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Our in-silico analysis identified Cystatin 6 (CST6), a cysteine protease inhibitor, as located on the placental surface where it might be released into maternal circulation. This study aimed to characterise CST6 and one of its high affinity targets, Legumain (LGMN), in preeclampsia and assess its biomarker potential by measuring levels in maternal circulation. Placental <jats:italic>CST6<\/jats:italic> mRNA expression was significantly increased in 78 pregnancies complicated by early-onset preeclampsia (delivering at &lt;\u200934 weeks\u2019 gestation) relative to 30 gestation matched controls <jats:italic>(P<\/jats:italic>\u2009&lt;\u20090.0001). <jats:italic>LGMN<\/jats:italic> mRNA expression was significantly decreased (<jats:italic>P<\/jats:italic>\u2009=\u20090.0309). Circulating CST6 was increased in 35 pregnancies complicated by early-onset preeclampsia (&lt;\u200934 weeks\u2019 gestation) relative to 27 gestation matched controls (<jats:italic>P<\/jats:italic>\u2009=\u20090.0261), and LGMN levels remained unchanged. At 36 weeks\u2019 gestation, circulating CST6 was significantly increased <jats:italic>(P\u2009=<\/jats:italic>\u20090.001), while LGMN was significantly decreased (<jats:italic>P<\/jats:italic>\u2009=\u20090.0135) in 21 pregnancies preceding diagnosis of preeclampsia at term, compared to 184 pregnancies that did not develop preeclampsia. Human trophoblast stem cells (hTSC) were differentiated into syncytiotrophoblast or extravillous trophoblast (EVT) to evaluate CST6 and LGMN expression in these trophoblast lineages. <jats:italic>CST6<\/jats:italic> and <jats:italic>LGMN<\/jats:italic> mRNA expression were significantly increased across 96\u00a0h after syncytiotrophoblast (<jats:italic>P<\/jats:italic>\u2009=\u20090.0066 and <jats:italic>P<\/jats:italic>\u2009=\u20090.0010 respectively) and EVT differentiation <jats:italic>(P\u2009=<\/jats:italic>\u20090.0618 and <jats:italic>P<\/jats:italic>\u2009=\u20090.0016 respectively), with the highest expression in syncytiotrophoblast. Computational analysis of two publicly available single-cell and single-nuclei RNA sequencing datasets correlated with the expression pattern observed <jats:italic>in vitro.<\/jats:italic> When syncytiotrophoblast cells were exposed to hypoxia (1% O<jats:sub>2<\/jats:sub> vs. 8% O<jats:sub>2<\/jats:sub>), <jats:italic>CST6<\/jats:italic> expression significantly increased (<jats:italic>P<\/jats:italic>\u2009=\u20090.0079), whilst <jats:italic>LGMN<\/jats:italic> expression was unchanged. The vascular endothelium may serve as an additional source of circulating CST6 and LGMN in preeclampsia.\u00a0 Induction of dysfunction in endothelial cells by TNF\u03b1, caused reduced <jats:italic>CST6<\/jats:italic> expression (<jats:italic>P<\/jats:italic>\u2009=\u20090.0036), whilst <jats:italic>LGMN<\/jats:italic> expression remained unchanged. Administering recombinant CST6 to endothelial cells enhanced markers of endothelial dysfunction and LGMN expression in the presence of TNF\u03b1. These findings indicate an inverse relationship between CST6 and LGMN in the placenta and maternal circulation in preeclampsia. We suggest elevated circulating levels of CST6 may be induced by placental hypoxia.\u00a0This study provides novel insight into the dysregulation of CST6 and LGMN in preeclampsia and introduces their potential roles in human pregnancy and associated pathology.  <\/jats:p>","DOI":"10.1038\/s41598-025-96823-9","type":"journal-article","created":{"date-parts":[[2025,4,15]],"date-time":"2025-04-15T11:31:16Z","timestamp":1744716676000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Cystatin 6 (CST6) and Legumain (LGMN) are potential mediators in the pathogenesis of preeclampsia"],"prefix":"10.1038","volume":"15","author":[{"given":"Stefan M.","family":"Botha","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lucy A.","family":"Bartho","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sunhild","family":"Hartmann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ping","family":"Cannon","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna","family":"Nguyen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tuong-Vi","family":"Nguyen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Natasha","family":"Pritchard","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ralf","family":"Dechend","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Olivia","family":"Nonn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephen","family":"Tong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tu\u2019uhevaha J.","family":"Kaitu\u2019u-Lino","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,4,15]]},"reference":[{"issue":"1","key":"96823_CR1","doi-asserted-by":"publisher","first-page":"8","DOI":"10.1038\/s41572-023-00417-6","volume":"9","author":"E Dimitriadis","year":"2023","unstructured":"Dimitriadis, E. et al. 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