Hystero-embryoscopy as a tool for RPL immunological research at the maternal-fetal interface

Introduction: An abnormal immune response at the fetal-maternal interface is expected in 50–60 % of unexplained Recurrent Pregnancy Loss (uRPL) cases. Detected immunophenotypes in uRPL could help in risk assessment, prognosis and therapy. The study of immune mechanisms at the fetal-maternal interface is crucial, but the technique used for research has limitations, including contamination and invasiveness, which can trigger immune responses. Hystero-embryoscopy may provide a precise method for studying the immunological factors involved in RPL at the maternal-fetal interface, reducing bias from sample collection techniques. Our aim is to assess its effectiveness in obtaining high-quality samples to study the immunological basis of RPL. Methods: This is a multicenter prospective study in which 10 women with a first-trimester ongoing miscarriage were enrolled and received surgical treatment at the University Hospital Federico II in Naples. Embryo-hysteroscopy was used to selectively obtain decidual and chorionic villous tissues separately, from the maternal-fetal interface. Transcriptome sequencing was performed at Regina Elena National Cancer Institute in Rome. Results: RNA integrity numbers (RIN) satisfied the minimum quality requirements (median RIN considering all samples was 8.4 ± 1.1) and RNA sequencing exhibited adequate sequencing depth (the mean of Uniquely Mapped Reads considering all samples was 52.1 ± 7.7), ensuring reliable downstream analysis. The bioinformatics analysis demonstrated the absence of cross-tissue contamination due to the clear separation between the transcriptional profiles of decidual and chorionic villous tissues: CD45 immunostaining and pathological validation confirmed these findings. Conclusions: These findings demonstrate that hystero-embryoscopy enables precise immunological profiling at the maternal-fetal interface while minimizing sample contamination.