*2.7. Histology*

Amnion biopsies were fixed for 24 h in 4% formalin and samples were embedded in para ffin. Immunohistochemistry against caspase 3 was performed with an anti-cleaved caspase 3 antibody 1:100 (Cell Signaling Technology, Danvers, MA, USA). Immunohistochemical negative controls were performed by replacing the primary antibody with bu ffer. Immunohistochemical sections were quantified with ImageJ software (National Institutes of Health, version 1.51j8, Bethesda, MD, USA). n = 3 (biological replicates).

#### *2.8. Transmission Electron Microscopy*

Biopsies were fixed with 2.5% glutaraldehyde and 2% paraformaldehyde for 2–3 h at room temperature and post-fixed with 1% OsO4 in 0.1 M cacodylate bu ffer. Dehydration and embedding in Epon resin were carried out according to standard protocols. Sections (70 nm) were contrasted with 2% uranyl acetate. Images were acquired with an electron microscope (Tecnai20, FEI Europe, Eindhoven, Netherlands) equipped with a 4K EagleCCD camera and processed with Adobe Photoshop. n = 2 (biological replicates).

#### *2.9. Reverse-Transcription Quantitative PCR Analysis*

Samples of hAM biopsies (8 mm diameter) were snap-frozen in liquid nitrogen and kept at −80 ◦C until further analysis. Total RNA extraction, mRNA reverse transcription and qPCR were performed by TAmiRNA GmbH (Vienna, city, Austria). n = 3 (biological replicates).

Total RNA extraction: total RNA was extracted from 10 amnion biopsies (8 mm diameter) using the miRNeasy Mini Kit (Qiagen, Hilden, Germany). Tissue was homogenized with 700 μL Qiazol; following incubation at room temperature for 5 min, 140 μL chloroform was added to the lysates, which were incubated for 3 min at room temperature and centrifuged at 12,000× *g* for 15 min at 4 ◦C. Precisely 350 μL of the upper aqueous phase was transferred to a miRNeasy mini column, and RNA was precipitated with 525 μL ethanol followed by automated washing with RPE and RWT bu ffer in a Qiacube liquid handling robot. Finally, total RNA was eluted in 30 μL nuclease free water and stored at −80 ◦C until further analysis.

mRNA reverse transcription and qPCR (RT-qPCR): messenger RNA quantification was performed using the TATAA Grandscript cDNA synthesis and SYBR Grandmaster mix kit (TATAA Biocenter, Göteborg, Sweden). Total RNA (500 ng) was used for reverse transcription and all steps were carried out according to recommendations by the manufacturer. PCR amplification was performed in a 96 well format in a Roche LC480 II instrument (Roche, Mannheim, Germany) with the following settings: 95 ◦C for 30 s followed by 45 cycles of 95 ◦C for 5 s, 63 ◦C for 15 s and 72 ◦C for 10 s and subsequent melting curve analysis. To calculate the cycle of quantification values (Cq-values), the second derivative method was used. Cq-values were subsequently normalized to the geometric mean of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-actin (ACTB), hypoxanthine phosphoribosyltransferase (HPRT1) and ubiquitin C (UBC) mRNA levels, by subtracting the gene of interest Cq-value from the respective geometric mean of the four references. Primer sequences of BAX and BCL-2 used for mRNA reverse-transcription quantitative PCR analysis are shown in Table S1.
