The human placenta expresses both HTRA1 and A1AT, and umbilical cord blood NIP levels correlate with plasma HTRA1 levels after birth. (A) Human placental tissue from both term and preterm gestations was subjected to immunofluorescence studies using primary antibodies targeting HTRA1 (green) and the C-terminus of A1AT (blue). DAPI was used as a nuclear counterstain (aqua). (Both 20× and 60× objectives were used; scale bars, 50 μm.) These images are representative of experiments performed by using 3 different placental samples from each group. Yellow arrows indicate juxtaposition of A1AT and HTRA1. (B) We further determined placental HTRA1 (red) and A1AT (green) protein expression via western blotting in placental extracts from both term and preterm gestations. The western blot is representative of 3 separate experiments in each group. We next assessed plasma concentrations of both HTRA1 and A1AT in plasma isolated from healthy adults and umbilical cord blood isolated from both healthy term neonates and preterm neonates. (C) The y-axis depicts plasma HTRA1 concentration ± SEM for adult plasma, term umbilical cord blood plasma, and preterm umbilical cord blood plasma. (D) The y-axis depicts plasma A1AT concentration ± SEM for adult plasma, term umbilical cord blood plasma, and preterm umbilical cord blood plasma. (E) Umbilical cord blood plasma and peripheral blood taken from the same neonate 3 days after delivery were subjected to western blotting using a primary antibody to detect the C-terminus of A1AT (green). nNIF, another NIP, was used as a positive control (Control). This western blot is representative of 3 separate experiments using plasma samples from 3 different neonates. (F) The plasma concentration was determined of HTRA1 in adult peripheral blood plasma, umbilical cord blood plasma, and plasma from neonates 3 days after birth. The y-axis depicts plasma HTRA1 concentration ± SEM. *P < .05 for umbilical cord blood vs both adult and day 3 neonatal plasma. **P < .01, ***P < .001. MW, molecular weight.