NAD(P)H oxidase enzymes are a major source of superoxide generation in vascular smooth muscle, vascular endothelium and in neutrophils. Superoxide has been implicated in signalling events leading to upregulation of antioxidant genes, angiogenesis, proliferation and matrix remodelling. Recently, it has been demonstrated that NAD(P)H oxidase is expressed in human placenta at term and is a major source of placental superoxide (Matsubara et al. 2001; Dechend et al. 2003). However it is not known whether the enzyme is active in placenta earlier in pregnancy.

The Institutional Medical Ethical Review Committee approved the study protocol. Superoxide production was assessed using a lucigenin chemiluminescence method in placentas obtained with informed consent from women undergoing termination of pregnancy (n = 19; mean gestational age ± S.E.M. is 11⁺⁶ ± 1⁺⁰ wks) and in placentas delivered by elective caesarean section after a full term uncomplicated pregnancy (n = 15; gestational age 38⁺⁶ ± 0⁺⁴ wks). NADPH was added to generate NAD(P)H oxidase mediated superoxide synthesis which was detected by lucigenin. Specificity for NAD(P)H oxidase was assessed using specific inhibitors (end-concentration; target) L-NAME (100 µmol/l; nitric oxide synthase), Rotenone (20 µmol/l; mitochondrial O₂-{special}synthesis), allopurinol (100 µmol/l; xanthine oxidase), DPI (10 µmol/l; NAD(P)H oxidase) and TIRON (50 mmol/l; O₂-{special} scavenger). The area under the curve (AUC) was calculated as a measure of total superoxide production (O₂-{special}_total) and expressed as arbitrary light units (ALU). Gestational differences in superoxide generation were calculated using linear regression with robust standard errors in STATA 8.

Placental superoxide synthesis (O₂-{special}_total) was higher in early pregnancy placentas compared with those obtained from women with full term pregnancy (geometric mean ± S.E.M.: 81.0 ± 6.4 versus 29.4 ± 3.5: P < 0.0001). The signal was unaffected by addition of L-NAME, rotenone, or allopurinol, but was totally inhibited by both DPI and TIRON. This indicates specificity of the signal for NAD(P)H oxidase. This study confirms that there is substantial basal NAD(P)H activity in normal human placenta in the third trimester but also shows that activity is higher in early pregnancy. Raised activity early in gestation could regulate expression of redox sensitive genes including VEGF and others, which may contribute to placental growth and trophoblast proliferation. Superoxide generation may also serve to upregulate antioxidant defences in the early placenta. The role of NAD(P)H oxidase in the placenta requires further investigation.

In conclusion we have demonstrated an elevated superoxide production by NAD(P)H oxidase in early human placenta tissue. The results justify further and more detailed investigation of the role of this enzyme in human placental physiology and pathophysiology.

This work was supported by Tommy’s the Baby Charity.