BeWo Spheroid Implantation Assay

The BeWo spheroid implantation model used here was adapted from previous studies [29 (link),30 (link),31 (link)]. Briefly, uterine epithelial cells were plated in 96-well plates at a concentration of 2.5 × 10⁴ cells/well and, after adherence, the cells were incubated for 1 h in either the control medium or media containing Boc-2 (1 μM), cyclosporine H (1 μM), or WRW4 (1 μM), and then incubated with AnxA1 (1.35 nM) throughout the implantation time. The spheroids (one spheroid/well) were gently transferred onto adhered uterine epithelial cells and this co-culture was maintained in a humid atmosphere at 5% CO₂ and 37 °C for 2 h. Following this incubation period, the wells were filled up to the brim with culture medium and the plates were sealed with an adhesive film for microplates, inverted, and then centrifuged at 30× g at RT for 5 min. After centrifugation, the plates were kept inverted while they were taken from the centrifuge and examined under a Leica DMi1 inverted microscope (Leica, Shinagawa, Tokyo, Japan) for the presence of the spheroids. The spheroids that disappeared during the centrifugation process were considered to be unattached, and the results were expressed as the percentage of attached spheroids.