Isolation of cfDNA from spent culture media and its association with implantation rate and maternal immunomodulation

Abstract

Objectives: This investigation aims to evaluate the association between the concentration of cell-free DNA (cfDNA) in the spent culture medium (SCM) with implantation rate and the maternal immune system in the in vitro fertilization (IVF). Method: In this study, 30 patients were selected based on the inclusion and exclusion criteria and their embryos were cultured until the blastocyst stage. For each patient, one blastocyst was graded according to Gardner's criteria and transferred to uterus. The concentration of cfDNA in embryo culture medium was evaluated by nanodrop spectrophotometry with a wavelength of 260 nm. The percentage of Th1, Th2, Th17, Treg, NK cells, and NK cells cytotoxicity in PBMC isolated from 30 patients in the 2end and 12th week after embryo transfer was investigated using flow cytometry technique. Results: According to the results, in the group of high quality embryos, the concentration of cfDNA was 24.62 ±9.201 and in the group of low quality embryos it was 21.58±9.768, and there was no significant relationship between the concentration of cfDNA and the quality of the embryos. The concentration of cfDNA in the β-HCG (-), β-HCG (+), and ongoing pregnancy groups were 20.70 ±9.224 ng/μL, 27.97 ± 7.990 ng/μL, and 28.91 ± 8.566 ng/μL, respectively. The ratio of Th1/Th2 and Th17/Treg reduced significantly (p<0.0001) in pregnant women, as well as the level of NK cells and NK cytotoxicity cells fell dramatically in the ongoing pregnancy group (p=0.0005 and 0.0048). And no significant relationship was observed between the cfDNA concentration and the amount of immunological cells in the three groups. Conclusion: Our investigation revealed that the concentration level of cfDNA in SCM could not be associated with implantation rate, prediction of ongoing pregnancy, and maternal immune system.