: Aim 1 is to examine the influence of hypoxia on differentiation of trophoblast amino acid transporters and the function of PPARg in the differentiation of hypoxic trophoblast. Aim 2 is to dissect the mechanisms of hypoxia-induced trophoblast apoptosis. 1) SIGNIFICANCE: This twice-revised proposal from an experienced investigator addresses a critical, understudied area of reproductive biology, which is how changes in oxygen tension might influence differentiation of trophoblast and its susceptibility to apoptosis. Because low oxygen tension is a normal feature of implantation and a pathological feature of later gestation it is of the utmost importance to learn how this condition impacts on placental development and function. If the investigator is able to separate oxygen-associated mechanisms regulating differentiation from oxygen-associated mechanisms related to apoptosis, much will have been achieved in understanding trophoblast biology. 2) APPROACH: The investigator of this project is highly knowledgeable regarding trophoblast structures and functions in human placentas and is skilled in culturing trophoblast cells and observing their behavior. Progress during the previous funding period as judged by the number of publications was excellent (13 papers, 18 abstracts). Several of these papers directly support the Aims of the proposed study although none were included as appendices. New experiments and attention to some of the reviewers' comments have resulted in total revision of this proposal on two separate occasions. However, many comments from the reviewers were still not addressed in the Introduction and a number of questions remain unanswered. In this densely written application containing 23 figures in the Preliminary Studies section, the investigator presents a major body of data on the effects of hypoxia. The application is better focused than before but still has two major goals, either of which could comprise a full application. Aim I has multiple parts, the first of which is somewhat unrelated to the others. The investigator proposes to study the effects of hypoxia on transport of neutral amino acids in cultured trophoblasts. This seems to have been done in large part (Preliminary Studies, Table 1). The second and third sets of experiments are exciting and novel. Here, PPARg is investigated for its potential role in hypoxia-induced failure of differentiation. The experiments are straightforward and entirely within the expertise of the investigative team, and should result in new information that is directly relevant to autocrine mechanisms underlying differentiation. However, it is not entirely clear whether these experiments will be done on both primary trophoblasts and the BeWo line or just on the BeWo line because of poor transfection efficiency in primary cells (P. 37, second para.). Nor is it certain why thromboxane was eliminated as a potential mediator. The investigator is well placed to investigate similar mechanisms in FGR pregnancies, and the set of experiments in this section can also be expected to be informative. In Aim II, the goal is to establish relationships between hypoxia and trophoblast apoptosis. Here, the investigator tackles an extremely difficult problem but does not present it clearly or concisely and does not explain some puzzling observations. For example, some in vitro and in vivo studies seem to point to a major apoptosis-inducing effect of hypoxia on cytotrophoblasts but not multinucleated syncytium-like trophoblast cells. Yet the recent publication in the AJOG reports that when cytotrophoblasts from human term placentas are cultured in low oxygen, viability remains unchanged through 72 hr of culture. It is not entirely clear how these in vitro results relate to the data shown in Figure 14, where hypoxic conditions increase TUNEL staining in cultured term cytotrophoblasts, or in Figure 17, where hypoxia increases Bax and decreases Bcl-2 at 24 hr or the observation that apoptosis in an FGR placenta is in the syncytial knots, not cytotrophoblast. In any event, a more enlightening discussion of this issue would be helpful; differentiation and apoptosis are often thought to be linked and trophoblast would be a notable exception. Despite these weaknesses and defects, experiments on the effects of hypoxia on Bcl-2, p53 and caspases may yield some interesting information. Novel experiments in this Aim include those directed toward trying to understand how apoptosis in a multinucleated cytoplasm might be different from apoptosis in single cells. This Aim uses sex-sorted nuclei under hypoxic conditions. However, it is difficult to see how the investigator expects to obtain syncytia when he has shown that low oxygen inhibits this phenomenon.