Methylation-specific PCR (MSP) assays
MSP assays were performed to assess the DNA methylation status. Briefly, genomic DNA was extracted from placentas and HTR-8/SVneo cells via DNA extraction kits (TIANGEN, Beijing, China) following the manufacturer’s instructions. The bisulfite conversion of DNA was carried out via EZ DNA methylation Gold (ZYMO Research Corporation, USA). Methylation-specific primers were subsequently designed to selectively amplify either methylated or unmethylated DNA sequences of the TP53BP2 promoter region. After amplification, the PCR products were analyzed via 2% gel electrophoresis to visualize the presence or absence of specific bands corresponding to methylated or unmethylated DNA, respectively. The primer sequences for the MSP assays are provided in Table S2.