble endoglin production from placenta. METHODS: Here we aimed to characterize the remaining four MT-MMPs (MMP-16, -17, -24 and -25) in severe early-onset pre-eclamptic placenta and assess their relative contribution to soluble endoglin production. RESULTS: Immunolocalisation studies revealed MMP-16, -24 and -25 were localized to the syncytiotrophoblast, the same site as endoglin, whilst MMP-17 was predominantly localized to fetal vessels and underlying stroma. MMP-17 protein was significantly (p < 0.05) up-regulated in pre-eclamptic placentas compared to gestationally matched pre-term controls, whilst MMP-25 mRNA was significantly (p < 0.05) down regulated. siRNA knockdown of MMP-16, -17, -24 and -25 in syncytialised BeWo cells did not alter soluble endoglin production in vitro. CONCLUSION: This is the first study to characterize MT-MMP protein localization in human placenta and indicates that MMP-14 is the only MT-MMP that contributes to soluble endoglin production in pre-eclampsia.