2.3. RNA Extraction and Quantitative Real-Time PCR Analysis

Total RNA was isolated from implantation sites using Trizol reagent (CWBIO, China) according to the manufacturer's instructions, and RNA quality was determined with a Nano-Drop spectrophotometer (Thermo Scientific, USA). One microgram of RNA was reverse transcribed with RevertAid 1st Strand cDNA Synthesis Kit (Thermo, USA). The sequence of the primers is supplied in [11–13], including specific genes ALK3, BMP2, insulin-like growth factor 1 (IGF-1), Mucin-1, iNOS, and β-actin. Gene expression of β-actin in each sample was quantified as a control. Complementary DNA (cDNA) was then used to amplify these primers by using SYBR Green (Takara, Japan) on an Applied Biosystems 7500 Real-Time PCR System (ABI, USA). The 2^−ΔΔct method was used to analyze gene expression levels as previously described [14].