Normal decidual samples with no previous pregnancy loss were obtained from elective terminations of apparently normal pregnancies. The elective termination was performed via dilation and curettage (D&C). For the decidua samples from abnormal pregnancies, the patients were advised to obtain an induced abortion after a clinical diagnosis of embryo demise. The diagnosis of embryo demise was following the guidelines from First Affiliated Hospital of University of Science and Technology of China, such as mean sac diameter of 25 mm or greater and no embryo or absence of an embryo with heartbeat 2 weeks or more after a scan that showed a gestational sac without a yolk sac. Fetal heart activity was assessed using Doppler ultrasound at 7–9 weeks of gestation. When abnormal fetal heart activity was observed, the patients were advised to be tested for serum β-human chorionic gonadotropin levels, with additional ultrasounds every other week. D&C was performed within 24 h of ultrasonographic documentation of fetal loss.

When decidua samples were obtained, chorionic villi and blood clots were identified and carefully separated from the maternal decidua. The chorionic villi were then sent for cytogenetic analysis. Normal embryo karyotypes were identified to enable the exclusion of genetic or mechanical causes for embryo demise. We also excluded patients with clinical symptoms of heavy bleeding and cramps prior to induced abortion. The mean gestational ages are 7.24 weeks in controls and 8.50 in RPL patients. All of the decidua samples were collected from the First Affiliated Hospital of the University of Science and Technology of China. Before surgery, informed consent was obtained from each patient. Ethical approvals were obtained from the ethics committee of the University of Science and Technology of China.

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