Key points Nitric oxide (NO) is a gasotransmitter with important physiological and pathophysiological roles in pregnancy. There is limited information available about the sources and metabolism of NO and its bioactive metabolites (NOx) in both normal and complicated pregnancies. The present study characterized and quantified endogenous NOx in human and mouse placenta following determination of the stability of exogenous NOx in placental homogenates. NOx have differential stability in placental homogenates. NO and iron nitrosyl species (FeNOs), are relatively unstable in placental homogenates from normal placentas. Exogenous NO, nitrite and nitrosothiols react with placental homogenates to form iron nitrosyl complexes. FeNOs were also detected endogenously in mouse and human placenta. NOx levels in placental villous tissue are increased in fetal growth restriction vs. placentas from women with normal pregnancies, particularly in fetal growth restriction associated with pre‐eclampsia. Villitis was not associated, however, with an increase in NOx levels in either normotensive or pre‐eclamptic placentas. The results call for further investigation of FeNOs in normal and complicated pregnancies. Abstract Nitric oxide (NO) is a gasotransmitter with important roles in pregnancy under both physiological and pathophysiological conditions. Although products of NO metabolism (NOx) also have significant bioactivity, little is known about the role of NO and NOx under conditions of aberrant placental inflammation during pregnancy. An ozone‐based chemiluminescence approach was used to investigate the stability and metabolic fate of NOx in human placental homogenates from uncomplicated pregnancies in healthy mothers compared to that in placental tissue from normotensive and pre‐eclamptic pregnancies complicated with fetal growth restriction (FGR) with and without villitis of unknown aetiology. We hypothesized that placental NOx would be increased in FGR vs. normal tissue, and be further increased in villitis vs. non‐villitis placentas. Findings indicate that nitrate, nitrite and nitrosothiols, but not NO or iron nitrosyl species (FeNOs), are relatively stable in placental homogenates from normal placentas, and that NO, nitrite and nitrosothiols react with placental homogenates to form iron nitrosyl complexes. Furthermore, NOx levels in placental villous tissue are increased in FGR vs. placentas from women with normal pregnancies, particularly in FGR associated with pre‐eclampsia. However, in contrast to our hypothesis, villitis was not associated with an increase in NOx levels in either normotensive or pre‐eclamptic placentas. Our results also strongly support the involvement of FeNOs in both mouse and human placenta, and call for their further study as a critical mechanistic link between pre‐eclampsia and fetal growth restriction.

Lmx1b is a LIM homeodomain transcription factor important for normal development of many organ systems including the cerebellum, eyes, limbs, and kidneys. In humans, haploinsufficiency of Lmx1b results in a Nail‐Patella syndrome, a disorder characterized by hypoplastic nails, bone deformities, glaucoma, and even kidney failure in severe cases. Despite its importance in developing these organ systems, the induction and regulation of Lmx1b is poorly understood. Previously using ChIP‐seq analysis, we identified two upstream, highly conserved Lmx1b‐associated regulatory modules (LARM1/2) bound by Lmx1b that were found to be associated with limb‐specific auto‐amplification of Lmx1b expression vital for limb dorsalization. We have subsequently identified another highly conserved region 10 kb upstream to these limb‐specific cis‐regulatory modules (CRMs), although it is not bound by Lmx1b. Yet, due to its active chromatin marks and close proximity to the Lmx1b locus, we hypothesized that this third regulatory module (LARM3) may also play a part in modulating Lmx1b expression. We generated a GFP‐reporter construct containing the isolated LARM3 sequence from mouse genomic DNA to test this hypothesis. To determine limb activity, this LARM3 construct was electoporated into the presumptive limb of Hamilton‐Hamburger stage 14 (HH14) chicken embryos. To determine activity in other organ systems such as the kidney and eye, the LARM3 construct was electroporated into a whole embryo (HH4) and in the presumptive lens placode (HH10), respectively. These embryos were viewed by fluorescent microscopy a few days after transfection to determine enhancer activity. LARM3 was found to be active in the limb mesoderm without a dorsoventral bias, showed punctate activity in the intermediate mesoderm that was consistent with mesonephric development (kidney), and was also active in the presumptive lens placode. The multi‐organ enhancer activity of LARM3 suggests a global role in regulating Lmx1b‐mediated development. Support or Funding Information Funded in part by the National Organization for Rare Diseases