# Determination of the Number of Circulating Small Extracellular Vesicles in Pregnancy Using the Novel Marker CD9

## TL;DR

This study explores the role of small extracellular vesicles in pregnancy and preeclampsia using a new marker, CD9, finding that their levels rise in established preeclampsia but not in early pregnancy.

## Contribution

The study introduces CD9 as a novel and effective marker for quantifying small extracellular vesicles in plasma.

## Key findings

- CD9 was consistently detected as a reliable marker for small EVs, unlike CD63 and TSG101.
- Small EV levels were significantly elevated in both early- and late-onset preeclampsia compared to controls.
- First-trimester EV levels did not differ between women who later developed preeclampsia and normal controls.

## Abstract

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.