This study was conducted at the outpatient clinics, wards, and delivery room of the Operating Unit of High Risk Pregnancy of the Fondazione Policlinico A. Gemelli IRCCS of Rome, Italy. The study was approved by the Ethics Committee of the Fondazione Policlinico A. Gemelli IRCCS of Rome (ID:4299 date of approval 23/01/2023) and conducted following the principles of the Declaration of Helsinki. Written informed consent was collected from all patients recruited in the study.

Pregnant women referred to our unit for diagnosis of PP in the period from January 2023 to June 2023 were recruited. The selection of patients diagnosed with PP was performed according to the ISUOG international guidelines [14]. The diagnosis and degree of PAS were defined by pathological assessment of paraffin-embedded inclusions of the placenta in toto, according to the International Federation of Obstetrics and Gynaecology (FIGO).

Women with uncomplicated pregnancy were recruited among those referred to our centre for regular outpatient obstetric check-ups. Exclusion criteria were maternal infectious diseases, autoimmune diseases, multiple pregnancies, pre-eclampsia, intrauterine growth restriction, type 1 and 2 diabetes, and congenital fetal abnormalities.

Three millilitres of venous blood were collected from each patient recruited in this study at diagnosis of PP by ultrasound, between 28 and 37 weeks of gestation, or in controls, matched for gestational age, via venipuncture from the antecubital fossa. Each sample was centrifuged at 1200 g for 10 min at 20 °C to remove the cellular component and obtain the plasma and was, then, frozen in 500 μl aliquots at − 80 °C until further use. All women recruited in this study delivered in our centre and data related to obstetric outcomes and pathological examination of the placenta were analysed.

Plasma samples were thawed at R/T. Each sample was diluted (1:1 vol/vol) with PBS, centrifuged at 3000 g for 30 min at 4 °C and, then, ultracentrifuged at 120,000 g for 90 min at 4 °C using an Optima XPN ultracentrifuge (Beckman Coulter). After ultracentrifugation, the supernatant was removed, and the pellet, containing extracellular vesicles of various sizes, was washed with PBS and centrifuged again at 120,000 g for 90 min at 4 °C. Finally, the supernatant was discarded, and the EV pellet was resuspended with 100 μl of PBS. To quantify STBEV, samples were first lysed with 0.25% NP40 for 60 min; the protein concentration was then determined by Bradford assay.

For the analysis of PLAP expression, STBEV samples isolated from the plasma of patients with PP (n = 35) and patients with normal placentation (n = 35) were lysed on ice for 30 min with NP40 at 0.25% for 60 min. For each sample, a total of 20 μg protein was loaded. Protein content normalisation was performed by ponceau. Samples were denatured at 95° and then subjected to separation by SDS/PAGE (Invitrogen). Subsequently, semi-dry transfer was performed on a PVDF membrane (Biorad, Hercules, CA, USA). Non-specific bands were blocked with TBS-T (20 mM Tris/HCl, 137 mM NaCl, 0.1%Tween-20, pH 7.6) containing 5% milk (Santa Cruz Biotechnology Inc., Dallas, TX, USA) for 1 h at R/T. The membranes were incubated in saline buffer and 0.05% Tween 20 (TBS-T) containing 1% BSA at 4 °C overnight with a murine monoclonal anti-PLAP antibody (NDOG2, University of Oxford, Oxford, UK; 1 μg/ml), specific for syncytiotrophoblast, recognising placental alkaline phosphatase (PLAP), and a rabbit monoclonal anti-CD63 antibody (Abcam, UK), a marker of exosomes.

The membranes were washed in TBS-T before incubation with horseradish peroxidase–conjugated secondary antibody (1:4000; Dako, Glostrup, Denmark) for 1 h at R/T. The antibody used was diluted in the blocking buffer. After washing, the membranes were treated with a chemiluminescence system (PierceTM, Thermo Fischer Scientific, Waltham, MA, USA) and exposed to Hyperfilm ECL (GE Healthcare Life Sciences, Cleveland, OH, USA). Western blot densitometric analysis was made using Nine Alliance software (Uvitec Alliance, Cambridge, UK).

The analysis of STBEV was performed by multi-colour flow cytometry using a CytoFLEX S cytometer (Beckman Coulter) equipped with violet laser (405 nm) excitation sources. This instrument is capable of recording the SSC (side scatter) for the blue laser (BSSC) and the violet laser (VSSC). The flow cytometer was calibrated using the Megamix-Plus FSC beads, which emit FITC of different sizes (100, 300, 500, and 900 nm). The number of STBEVs was measured using the instrument’s cell counting function based on a calibrated peristaltic pump per loaded sample. STBEVs were marked with CellTraceTM Calcein Violet (ThermoFisher Scientific, Waltham, MA, USA) to distinguish intact vesicles from cell debris.

Before the STBEV samples were run, the filtered PBS was analysed three times for 2 min to remove the background (approximately 100 events per second). To confirm the placental origin of the vesicles, STBEVs were labelled with an anti-PLAP (placental anti-alkaline phosphatase) PE (phycoerythrin)–conjugated murine monoclonal antibody (NDOG2, University of Oxford, Oxford, UK) or its control IgG1 PE-conjugated (Biolegend UK Ltd., Cambridge, UK). PLAP protein is mainly expressed in placental tissue (https://www.proteinatlas.org/) and it is commonly used to distinguish vesicles released from the placenta from those derived from other cell types [15]. PLAP-positive vesicles were normalised to positive calcein events. EVs isolated from colorectal cancer cell culture medium were used as a negative control to verify PLAP positivity. Before use, all antibodies were filtered through Nanosep 0.2 mm centrifuge devices (Pall Life Sciences) to minimise interference from background particles. All antibodies were also titrated to ensure they were used at optimal concentrations. Fluorochrome compensation for the multi-coloured STBEV cytofluorometer was performed using BD Compbeads (BD Biosciences), labelled with fluorescence-conjugated antibodies.

All samples were incubated for 1 h at 4 °C in the dark with the anti-PLAP PE-conjugated antibody (NDOG2, University of Oxford, Oxford, UK) or its control IgG1 PE-conjugated (Biolegend UK Ltd., Cambridge, UK). Subsequently, filtered PBS was added to the labelled samples to reach 500 μl and evaluated immediately in the cytofluorometer (Beckman Coulter). The gates were initially organised so that ≤ 1% of the positive STBEVs fell within the negative controls. Data were acquired and analysed by CytExpert 2.2TM software (version 2.2, CytoFLEX S, Beckman Coulter, Milan, Italy).

The clinical data are shown as the mean ± standard deviation (SD) or percentage (%), depending on the type of variable, and were analysed using Student’s T test. All data was analysed for normal distribution using the Shapiro–Wilk test and analysed by the Mann–Whitney U test performed with Prism software version 9.0. For all analyses, p < 0.05 was considered significant.