Small extracellular vesicles (small EVs) play pivotal roles in intercellular communication and pregnancy maintenance, but their clinical significance in preeclampsia (PE) remains unclear. We obtained plasma samples from non-pregnant women, healthy pregnant women, and patients with early-onset (EoPE) and late-onset PE (LoPE). Small EVs were isolated using ultracentrifugation and validated using transmission electron microscopy and nanoparticle tracking analysis; in addition, Western blotting was performed to identify suitable surface markers for plasma-derived small EVs. In our analysis, we consistently detected cluster of differentiation 9 (CD9), whereas classical markers such as cluster of differentiation 63 (CD63) and tumor susceptibility gene 101 (TSG101) were absent. In a prospective, nested case–control study, we analyzed first-trimester samples by using a CD9-based ELISA for small-EV quantification. The number of small EVs did not significantly differ between non-pregnant and healthy pregnant women regardless of the gestational age. However, EVs were significantly elevated in both EoPE (3.5-fold) and LoPE (1.5-fold) compared with matched controls. First-trimester EV levels did not show differences between women who later developed PE and normal controls. These findings indicate that CD9 is a promising marker for plasma-derived small EVs and that an elevated number of small EVs is associated with established PE but has limited predictive value in early pregnancy. Further studies are required to elucidate the cellular origin and clinical implications of small EVs in PE.

Background: Small extracellular vesicles (small EVs) play pivotal roles in intercellular communication and pregnancy maintenance. Their clinical significance in preeclampsia (PE) remains unclear. Methods: Plasma samples were obtained from non-pregnant women, healthy pregnant women, and patients with early-onset (EoPE) and late-onset PE (LoPE). Small EVs were isolated by ultracentrifugation and validated using transmission electron microscopy and nanoparticle tracking analysis. Western blotting was performed to identify suitable surface markers for plasma-derived small EVs. The quantification was conducted using a cluster of differentiation 9 (CD9)-based ELISA. A prospective nested case-control study analyzed >1,400 first-trimester samples. Results: CD9 was consistently detected in plasma-derived small EVs, whereas classical markers such as cluster of differentiation 63 (CD63) and tumor susceptibility gene 101 (TSG101) were absent. the number of small EVs did not significantly differ between non-pregnant and healthy pregnant women regardless of the gestational age. However, EVs were significantly elevated in both EoPE (3.5-fold) and LoPE (1.5-fold) compared with matched controls. First-trimester EV levels did not show the difference between women who later developed PE and normal controls. Conclusions: CD9 is a promising marker for plasma-derived small EVs. Elevated numbers of small EVs are associated with established PE but show limited predictive value in early pregnancy. Further studies are required to elucidate the cellular origin and clinical implications of small EVs in PE.