Dysregulation of the Heparanase/TFPI-2/Heparan Sulfate Axis in Recurrent Pregnancy Loss

Recurrent pregnancy loss (RPL) is a clinically heterogeneous reproductive disorder, and in many women the underlying mechanism remains unexplained by standard thrombophilia evaluation. Pregnancy is characterized by a tightly regulated hemostatic balance at the maternal-placental interface. Heparanase, tissue factor pathway inhibitor 2 (TFPI-2), and heparan sulfate (HS) are expressed in placental and hemostatic pathways and may contribute to coagulation-related pregnancy complications. We investigated whether the heparanase/TFPI-2/HS axis is altered in women with RPL. Blood samples were obtained from 69 women with RPL and 47 control women with at least two normal deliveries and no history of pregnancy-related vascular complications. Samples were collected at least three months after pregnancy and in the absence of hormonal therapy. Plasma and white blood cell samples were analyzed using an enzyme-linked immunosorbent assay, Western blotting, real-time polymerase chain reaction, co-immunoprecipitation, and Sanger sequencing. Mechanistic experiments using heparanase-derived peptide 16 and TFPI-2-derived peptide 6 were performed in MCF-7 and JAR cells. We found that women with RPL had increased plasma heparanase levels, heparanase procoagulant activity, HS levels, and heparanase mRNA expression compared with controls. TFPI-2 protein and mRNA expression were also elevated. The heparanase rs4693608 AA genotype was more frequent in women with RPL and, together with rs4364254 TT, was associated with increased heparanase expression and procoagulant activity. Although TFPI-2 levels were higher in RPL, co-immunoprecipitation showed reduced heparanase/TFPI-2 complex formation, suggesting impaired inhibitory regulation. HS modulated heparanase binding to TF and TFPI-2 in a concentration-dependent, bell-shaped manner. Heparanase-derived peptide 16 increased TFPI-2 expression, supporting a regulatory feedback loop. In conclusion, RPL is associated with dysregulation of the heparanase/TFPI-2/HS axis, including increased heparanase expression and activity, altered heparanase/TFPI-2 interaction, and heparanase-related genetic variation. These findings suggest a coagulation-related reproductive mechanism that may contribute to pregnancy loss and warrants validation in larger prospective studies.