Abnormalities in amniotic fluid (AF) volume occur in 5-10% of pregnancies in the US and are often associated with increased maternal and fetal morbidity that compromises pregnancy outcome with potential long term adverse consequences. This dramatically increases the need for clinical intervention and thus costs of obstetrical management. Approximately 50% of symptomatic oligohydramnios and polyhydramnios have unknown etiology. However despite the high incidence, there remains no effective therapy for correcting the inappropriate AF volume thereby improving neonatal survival. This is primarily due to the lack of understanding of the mechanisms that regulate AF volume and the causes of the abnormalities that ultimately lead to excessive or reduced AF volumes. The limited amount of research on this subject is largely because of the difficulties in designing studies on relatively unknown and complex regulatory mechanisms without an appropriate human model. Studies in experimental animals have generated most of the current knowledge on AF transport characteristics across the amnion, the rate-limiting layer for transport. However, a critical question remains as to the applicability of these animal data to the human, and the relevance of using animal models for investigations of human biology. To address this deficiency, we propose to develop a human model for studies of AF volume regulation. We have recently developed an in vitro ovine amnion cell model for the study of AF transport across amnion cell monolayers that are readily accessible to experimental manipulations. Our goal is to adapt this cell culture system for transport studies in human amnion cells. The present application is designed to investigate the validity of this in vitro human amnion cell model for studies of AF transport mechanisms in normal pregnancies and in oligohydramnios and polyhydramnios.
In Specific Aim 1, the active and passive components of AF transport across human amnion cell monolayers as a function of solute molecular weight will be determined;the involvement of caveolae in active transcellular transport will be investigated;the role of VEGF165 and its inhibitory isoform in regulating active transport will be explored;and alterations in transport characteristics in amnion cells from oligohydramnios and polyhydramnios will be elucidated.
In Specific Aim 2, the VEGF165 regulation of passive diffusion across water channels aquaporins 1, 3 and 9 in human amnion cells will be examined. Whether passive diffusion across aquaporin channels in amnion cells from oligohydramnios and polyhydramnios is altered will be analyzed.
In Specific Aim 3, the role of VEGF165 as the regulatory factor in the cell signaling events that mediate active caveolar transcytosis will be investigated in amnion cells from normal pregnancy, and from oligohydramnios and polyhydramnios. Overall, the studies will reveal whether this human amnion cell culture model is a suitable model for the investigation of AF volume regulatory mechanisms in human pregnancy, and test the feasibility of using this model to decipher the etiology underlying abnormal AF volume in oligohydramnios and polyhydramnios.
The proposed studies will establish and characterize a human amnion cell model for the investigation of mechanisms mediating amniotic fluid volume regulation in human pregnancy. Knowledge gained from studies using this model will allow the elucidation of the etiology of oligohydramnios and polyhydramnios, and thus provide the scientific basis for designing management protocols and treatment paradigms for amniotic fluid abnormalities.